U.S. patent application number 10/996573 was filed with the patent office on 2005-07-14 for polymer arrays.
Invention is credited to Fodor, Stephen P.A., Pirrung, Michael C., Read, J. Leighton, Stryer, Lubert.
Application Number | 20050153362 10/996573 |
Document ID | / |
Family ID | 34744048 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050153362 |
Kind Code |
A1 |
Pirrung, Michael C. ; et
al. |
July 14, 2005 |
Polymer arrays
Abstract
The present invention provides methods and apparatus for
sequencing, fingerprinting and mapping biological macromolecules,
typically biological polymers. The methods make use of a plurality
of sequence specific recognition reagents which can also be used
for classification of biological samples, and to characterize their
sources.
Inventors: |
Pirrung, Michael C.; (Chapel
Hill, NC) ; Stryer, Lubert; (Stanford, CA) ;
Fodor, Stephen P.A.; (Palo Alto, CA) ; Read, J.
Leighton; (Palo Alto, CA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
34744048 |
Appl. No.: |
10/996573 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10996573 |
Nov 23, 2004 |
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09653761 |
Sep 1, 2000 |
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09653761 |
Sep 1, 2000 |
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09557875 |
Apr 24, 2000 |
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6610482 |
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09557875 |
Apr 24, 2000 |
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09056927 |
Apr 8, 1998 |
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6197506 |
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09056927 |
Apr 8, 1998 |
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08670118 |
Jun 25, 1996 |
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5800992 |
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08670118 |
Jun 25, 1996 |
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08168904 |
Dec 15, 1993 |
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08168904 |
Dec 15, 1993 |
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07624114 |
Dec 6, 1990 |
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07624114 |
Dec 6, 1990 |
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07362901 |
Jun 7, 1989 |
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07624114 |
Dec 6, 1990 |
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07492462 |
Mar 7, 1990 |
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5143854 |
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09557875 |
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08348471 |
Nov 30, 1994 |
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6420169 |
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08348471 |
Nov 30, 1994 |
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07805727 |
Dec 6, 1991 |
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5424186 |
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07805727 |
Dec 6, 1991 |
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07624120 |
Dec 6, 1990 |
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07624120 |
Dec 6, 1990 |
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07492462 |
Mar 7, 1990 |
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5143854 |
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07492462 |
Mar 7, 1990 |
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07362901 |
Jun 7, 1989 |
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Current U.S.
Class: |
435/7.1 ;
435/287.2 |
Current CPC
Class: |
Y10S 436/807 20130101;
B01J 2219/00436 20130101; B82Y 30/00 20130101; G03F 7/265 20130101;
G03F 7/00 20130101; C07K 7/06 20130101; C12Q 1/6816 20130101; C40B
60/14 20130101; B01J 2219/00626 20130101; C12Q 1/6837 20130101;
B01J 2219/00612 20130101; B01J 2219/00659 20130101; C07K 17/14
20130101; C12Q 1/6809 20130101; G03F 7/38 20130101; C12Q 1/6827
20130101; B01J 2219/00637 20130101; B01J 2219/00621 20130101; B01J
2219/00641 20130101; G01N 21/6452 20130101; C07H 19/04 20130101;
C07H 19/10 20130101; B01J 2219/00468 20130101; B01J 2219/00608
20130101; C07D 317/62 20130101; B01J 2219/005 20130101; B01J
2219/00529 20130101; B01J 2219/00596 20130101; B01J 2219/00695
20130101; C12Q 1/6874 20130101; B82Y 10/00 20130101; C07D 263/44
20130101; B01J 2219/00459 20130101; B01J 2219/00585 20130101; G01N
21/6428 20130101; B01J 2219/00527 20130101; B01J 2219/00689
20130101; B01J 2219/00725 20130101; C07B 2200/11 20130101; B01J
2219/00434 20130101; C07H 21/00 20130101; C07K 1/042 20130101; B01J
2219/00432 20130101; C07C 229/14 20130101; C07K 1/047 20130101;
C40B 40/06 20130101; B01J 2219/00648 20130101; B01J 2219/00711
20130101; C07C 229/16 20130101; G11C 13/0014 20130101; B01J
2219/00315 20130101; B01J 2219/00531 20130101; B01J 2219/00605
20130101; B01J 2219/00644 20130101; B01J 2219/00722 20130101; C07K
17/06 20130101; B01J 2219/0059 20130101; B01J 19/0046 20130101;
C07K 1/045 20130101; C40B 40/10 20130101; B01J 2219/00475 20130101;
G11C 13/0019 20130101; B01J 2219/00617 20130101; G01N 15/1475
20130101; B01J 2219/0061 20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2 |
International
Class: |
C12Q 001/68; G01N
033/53; C12M 001/34 |
Claims
What is claimed is:
1. A substrate with a surface comprising a plurality of
polypeptides with different, known sequences bound to the surface
in positionally defined locations, at a density exceeding 400
different polypeptides occupying a total area of less than 1
cm.sup.2 on said substrate, each of said polypeptides having a
different polypeptide sequence.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/653,761, filed Sep. 1, 2000, which is a continuation of U.S.
Ser. No. 09/557,875, filed Apr. 24, 2000 (the teachings of both of
which are incorporated herein by reference), which is a
continuation of U.S. Ser. No. 09/056,927, filed Apr. 8, 1998, now
U.S. Pat. No. 6,197,506, which is a continuation of U.S. Ser. No.
08/670,118, filed Jun. 25, 1996, now U.S. Pat. No. 5,800,992, which
is a divisional of U.S. Ser. No. 08/168,904, filed Dec. 15, 1993,
now abandoned, which is a continuation of U.S. Ser. No. 07/624,114,
filed Dec. 6, 1990, now abandoned, which is a continuation-in-part
of U.S. Ser. No. 07/362,901, filed on Jun. 7, 1989, now abandoned;
and U.S. Ser. No. 07/492,462, filed on Mar. 7, 1990, now U.S. Pat.
No. 5,143,854. Application Ser. No. 09/557,875, filed on Apr. 24,
2000, is also a continuation-in-part of U.S. Ser. No. 08/348,471,
filed Nov. 30, 1994, which is a continuation of U.S. Ser. No.
07/805,727, filed Dec. 6, 1991, now U.S. Pat. No. 5,424,186, which
is a continuation-in-part of U.S. Ser. No. 07/624,120, filed Dec.
6, 1990, now abandoned, which is a continuation-in-part of U.S.
Ser. No. 07/492,462, filed Mar. 7, 1990, now U.S. Pat. No.
5,143,854, which is a continuation-in-part of U.S. Ser. No.
07/362,901, filed Jun. 7, 1989, now abandoned. Additional commonly
assigned applications Barrett et al., U.S. Ser. No. 07/435,316
(caged biotin parent), filed Nov. 13, 1989; and Barrett et al.,
U.S. Ser. No. 07/612,671 (caged biotin CIP), filed Nov. 13, 1990
are also incorporated herein by reference. Additional applications
Pirrung et al., U.S. Ser. No. 07/624,120 (now abandoned), a
divisional of which has issued as U.S. Pat. No. 5,744,101, and
Dower et al., U.S. Ser. No. 07/626,730 (now U.S. Pat. No.
5,547,839), which are also commonly assigned and filed Dec. 6,
1990, are also hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the sequencing,
fingerprinting, and mapping of polymers, particularly biological
polymers. The inventions may be applied, for example, in the
sequencing, fingerprinting, or mapping of nucleic acids,
polypeptides, oligosaccharides, and synthetic polymers.
[0003] The relationship between structure and function of
macromolecules is of fundamental importance in the understanding of
biological systems. These relationships are important to
understanding, for example, the functions of enzymes, structural
proteins, and signaling proteins, ways in which cells communicate
with each other, as well as mechanisms of cellular control and
metabolic feedback.
[0004] Genetic information is critical in continuation of life
processes. Life is substantially informationally based and its
genetic content controls the growth and reproduction of the
organism and its complements. Polypeptides, which are critical
features of all living systems, are encoded by the genetic material
of the cell. In particular, the properties of enzymes, functional
proteins, and structural proteins are determined by the sequence of
amino acids which make them up. As structure and function are
integrally related, many biological functions may be explained by
elucidating the underlying structural features which provide those
functions. For this reason, it has become very important to
determine the genetic sequences of nucleotides which encode the
enzymes, structural proteins, and other effectors of biological
functions. In addition to segments of nucleotides which encode
polypeptides, there are many nucleotide sequences which are
involved in control and regulation of gene expression.
[0005] The human genome project is directed toward determining the
complete sequence of the genome of the human organism. Although
such a sequence would not correspond to the sequence of any
specific individual, it would provide significant information as to
the general organization and specific sequences contained within
segments from particular individuals. It would also provide mapping
information which is very useful for further detailed studies.
However, the need for highly rapid, accurate, and inexpensive
sequencing technology is nowhere more apparent than in a demanding
sequencing project such as this. To complete the sequencing of a
human genome would require the determination of approximately
3.times.10.sup.9, or 3 billion base pairs.
[0006] The procedures typically used today for sequencing include
the Sanger dideoxy method, see, e.g., Sanger et al. (1977) Proc.
Natl. Acad. Sci. USA, 74: 5463-5467, or the Maxam and Gilbert
method, see, e.g., Maxam et al., (1980) Methods in Enzymology, 65:
499-559. The Sanger method utilizes enzymatic elongation procedures
with chain terminating nucleotides. The Maxam and Gilbert method
uses chemical reactions exhibiting specificity of reaction to
generate nucleotide specific cleavages. Both methods require a
practitioner to perform a large number of complex manual
manipulations. These manipulations usually require isolating
homogeneous DNA fragments, elaborate and tedious preparing of
samples, preparing a separating gel, applying samples to the gel,
electrophoresing the samples into this gel, working up the finished
gel, and analyzing the results of the procedure.
[0007] Thus, a less expensive, highly reliable, and labor efficient
means for sequencing biological macromolecules is needed. A
substantial reduction in cost and increase in speed of nucleotide
sequencing would be very much welcomed. In particular, an automated
system would improve the reproducibility and accuracy of
procedures. The present invention satisfies these and other
needs.
SUMMARY OF THE INVENTION
[0008] The present invention provides improved methods useful for
de novo sequencing of an unknown polymer sequence, for verification
of known sequences, for fingerprinting polymers, and for mapping
homologous segments within a sequence. By reducing the number of
manual manipulations required and automating most of the steps, the
speed, accuracy, and reliability of these procedures are greatly
enhanced.
[0009] The production of a substrate having a matrix of
positionally defined regions with attached reagents exhibiting
known recognition specificity can be used for the sequence analysis
of a polymer. Although most directly applicable to sequencing, the
present invention is also applicable to fingerprinting, mapping,
and general screening of specific interactions. The VLSIPS.TM.
Technology (Very Large Scale Immobilized Polymer Synthesis)
substrates will be applied to evaluating other polymers, e.g.,
carbohydrates, polypeptides, hydrocarbon synthetic polymers, and
the like. For these nonpolynucleotides, the sequence specific
reagents will usually be antibodies specific for a particular
subunit sequence.
[0010] The present invention also provides a means to automate
sequencing manipulations. The automation of the substrate
production method and of the scan and analysis steps minimizes the
need for human intervention. This simplifies the tasks and promotes
reproducibility.
[0011] The present invention provides a composition comprising a
plurality of positionally distinguishable sequence specific
reagents attached to a solid substrate, which reagents are capable
of specifically binding to a predetermined subunit sequence of a
preselected multi-subunit length having at least three subunits,
said reagents representing substantially all possible sequences of
said preselected length. In some embodiments, the subunit sequence
is a polynucleotide or a polypeptide, in others the preselected
multi-subunit length is five subunits and the subunit sequence is a
polynucleotide sequence. In other embodiments, the specific reagent
is an oligonucleotide of at least about five nucleotides.
Alternatively, the specific reagent is a monoclonal antibody.
Usually the specific reagents are all attached to a single solid
substrate, and the reagents comprise about 3000 different
sequences. In other embodiments, the reagents represents at least
about 25% of the possible subsequences of said preselected length.
Usually, the reagents are localized in regions of the substrate
having a density of at least 25 regions per square centimeter, and
often the substrate has a surface area of less than about 4 square
centimeters.
[0012] The present invention also provides methods for analyzing a
sequence of a polynucleotide or a polypeptide, said method
comprising the step of:
[0013] a) exposing said polynucleotide or polypeptide to a
composition as described.
[0014] It also provides useful methods for identifying or comparing
a target sequence with a reference, said method comprising the step
of:
[0015] a) exposing said target sequence to a composition as
described;
[0016] b) determining the pattern of positions of the reagents
which specifically interact with the target sequence; and
[0017] c) comparing the pattern with the pattern exhibited by the
reference when exposed to the composition.
[0018] The present invention also provides methods for sequencing a
segment of a polynucleotide comprising the steps of:
[0019] a) combining:
[0020] i) a substrate comprising a plurality of chemically
synthesized and positionally distinguishable oligonucleotides
capable of recognizing defined oligonucleotide sequences; and
[0021] ii) a target polynucleotide; thereby forming high fidelity
matched duplex structures of complementary subsequences of known
sequence; and
[0022] b) determining which of said reagents have specifically
interacted with subsequences in said target polynucleotide.
[0023] In one embodiment, the segment is substantially the entire
length of said polynucleotide.
[0024] The invention also provides methods for sequencing a
polymer, said method comprising the steps of:
[0025] a) preparing a plurality of reagents which each specifically
bind to a subsequence of preselected length;
[0026] b) positionally attaching each of said reagents to one or
more solid phase substrates, thereby producing substrates of
positionally definable sequence specific probes;
[0027] c) combining said substrates with a target polymer whose
sequence is to be determined; and
[0028] d) determining which of said reagents have specifically
interacted with subsequences in said target polymer.
[0029] In one embodiment, the substrates are beads. Preferably, the
plurality of reagents comprise substantially all possible
subsequences of said preselected length found in said target. In
another embodiment, the solid phase substrate is a single substrate
having attached thereto reagents recognizing substantially all
possible subsequences of preselected length found in said
target.
[0030] In another embodiment, the method further comprises the step
of analyzing a plurality of said recognized subsequences to
assemble a sequence of said target polymer. In a bead embodiment,
at least some of the plurality of substrates have one subsequence
specific reagent attached thereto, and the substrates are coded to
indicate the sequence specificity of said reagent.
[0031] The present invention also embraces a method of using a
fluorescent nucleotide to detect interactions with oligonucleotide
probes of known sequence, said method comprising:
[0032] a) attaching said nucleotide to a target unknown
polynucleotide sequence; and
[0033] b) exposing said target polynucleotide sequence to a
collection of positionally defined oligonucleotide probes of known
sequences to determine the sequences of said probes which interact
with said target.
[0034] In a further refinement, an additional step is included
of:
[0035] a) collating said known sequences to determine the overlaps
of said known sequences to determine the sequence of said target
sequence.
[0036] A method of mapping a plurality of sequences relative to one
another is also provided, the method comprising:
[0037] a) preparing a substrate having a plurality of positionally
attached sequence specific probes attached;
[0038] b) exposing each of said sequences to said substrate,
thereby determining the patterns of interaction between said
sequence specific probes and said sequences; and
[0039] c) determining the relative locations of said sequence
specific probe interactions on said sequences to determine the
overlaps and order of said sequences.
[0040] In one refinement, the sequence specific probes are
oligonucleotides, applicable to where the target sequences are
nucleic acid sequences.
[0041] In the nucleic acid sequencing application, the steps of the
sequencing process comprise:
[0042] a) producing a matrix substrate having known positionally
defined regions of known sequence specific oligonucleotide
probes;
[0043] b) hybridizing a target polynucleotide to the positions on
the matrix so that each of the positions which contain
oligonucleotide probes complementary to a sequence on the target
hybridize to the target molecule;
[0044] c) detecting which positions have bound the target, thereby
determining sequences which are found on the target; and
[0045] d) analyzing the known sequences contained in the target to
determine sequence overlaps and assembling the sequence of the
target therefrom.
[0046] The enablement of the sequencing process by hybridization is
based in large part upon the ability to synthesize a large number
(e.g., to virtually saturate) of the possible overlapping sequence
segments and distinguishing those probes which hybridize with
fidelity from those which have mismatched bases, and to analyze a
highly complex pattern of hybridization results to determine the
overlap regions.
[0047] The detecting of the positions which bind the target
sequence would typically be through a fluorescent label on the
target. Although a fluorescent label is probably most convenient,
other sorts of labels, e.g., radioactive, enzyme linked, optically
detectable, or spectroscopic labels may be used. Because the
oligonucleotide probes are positionally defined, the location of
the hybridized duplex will directly translate to the sequences
which hybridize. Thus, analysis of the positions provides a
collection of subsequences found within the target sequence. These
subsequences are matched with respect to their overlaps so as to
assemble an intact target sequence.
[0048] In one preferred embodiment, linker molecules are provided
on a substrate. A terminal end of the linker molecules is provided
with a reactive functional group protected with a photoremovable
protective group. Using lithographic methods, the photoremovable
protective group is exposed to light and removed from the linker
molecules in first selected regions. The substrate is then washed
or otherwise contacted with a first monomer that reacts with
exposed functional groups on the linker molecules. In a preferred
embodiment, the monomer is an amino acid containing a
photoremovable protective group at its amino or carboxy terminus
and the linker molecule terminates in an amino or carboxy acid
group bearing a photoremovable protective group.
[0049] A second set of selected regions is, thereafter, exposed to
light and the photoremovable protective group on the linker
molecule/protected amino acid is removed at the second set of
regions. The substrate is then contacted with a second monomer
containing a photoremovable protective group for reaction with
exposed functional groups. This process is repeated to selectively
apply monomers until polymers of a desired length and desired
chemical sequence are obtained. Photolabile groups are then
optionally removed and the sequence is, thereafter, optionally
capped. Side chain protective groups, if present, are also
removed.
[0050] An improved method and apparatus for the preparation of
polymers is disclosed. The method and apparatus may be applied to
synthesize a variety of polymers at known locations on a substrate.
The method could be used to synthesize up to about 10.sup.6 or more
different sequences per cm.sup.2 at known locations in some
embodiments.
[0051] The method enables greater ease in peptide synthesis because
the physical separation of reagents is not required when growing
polymer chains. The chains themselves are separated by different
physical locations on the substrate, but the entire substrate is
exposed to the various reagents as the synthesis is conducted.
Differential reactive functional groups to, e.g., light, electric
currents, or another spatially localized activator. Remaining areas
on the substrate remain unreacted.
[0052] By using the lithographic techniques disclosed herein, it is
possible to direct light to relatively small and precisely known
locations on the substrate. It is, therefore, possible to
synthesize polymers of a known chemical sequence at known locations
on the substrate.
[0053] The resulting substrate will have a variety of uses
including, for example, screening large numbers of polymers for
biological activity. To screen for biological activity, the
substrate is exposed to one or more receptors such as antibody
whole cells, receptors on vesicles, lipids, or any one of a variety
of other receptors. The receptors are preferably labeled with, for
example, a fluorescent marker, radioactive marker, or a labeled
antibody reactive with the receptor. The location of the marker on
the substrate is detected with, for example, photon detection or
autoradiographic techniques. Through knowledge of the sequence of
the material at the location where binding is detected, it is
possible to quickly determine which sequence binds with the
receptor and, therefore, the technique can be used to screen large
numbers of peptides. Other possible applications of the inventions
herein include diagnostics in which various antibodies for
particular receptors would be placed on a substrate and, for
example, blood sera would be screened for immune deficiencies.
Still further applications include, for example, selective "doping"
of organic materials in semiconductor devices, and the like.
[0054] In connection with one aspect of the invention an improved
reactor system for synthesizing polymers is also disclosed. The
reactor system includes a substrate mount which engages a substrate
around a periphery thereof. The substrate mount provides for a
reactor space between the substrate and the mount through or into
which reaction fluids are pumped or flowed. A mask is placed on or
focused on the substrate and illuminated so as to deprotect
selected regions of the substrate in the reactor space. A monomer
is pumped through the reactor space or otherwise contacted with the
substrate and reacts with the deprotected regions. By selectively
deprotecting regions on the substrate and flowing predetermined
monomers through the reactor space, desired polymers at known
locations may be synthesized.
[0055] Improved detection apparatus and methods are also disclosed.
The detection method and apparatus utilize a substrate having a
large variety of polymer sequences at known locations on a surface
thereof. The substrate is exposed to a fluorescently labeled
receptor which binds to one or more of the polymer sequences. The
substrate is placed in a microscope detection apparatus for
identification of locations where binding takes place. The
microscope detection apparatus includes a monochromatic or
polychromatic light source for directing light at the substrate,
means for detecting fluoresced light from the substrate, and means
for determining a location of the fluoresced light. The means for
detecting light fluoresced on the substrate may in some embodiments
include a photon counter. The means for determining a location of
the fluoresced light may include an x/y translation table for the
substrate. Translation of the slide and data collection are
recorded and managed by an appropriately programmed digital
computer.
[0056] A further understanding of the nature and advantages of the
inventions herein may be realized by reference to the remaining
portions of the specification and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 illustrates a flow chart for sequence, fingerprint,
or mapping analysis.
[0058] FIG. 2 illustrates the proper function of a VLSIPS.TM.
Technology nucleotide synthesis.
[0059] FIG. 3 illustrates the proper function of a VLSIPS.TM.
Technology nucleotide synthesis.
[0060] FIGS. 4A-4M illustrate the process of a VLSIPS trinucleotide
synthesis.
[0061] FIG. 5 illustrates masking and irradiation of a substrate at
a first location. The substrate is shown in cross-section;
[0062] FIG. 6 illustrates the substrate after application of a
monomer "A";
[0063] FIG. 7 illustrates irradiation of the substrate at a second
location;
[0064] FIG. 8 illustrates the substrate after application of
monomer "B";
[0065] FIG. 9 illustrates irradiation of the "A" monomer;
[0066] FIG. 10 illustrates the substrate after a second application
of "B";
[0067] FIG. 11 illustrates a completed substrate;
[0068] FIGS. 12A and 12B illustrate alternative embodiments of a
reactor system for forming a plurality of polymers on a
substrate;
[0069] FIG. 13 illustrates a detection apparatus for locating
fluorescent markers on the substrate;
[0070] FIGS. 14A-14M illustrate the method as it is applied to the
production of the trimers of monomers "A" and "B";
[0071] FIGS. 15A and 15B are fluorescence traces for standard
fluorescent beads;
[0072] FIGS. 16A and 16B are fluorescence curves for NVOC slides
not exposed and exposed to light respectively;
[0073] FIGS. 17A to 17D are fluorescence plots of slides exposed
through 100 .mu.m, 50 .mu.m, 20 .mu.m, and 10 .mu.m masks;
[0074] FIG. 18 illustrates fluorescence of a slide with the peptide
YGGFL on selected regions of its surface which has been exposed to
labeled Herz antibody specific for this sequence;
[0075] FIGS. 19A to 19D illustrate formation of and a fluorescence
plot of a slide with a checkerboard pattern of YGGFL and GGFL
exposed to labeled Herz antibody. FIG. 19C illustrates a
500.times.500 .mu.m mask which has been focused on the substrate
according to FIG. 12A while FIG. 19D illustrates a 50.times.50
.mu.m mask placed in direct contact with the substrate in accord
with FIG. 12B;
[0076] FIG. 20 is a fluorescence plot of YGGFL and PGGFL
synthesized in a 50 .mu.m checkerboard pattern;
[0077] FIG. 21 is a fluorescence plot of YPGGFL and YGGFL
synthesized in a 50 .mu.m checkerboard pattern;
[0078] FIGS. 22A and 22B illustrate the mapping of sixteen
sequences synthesized on two different glass slides;
[0079] FIG. 23 is a fluorescence plot of the slide illustrated in
FIG. 22A; and
[0080] FIG. 24 is a fluorescence plot of the slide illustrated in
FIG. 14B.
GLOSSARY
[0081] The following terms are intended to have the following
general meanings as they are used herein:
[0082] 1. Complementary: Refers to the topological compatibility or
matching together of interacting surfaces of a ligand molecule and
its receptor. Thus, the receptor and its ligand can be described as
complementary, and furthermore, the contact surface characteristics
are complementary to each other.
[0083] 2. Epitope: The portion of an antigen molecule which is
delineated by the area of interaction with the subclass of
receptors known as antibodies.
[0084] 3. Ligand: A ligand is a molecule that is recognized by a
particular receptor. Examples of ligands that can be investigated
by this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,
proteins, and monoclonal antibodies.
[0085] 4. Monomer: A member of the set of small molecules which can
be joined together to form a polymer. The set of monomers includes
but is not restricted to, for example, the set of common L-amino
acids, the set of D-amino acids, the set of synthetic amino acids,
the set of nucleotides and the set of pentoses and hexoses. As used
herein, monomers refers to any member of a basis set for synthesis
of a polymer. For example, dimers of L-amino acids form a basis set
of 400 monomers for synthesis of polypeptides. Different basis sets
of monomers may be used at successive steps in the synthesis of a
polymer.
[0086] 5. Peptide: A polymer in which the monomers are alpha amino
acids and which are joined together through amide bonds and
alternatively referred to as a polypeptide. In the context of this
specification it should be appreciated that the amino acids may be
the L-optical isomer or the D-optical isomer. Peptides are more
than two amino acid monomers long, and often more than 20 amino
acid monomers long. Standard abbreviations for amino acids are used
(e.g., P for proline). These abbreviations are included in Stryer,
Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes.
[0087] 6. Radiation: Energy which may be selectively applied
including energy having a wavelength of between 10.sup.-14 and
10.sup.4 meters including, for example, electron beam radiation,
gamma radiation, x-ray radiation, ultra-violet radiation, visible
light, infrared radiation, microwave radiation, and radio waves.
"Irradiation" refers to the application of radiation to a
surface.
[0088] 7. Receptor: A molecule that has an affinity for a given
ligand. Receptors may be naturally-occurring or manmade molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Receptors may be attached,
covalently or noncovalently, to a binding member, either directly
or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles. Receptors are sometimes
referred to in the art as anti-ligands. As the term receptors is
used herein, no difference in meaning is intended. A "Ligand
Receptor Pair" is formed when two macromolecules have combined
through molecular recognition to form a complex.
[0089] Other examples of receptors which can be investigated by
this invention include but are not restricted to:
[0090] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in a new class
of antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.
[0091] b) Enzymes: For instance, the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters;
determination of ligands which bind to certain receptors to
modulate the action of the enzymes which cleave the different
neurotransmitters is useful in the development of drugs which can
be used in the treatment of disorders of neurotransmission.
[0092] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the development of vaccines of which the immunogen is based on one
or more of such sequences or lead to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for autoimmune diseases (e.g., by blocking the binding of
the "self" antibodies).
[0093] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
[0094] e) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides are described in, for example,
U.S. application Ser. No. 404,920, which is incorporated herein by
reference for all purposes.
[0095] f) Hormone receptors: For instance, the receptors for
insulin and growth hormone. Determination of the ligands which bind
with high affinity to a receptor is useful in the development of,
for example, an oral replacement of the daily injections which
diabetics must take to relieve the symptoms of diabetes, and in the
other case, a replacement for the scarce human growth hormone which
can only be obtained from cadavers or by recombinant DNA
technology. Other examples are the vasoconstrictive hormone
receptors; determination of those ligands which bind to a receptor
may lead to the development of drugs to control blood pressure.
[0096] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0097] 8. Substrate: A material having a rigid or semi-rigid
surface. In many embodiments, at least one surface of the substrate
will be substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
polymers with, for example, wells, raised regions, etched trenches,
or the like. According to other embodiments, small beads may be
provided on the surface which may be released upon completion of
the synthesis.
[0098] 9. Protective Group: A material which is bound to a monomer
unit and which may be spatially removed upon selective exposure to
an activator such as electromagnetic radiation. Examples of
protective groups with utility herein include Nitroveratryloxy
carbonyl, Nitrobenzyloxy carbonyl, Dimethyl dimethoxybenzyloxy
carbonyl, 5-Bromo-7-nitroindolinyl, o-Hydroxy-.alpha.-methyl
cinnamoyl, and 2-Oxymethylene anthraquinone. Other examples of
activators include ion beams, electric fields, magnetic fields,
electron beams, x-ray, and the like.
[0099] 10. Predefined Region: A predefined region is a localized
area on a surface which is, was, or is intended to be activated for
formation of a polymer. The predefined region may have any
convenient shape, e.g., circular, rectangular, elliptical,
wedge-shaped, etc. For the sake of brevity herein, "predefined
regions" are sometimes referred to simply as "regions."
[0100] 11. Substantially Pure: A polymer is considered to be
"substantially pure" within a predefined region of a substrate when
it exhibits characteristics that distinguish it from other
predefined regions. Typically, purity will be measured in terms of
biological activity or function as a result of uniform sequence.
Such characteristics will typically be measured by way of binding
with a selected ligand or receptor.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0101] I. Overall Description
[0102] A. general
[0103] B. VLSIPS.TM. Technology substrates
[0104] C. binary masking
[0105] D. applications
[0106] E. detection methods and apparatus
[0107] F. data analysis
[0108] II. Theoretical Analysis
[0109] A. simple n-mer structure; theory
[0110] B. complications
[0111] C. non-polynucleotide embodiments
[0112] III. Polynucleotide Sequencing
[0113] A. preparation of substrate matrix
[0114] B. labeling target polynucleotide
[0115] C. hybridization conditions
[0116] D. detection; VLSIPS.TM. Technology scanning
[0117] E. analysis
[0118] F. substrate reuse
[0119] G. non-polynucleotide aspects
[0120] IV. Fingerprinting
[0121] A. general
[0122] B. preparation of substrate matrix
[0123] C. labeling target nucleotides
[0124] D. hybridization conditions
[0125] E. detection; VLSIPS.TM. Technology scanning
[0126] F. analysis
[0127] G. substrate reuse
[0128] H. non-polynucleotide aspects
[0129] V. Mapping
[0130] A. general
[0131] B. preparation of substrate matrix
[0132] C. labeling
[0133] D. hybridization/specific interaction
[0134] E. detection
[0135] F. analysis
[0136] G. substrate reuse
[0137] H. non-polynucleotide aspects
[0138] VI. Additional Screening
[0139] A. specific interactions
[0140] B. sequence comparisons
[0141] C. categorizations
[0142] D. statistical correlations
[0143] VII. Formation of Substrate
[0144] A. instrumentation
[0145] B. binary masking
[0146] C. synthetic methods
[0147] D. surface immobilization
[0148] VIII. Hybridization/Specific Interaction
[0149] A. general
[0150] B. important parameters
[0151] IX. Detection Methods
[0152] A. labeling techniques
[0153] B. scanning system
[0154] X. Data Analysis
[0155] A. general
[0156] B. hardware
[0157] C. software
[0158] XI. Substrate Reuse
[0159] A. removal of label
[0160] B. storage and preservation
[0161] C. processes to avoid degradation of oligomers
[0162] XII. Integrated Sequencing Strategy
[0163] A. initial mapping strategy
[0164] B. selection of smaller clones
[0165] C. actual sequencing procedures
[0166] XIII. Commercial Applications
[0167] A. sequencing
[0168] B. fingerprinting
[0169] C. mapping
DETAILED DESCRIPTION OF THE INVENTION
[0170] A description of preferred embodiments of the invention
follows.
[0171] A. General
[0172] The present invention relies in part on the ability to
synthesize or attach specific recognition reagents at known
locations on a substrate, typically a single substrate. In
particular, the present invention provides the ability to prepare a
substrate having a very high density matrix pattern of positionally
defined specific recognition reagents. The reagents are capable of
interacting with their specific targets while attached to the
substrate, e.g., solid phase interactions, and by appropriate
labeling of these targets, the sites of the interactions between
the target and the specific reagents may be derived. Because the
reagents are positionally defined, the sites of the interactions
will define the specificity of each interaction. As a result, a map
of the patterns of interactions with specific reagents on the
substrate is convertible into information on the specific
interactions taking place, e.g., the recognized features. Where the
specific reagents recognize a large number of possible features,
this system allows the determination of the combination of specific
interactions which exist on the target molecule. Where the number
of features is sufficiently large, the identical same combination,
or pattern, of features is sufficiently unlikely that a particular
target molecule may often be uniquely defined by its features. In
the extreme, the features may actually be the subunit sequence of
the target molecule, and a given target sequence may be uniquely
defined by its combination of features.
[0173] In particular, the methodology is applicable to sequencing
polynucleotides. The specific sequence recognition reagents will
typically be oligonucleotide probes which hybridize with
specificity to subsequences found on the target sequence. A
sufficiently large number of those probes allows the fingerprinting
of a target polynucleotide or the relative mapping of a collection
of target polynucleotides, as described in greater detail
below.
[0174] In the high resolution fingerprinting provided by a
saturating collection of probes which include all possible
subsequences of a given size, e.g., 10-mers, collating of all the
subsequences and determination of specific overlaps will be derived
and the entire sequence can usually be reconstructed.
[0175] Although a polynucleotide sequence analysis is a preferred
embodiment, for which the specific reagents are most easily
accessible, the invention is also applicable to analysis of other
polymers, including polypeptides, carbohydrates, and synthetic
polymers, including alpha-, beta-, and omega-amino acids,
polyurethanes, polyesters, polycarbonates, polyureas, polyamides,
polyethyleneimines, polyarylene sulfides, polysiloxanes,
polyimides, polyacetates, and mixed polymers. Various optical
isomers, e.g., various D- and L-forms of the monomers, may be
used.
[0176] Sequence analysis will take the form of complete sequence
determination, to the level of the sequence of individual subunits
along the entire length of the target sequence. Sequence analysis
also takes the form of sequence homology, e.g., less than absolute
subunit resolution, where "similarity" in the sequence will be
detectable, or the form of selective sequences of homology
interspersed at specific or irregular locations.
[0177] In either case, the sequence is determinable at selective
resolution or at particular locations. Thus, the hybridization
method will be useful as a means for identification, e.g., a
"fingerprint," much like a Southern hybridization method is used.
It is also useful to map particular target sequences.
[0178] B. VLSIPS.TM. Technology
[0179] The invention is enabled by the development of technology to
prepare substrates on which specific reagents may be either
positionally attached or synthesized. In particular, the very large
scale-immobilized polymer synthesis VLSIPS.TM. Technology allows
for the very high density production of an enormous diversity of
reagents mapped out in a known matrix pattern on a substrate. These
reagents specifically recognize subsequences in a target polymer
and bind thereto, producing a map of positionally defined regions
of interaction. These map positions are convertible into actual
features recognized, and thus would be present in the target
molecule of interest.
[0180] As indicated, the sequence specific recognition reagents
will often be oligonucleotides which hybridize with fidelity and
discrimination to the target sequence. For use with other polymers,
monoclonal or polyclonal antibodies having high sequence
specificity will often be used.
[0181] In the generic sense, the VLSIPS.TM. Technology allows the
production of a substrate with a high density matrix of
positionally mapped regions with specific recognition reagents
attached at each distinct region. By use of protective groups which
can be positionally removed, or added, the regions can be activated
or deactivated for addition of particular reagents or compounds.
Details of the protection are described below and in related
application Pirrung et al. (1992) U.S. Pat. No. 5,143,854. In a
preferred embodiment, photosensitive protecting agents will be used
and the regions of activation or deactivation may be controlled by
electro-optical and optical methods, similar to many of the
processes used in semiconductor wafer and chip fabrication.
[0182] In the nucleic acid nucleotide sequencing application, a
VLSIPS.TM. Technology substrate is synthesized having positionally
defined oligonucleotide probes. See Pirrung et al. (1992) U.S. Pat.
No. 5,143,854; and U.S. Pat. No. 5,489,678. By use of masking
technology and photosensitive synthetic subunits, the VLSIPS.TM.
Technology apparatus allows for the stepwise synthesis of polymers
according to a positionally defined matrix pattern. Each
oligonucleotide probe will be synthesized at known and defined
positional locations on the substrate. This forms a matrix pattern
of known relationship between position and specificity of
interaction. The VLSIPS.TM. Technology allows the production of a
very large number of different oligonucleotide probes to be
simultaneously and automatically synthesized including numbers in
excess of about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
or even more, and at densities of at least about 10.sup.2,
10.sup.3/cm.sup.2, 10.sup.4/cm.sup.2, 10.sup.5/cm.sup.2 and up to
106/cm.sup.2 or more. This application discloses methods for
synthesizing polymers on a silicon or other suitably derivatized
substrate, methods and chemistry for synthesizing specific types of
biological polymers on those substrates, apparatus for scanning and
detecting whether interaction has occurred at specific locations on
the substrate, and various other technologies related to the use of
a high density very large scale immobilized polymer substrate. In
particular, sequencing, fingerprinting, and mapping applications
are discussed herein in detail, though related technologies are
described in simultaneously filed applications U.S. Ser. No.
07/624,120, now abandoned and U.S. Pat. No. 5,427,408, each of
which is hereby incorporated herein by reference.
[0183] In other embodiments, antibody probes will be generated
which specifically recognize particular subsequences found on a
polymer. Antibodies would be generated which are specific for
recognizing a three contiguous amino acid sequence, and monoclonal
antibodies may be preferred. Optimally, these antibodies would not
recognize any sequences other than the specific three amino acid
stretch desired and the binding affinity should be insensitive to
flanking or remote sequences found on a target molecule. Likewise,
antibodies specific for particular carbohydrate linkages or
sequences will be generated. A similar approach could be used for
preparing specific reagents which recognize other polymer subunit
sequences. These reagents would typically be site specifically
localized to a substrate matrix pattern where the regions are
closely packed.
[0184] These reagents could be individually attached at specific
sites on the substrate in a matrix by an automated procedure where
the regions are positionally targeted by some other specific
mechanism, e.g., one which would allow the entire collection of
reagents to be attached to the substrate in a single reaction. Each
reagent could be separately attached to a specific oligonucleotide
sequence by an automated procedure. This would produce a collection
of reagents where, e.g., each monoclonal antibody would have a
unique oligonucleotide sequence attached to it. By virtue of a
VLSIPS.TM. Technology substrate which has different complementary
oligonucleotides synthesized on it, each monoclonal antibody would
specifically be bound only at that site on the substrate where the
complementary oligonucleotide has been synthesized. A cross-linking
step would fix the reagent to the substrate. See, e.g., Dattagupta
et al. (1985) U.S. Pat. No. 4,542,102 and (1987) U.S. Pat. No.
4,713,326; and Chatteijee, M. et al. (1990) J. Am. Chem. Soc., 112:
6397-6399, which are hereby incorporated herein by reference. This
allows a high density positionally specific collection of specific
recognition reagents, e.g., monoclonal antibodies, to be
immobilized to a solid substrate using an automated system.
[0185] The regions which define particular reagents will usually be
generated by selective protecting groups which may be activated or
deactivated. Typically the protecting group will be bound to a
monomer subunit or spatial region, and can be spatially affected by
an activator, such as electromagnetic radiation. Examples of
protective groups with utility herein include nitroveratryl
oxycarbonyl (NVOC), nitrobenzyl oxycarbony (NBOC), dimethyl
dimethoxy benzyloxy carbonyl, 5-bromo-7-nitroindolinyl,
o-hydroxy-alpha-methyl cinnamoyl, and 2-oxymethylene anthraquinone.
Examples of activators include ion beams, electric fields, magnetic
fields, electron beams, x-ray, and other forms of electromagnetic
radiation.
[0186] The present invention provides methods and apparatus for the
preparation and use of a substrate having a plurality of polymer
sequences in predefined regions. The invention is described herein
primarily with regard to the preparation of molecules containing
sequences of amino acids, but could readily be applied in the
preparation of other polymers. Such polymers include, for example,
both linear and cyclic polymers of nucleic acids, polysaccharides,
phospholipids, and peptides having either alpha-, beta-, or
omega-amino acids, hetero-polymers in which a known drug is
covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or
other polymers which will be apparent upon review of this
disclosure. In a preferred embodiment, the invention herein is used
in the synthesis of peptides.
[0187] The prepared substrate may, for example, be used in
screening a variety of polymers as ligands for binding with a
receptor, although it will be apparent that the invention could be
used for the synthesis of a receptor for binding with a ligand. The
substrate, disclosed herein will have a wide variety of other uses.
Merely by way of example, the invention herein can be used in
determining peptide and nucleic acid sequences which bind to
proteins, finding sequence-specific binding drugs, identifying
epitopes recognized by antibodies, and evaluation of a variety of
drugs for clinical and diagnostic applications, as well as
combinations of the above.
[0188] The invention preferably provides for the use of a substrate
"S" with a surface. Linker molecules "L" are optionally provided on
a surface of the substrate. The purpose of the linker molecules, in
some embodiments, is to facilitate receptor recognition of the
synthesized polymers.
[0189] Optionally, the linker molecules may be chemically protected
for storage purposes. A chemical storage protective group such as
t-BOC (t-butoxycarbonyl) may be used in some embodiments. Such
chemical protective groups would be chemically removed upon
exposure to, for example, acidic solution and would serve to
protect the surface during storage and be removed prior to polymer
preparation.
[0190] On the substrate or a distal end of the linker molecules, a
functional group with a protective group P.sub.0 is provided. The
protective group P.sub.0 may be removed upon exposure to radiation,
electric fields, electric currents, or other activators to expose
the functional group.
[0191] In a preferred embodiment, the radiation is ultraviolet
(UV), infrared (IR), or visible light. As more fully described
below, the protective group may alternatively be an
electrochemically-sensitive group which may be removed in the
presence of an electric field. In still further alternative
embodiments, ion beams, electron beams, or the like may be used for
deprotection.
[0192] In some embodiments, the exposed regions and, therefore, the
area upon which each distinct polymer sequence is synthesized are
smaller than about 1 cm.sup.2 or less than 1 mm.sup.2. In preferred
embodiments the exposed area is less than about 10,000 .mu.m.sup.2
or, more preferably, less than 100 .mu.m.sup.2 and may, in some
embodiments, encompass the binding site for as few as a single
molecule. Within these regions, each polymer is preferably
synthesized in a substantially pure form.
[0193] Concurrently or after exposure of a known region of the
substrate to light, the surface is contacted with a first monomer
unit M.sub.1 which reacts with the functional group which has been
exposed by the deprotection step. The first monomer includes a
protective group P.sub.1. P.sub.1 may or may not be the same as
P.sub.0.
[0194] Accordingly, after a first cycle, known first regions of the
surface may comprise the sequence:
[0195] S--L--M.sub.1--P.sub.1
[0196] while remaining regions of the surface comprise the
sequence:
[0197] S--L--P.sub.0.
[0198] Thereafter, second regions of the surface (which may include
the first region) are exposed to light and contacted with a second
monomer M.sub.2 (which may or may not be the same as M.sub.1)
having a protective group P.sub.2. P.sub.2 may or may not be the
same as P.sub.0 and P.sub.1. After this second cycle, different
regions of the substrate may comprise one or more of the following
sequences:
[0199] S--L--M.sub.1--M.sub.2--P.sub.2
[0200] S--L--M.sub.2--P.sub.2
[0201] S--L-M.sub.1--P.sub.1
[0202] and/or
[0203] S--L--P.sub.0
[0204] The above process is repeated until the substrate includes
desired polymers of desired lengths. By controlling the locations
of the substrate exposed to light and the reagents exposed to the
substrate following exposure, the location of each sequence will be
known.
[0205] Thereafter, the protective groups are removed from some or
all of the substrate and the sequences are, optionally, capped with
a capping unit C. The process results in a substrate having a
surface with a plurality of polymers of the following general
formula:
S--[L]--(M.sub.i)--(M.sub.j)--(M.sub.k) . . . (M.sub.x)--[C]
[0206] where square brackets indicate optional groups, and M.sub.i
. . . M.sub.x indicates any sequence of monomers. The number of
monomers could cover a wide variety of values, but in a preferred
embodiment they will range from 2 to 100.
[0207] In some embodiments a plurality of locations on the
substrate polymers are to contain a common monomer subsequence. For
example, it may be desired to synthesize a sequence
S--M.sub.1--M.sub.2--M.sub.3 at first locations and a sequence
S--M.sub.1--M.sub.2--M.sub.3 at second locations. The process would
commence with irradiation of the first locations followed by
contacting with M.sub.1--P, resulting in the sequence S--M.sub.1--P
at the first location. The second location would then be irradiated
and contacted with M.sub.4--P, resulting in the sequence
S--M.sub.4--P at the second locations. Thereafter both the first
and second locations would be irradiated and contacted with the
dimer M.sub.2--M.sub.3, resulting in the sequence
S--M.sub.1--M.sub.2--M.sub.3 at the first locations and
S--M.sub.4--M.sub.2--M.sub.3 at the second locations. Of course,
common subsequences of any length could be utilized including those
in a range of 2 or more monomers, 2 to 100 monomers, 2 to 20
monomers, and a most preferred range of 2 to 3 monomers.
[0208] According to other embodiments, a set of masks is used for
the first monomer layer and, thereafter, varied light wavelengths
are used for selective deprotection. For example, in the process
discussed above, first regions are first exposed through a mask and
reacted with a first monomer having a first protective group
P.sub.1, which is removable upon exposure to a first wavelength of
light (e.g., IR). Second regions are masked and reacted with a
second monomer having a second protective group P.sub.2, which is
removable upon exposure to a second wavelength of light (e.g., UV).
Thereafter, masks become unnecessary in the synthesis because the
entire substrate may be exposed alternatively to the first and
second wavelengths of light en the deprotection cycle.
[0209] The polymers prepared on a substrate according to the above
methods will have a variety of uses including, for example,
screening for biological activity. In such screening activities,
the substrate containing the sequences is exposed to an unlabeled
or labeled receptor such as an antibody, receptor on a cell,
phospholipid vesicle, or any one of a variety of other receptors.
In one preferred embodiment the polymers are exposed to a first,
unlabeled receptor of interest and, thereafter, exposed to a
labeled receptor-specific recognition element, which is, for
example, an antibody. This process will provide signal
amplification in the detection stage.
[0210] The receptor molecules may bind with one or more polymers on
the substrate. The presence of the labeled receptor and, therefore,
the presence of a sequence which binds with the receptor is
detected in a preferred embodiment through the use of
autoradiography, detection of fluorescence with a charge-coupled
device, fluorescence microscopy, or the like. The sequence of the
polymer at the locations where the receptor binding is detected may
be used to determine all or part of a sequence which is
complementary to the receptor.
[0211] Use of the invention herein is illustrated primarily with
reference to screening for biological activity. The invention will,
however, find many other uses. For example, the invention may be
used in information storage (e.g., on optical disks), production of
molecular electronic devices, production of stationary phases in
separation sciences, production of dyes and brightening agents,
photography, and in immobilization of cells, proteins, lectins,
nucleic acids, polysaccharides and the like in patterns on a
surface via molecular recognition of specific polymer sequences. By
synthesizing the same compound in adjacent, progressively differing
concentrations, a gradient will be established to control
chemotaxis or to develop diagnostic dipsticks which, for example,
titrate an antibody against an increasing amount of antigen. By
synthesizing several catalyst molecules in close proximity, more
efficient multistep conversions may be achieved by "coordinate
immobilization." Coordinate immobilization also may be used for
electron transfer systems, as well as to provide both structural
integrity and other desirable properties to materials such as
lubrication, wetting, etc.
[0212] According to alternative embodiments, molecular
biodistribution or pharmacokinetic properties may be examined. For
example, to assess resistance to intestinal or serum proteases,
polymers may be capped with a fluorescent tag and exposed to
biological fluids of interest.
[0213] III. Polymer Synthesis
[0214] FIG. 1 illustrates one embodiment of the invention disclosed
herein in which a substrate 2 is shown in cross-section.
Essentially, any conceivable substrate may be employed in the
invention. The substrate may be biological, nonbiological, organic,
inorganic, or a combination of any of these, existing as particles,
strands, precipitates, gels, sheets, tubing, spheres, containers,
capillaries, pads, slices, films, plates, slides, etc. The
substrate may have any convenient shape, such as a disc, square,
sphere, circle, etc. The substrate is preferably flat but may take
on a variety of alternative surface configurations. For example,
the substrate may contain raised or depressed regions on which the
synthesis takes place. The substrate and its surface preferably
form a rigid support on which to carry out the reactions described
herein. The substrate and its surface is also chosen to provide
appropriate light-absorbing characteristics. For instance, the
substrate may be a polymerized Langmuir Blodgett film,
functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2, SiN.sub.4,
modified silicon, or any one of a wide variety of gels or polymers
such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,
polystyrene, polycarbonate, or combinations thereof. Other
substrate materials will be readily apparent to those of skill in
the art upon review of this disclosure. In a preferred embodiment
the substrate is flat glass or single-crystal silicon with surface
relief features of less than 10 .ANG..
[0215] According to some embodiments, the surface of the substrate
is etched using well known techniques to provide for desired
surface features. For example, by way of the formation of trenches,
v-grooves, mesa structures, or the like, the synthesis regions may
be more closely placed within the focus point of impinging light,
be provided with reflective "mirror" structures for maximization of
light collection from fluorescent sources, or the like.
[0216] Surfaces on the solid substrate will usually, though not
always, be composed of the same material as the substrate. Thus,
the surface may be composed of any of a wide variety of materials,
for example, polymers, plastics, resins, polysaccharides, silica or
silica-based materials, carbon, metals, inorganic glasses,
membranes, or any of the above-listed substrate materials. In some
embodiments the surface may provide for the use of caged binding
members which are attached firmly to the surface of the substrate
in accord with the teaching of copending application Ser. No.
404,920, previously incorporated herein by reference. Preferably,
the surface will contain reactive groups, which could be carboxyl,
amino, hydroxyl, or the like. Most preferably, the surface will be
optically transparent and will have surface Si--OH functionalities,
such as are found on silica surfaces.
[0217] The surface 4 of the substrate is preferably provided with a
layer of linker molecules 6, although it will be understood that
the linker molecules are not required elements of the invention.
The linker molecules are preferably of sufficient length to permit
polymers in a completed substrate to interact freely with molecules
exposed to the substrate. The linker molecules should be 6-50 atoms
long to provide sufficient exposure. The linker molecules may be,
for example, aryl acetylene, ethylene glycol oligomers containing
2-10 monomer units, diamines, diacids, amino acids, or combinations
thereof. Other linker molecules may be used in light of this
disclsoure.
[0218] According to alternative embodiments, the linker molecules
are selected based upon their hydrophilic/hydrophobic properties to
improve presentation of synthesized polymers to certain receptors.
For example, in the case of a hydrophilic receptor, hydrophilic
linker molecules will be preferred so as to permit the receptor to
more closely approach the synthesized polymer.
[0219] According to another alternative embodiment, linker
molecules are also provided with a photocleavable group at an
intermediate position. The photocleavable group is preferably
cleavable at a wavelength different from the protective group. This
enables removal of the various polymers following completion of the
synthesis by way of exposure to the different wavelengths of
light.
[0220] The linker molecules can be attached to the substrate via
carbon-carbon bonds using, for example,
(poly)trifluorochloroethylene surfaces, or preferably, by siloxane
bonds (using, for example, glass or silicon oxide surfaces).
Siloxane bonds with the surface of the substrate may be formed in
one embodiment via reactions of linker molecules bearing
trichlorosilyl groups. The linker molecules may optionally be
attached in an ordered array, i.e., as parts of the head groups in
a polymerized Langmuir Blodgett film. In alternative embodiments,
the linker molecules are adsorbed to the surface of the
substrate.
[0221] The linker molecules and monomers used herein are provided
with a functional group to which is bound a protective group.
Preferably, the protective group is on the distal or terminal end
of the linker molecule opposite the substrate. The protective group
may be either a negative protective group (i.e., the protective
group renders the linker molecules less reactive with a monomer
upon exposure) or a positive protective group (i.e., the protective
group renders the linker molecules more reactive with a monomer
upon exposure). In the case of negative protective groups an
additional step of reactivation will be required. In some
embodiments, this will be done by heating.
[0222] The protective group on the linker molecules may be selected
from a wide variety of positive light-reactive groups preferably
including nitro aromatic compounds such as o-nitrobenzyl
derivatives or benzylsulfonyl. In a preferred embodiment,
6-nitroveratryloxy-carbonyl (NVOC), 2-nitrobenzyloxycarbonyl (NBOC)
or alpha,alpha-dimethyl-dimethoxybenzylox- ycarbonyl (DDZ) is used.
In one embodiment, a nitro aromatic compound containing a benzylic
hydrogen ortho to the nitro group is used, i.e., a chemical of the
form: 1
[0223] where R.sub.1 is alkoxy, alkyl, halo, aryl, alkenyl, or
hydrogen; R.sub.2 is alkoxy, alkyl, halo, aryl, nitro, or hydrogen;
R.sub.3 is alkoxy, alkyl, halo, nitro, aryl, or hydrogen; R.sub.4
is alkoxy, alkyl, hydrogen, aryl, halo, or nitro; and R.sub.5 is
alkyl, alkynyl, cyano, alkoxy, hydrogen, halo, aryl, or alkenyl.
Other materials which may be used include o-hydroxy-alpha-methyl
cinnamoyl derivatives. Photoremovable protective groups are
described in, for example, Patchornik, J. Am. Chem. Soc., (1970)
92: 6333 and Amit et al., J. Org. Chem. (1974) 39: 192, both of
which are incorporated herein by reference.
[0224] In an alternative embodiment the positive reactive group is
activated for reaction with reagents in solution. For example, a
5-bromo-7-nitro indoline group, when bound to a carbonyl, undergoes
reaction upon exposure to light at 420 nm.
[0225] In a second alternative embodiment, the reactive group on
the linker molecule is selected from a wide variety of negative
light-reactive groups including a cinammate group.
[0226] Alternatively, the reactive group is activated or
deactivated by electron beam lithography, x-ray lithography, or any
other radiation. Suitable reactive groups for electron beam
lithography include sulfonyl. Other methods may be used including,
for example, exposure to a current source. Other reactive groups
and methods of activation may be used in light of this
disclosure.
[0227] As shown in FIG. 5, the linking molecules are preferably
exposed to, for example, light through a suitable mask 8 using
photolithographic techniques of the type known in the semiconductor
industry and described in, for example, Sze, VLSI Technology,
McGraw-Hill (1983), and Mead et al., Introduction to VLSI Systems,
Addison-Wesley (1980), which are incorporated herein by reference
for all purposes. The light may be directed at either the surface
containing the protective groups or at the back of the substrate,
so long as the substrate is transparent to the wavelength of light
needed for removal of the protective groups. In the embodiment
shown in FIG. 5, light is directed at the surface of the substrate
containing the protective groups. FIG. 5 illustrates the use of
such masking techniques as they are applied to a positive reactive
group so as to activate linking molecules and expose functional
groups in areas 10a and 10b.
[0228] The mask 8 is in one embodiment a transparent support
material selectively coated with a layer of opaque material.
Portions of the opaque material are removed, leaving opaque
material in the precise pattern desired on the substrate surface.
The mask is brought into close proximity with, imaged on, or
brought directly into contact with the substrate surface as shown
in FIG. 5. "Openings" in the mask correspond to locations on the
substrate where it is desired to remove photoremovable protective
groups from the substrate. Alignment may be performed using
conventional alignment techniques in which alignment marks (not
shown) are used to accurately overlay successive masks with
previous patterning steps, or more sophisticated techniques may be
used. For example, interferometric techniques such as the one
described in Flanders et al., "A New Interferometric Alignment
Technique," App. Phys. Lett., (1977) 31: 426-428, which is
incorporated herein by reference, may be used.
[0229] To enhance contrast of light applied to the substrate, it is
desirable to provide contrast enhancement materials between the
mask and the substrate according to some embodiments. This contrast
enhancement layer may comprise a molecule which is decomposed by
light such as quinone diazide or a material which is transiently
bleached at the wavelength of interest. Transient bleaching of
materials will allow greater penetration where light is applied,
thereby enhancing contrast. Alternatively, contrast enhancement may
be provided by way of a cladded fiber optic bundle.
[0230] The light may be from a conventional incandescent source, a
laser, a laser diode, or the like. If non-collimated sources of
light are used it may be desirable to provide a thick- or
multi-layered mask to prevent spreading of the light onto the
substrate. It may, further, be desirable in some embodiments to
utilize groups which are sensitive to different wavelengths to
control synthesis. For example, by using groups which are sensitive
to different wavelengths, it is possible to select branch positions
in the synthesis of a polymer or eliminate certain masking steps.
Several reactive groups along with their corresponding wavelengths
for deprotection are provided in Table 1.
1 TABLE 1 Approximate Group Deprotection Wavelength
Nitroveratryloxy carbonyl (NVOC) UV (300-400 nm) Nitrobenzyloxy
carbonyl (NBOC) UV (300-350 nm) Dimethyl dimethoxybenzyloxy
carbonyl UV (280-300 nm) 5-Bromo-7-nitroindolinyl UV (420 nm)
o-Hydroxy-alpha-methyl cinnamoyl UV (300-350 nm) 2-Oxymethylene
anthraquinone UV (350 nm)
[0231] While the invention is illustrated primarily herein by way
of the use of a mask to illuminate selected regions the substrate,
other techniques may also be used. For example, the substrate may
be translated under a modulated laser or diode light source. Such
techniques are discussed in, for example, U.S. Pat. No. 4,719,615
(Feyrer et al.), which is incorporated herein by reference. In
alternative embodiments a laser galvanometric scanner is utilized.
In other embodiments, the synthesis may take place on or in contact
with a conventional liquid crystal (referred to herein as a "light
valve") or fiber optic light sources. By appropriately modulating
liquid crystals, light may be selectively controlled so as to
permit light to contact selected regions of the substrate.
Alternatively, synthesis may take place on the end of a series of
optical fibers to which light is selectively applied. Other means
of controlling the location of light exposure will be apparent to
those of skill in the art.
[0232] The substrate may be irradiated either in contact or not in
contact with a solution (not shown) and is, preferably, irradiated
in contact with a solution. The solution contains reagents to
prevent the by-products formed by irradiation from interfering with
synthesis of the polymer according to some embodiments. Such
by-products might include, for example, carbon dioxide,
nitrosocarbonyl compounds, styrene derivatives, indole derivatives,
and products of their photochemical reactions. Alternatively, the
solution may contain reagents used to match the index of refraction
of the substrate. Reagents added to the solution may further
include, for example, acidic or basic buffers, thiols, substituted
hydrazines and hydroxylamines, reducing agents (e.g., NADH) or
reagents known to react with a given functional group (e.g., aryl
nitroso+glyoxylic acid.fwdarw.aryl formhydroxamate+CO.sub.2).
[0233] Either concurrently with or after the irradiation step, the
linker molecules are washed or otherwise contacted with a first
monomer, illustrated by "A" in regions 12a and 12b in FIG. 6. The
first monomer reacts with the activated functional groups of the
linkage molecules which have been exposed to light. The first
monomer, which is preferably an amino acid, is also provided with a
photoprotective group. The photoprotective group on the monomer may
be the same as or different than the protective group used in the
linkage molecules, and may be selected from any of the
above-described protective groups. In one embodiment, the
protective groups for the A monomer is selected from the group NBOC
and NVOC.
[0234] As shown in FIG. 7, the process of irradiating is thereafter
repeated, with a mask repositioned so as to remove linkage
protective groups and expose functional groups in regions 14a and
14b which are illustrated as being regions which were protected in
the previous masking step. As an alternative to repositioning of
the first mask, in many embodiments a second mask will be utilized.
In other alternative embodiments, some steps may provide for
illuminating a common region in successive steps. As shown in FIG.
7, it may be desirable to provide separation between irradiated
regions. For example, separation of about 1-5 .mu.m may be
appropriate to account for alignment tolerances.
[0235] As shown in FIG. 8, the substrate is then exposed to a
second protected monomer "B" producing B regions 16a and 16b.
Thereafter, the substrate is again masked so as to remove the
protective groups and expose reactive groups on A region 12a and B
region 16b. The substrate is again exposed to monomer B, resulting
in the formation of the structure shown in FIG. 10. The diners B-A
and B--B have been produced on the substrate.
[0236] A subsequent series of masking and contacting steps similar
to those described above with A (not shown) provides the structure
shown in FIG. 11. The process provides all possible dimers of B and
A, i.e., B-A, A-B, A--A, and B--B.
[0237] The substrate, the area of synthesis, and the area for
synthesis of each individual polymer could be of any size or shape.
For example, squares, ellipsoids, rectangles, triangles, circles,
or portions thereof, along with irregular geometric shapes, may be
utilized. Duplicate synthesis areas may also be applied to a single
substrate for purposes of redundancy.
[0238] In one embodiment the regions 12 and 16 on the substrate
will have a surface area of between about 1 cm.sup.2 and 10.sup.-10
cm.sup.2. In some embodiments the regions 12 and 16 have areas of
less than about 10.sup.-1 cm.sup.2, 10.sup.-2 cm.sup.2, 10.sup.-3
cm.sup.2, 10.sup.-4 cm.sup.2, 10.sup.-5 cm.sup.2, 10.sup.-6
cm.sup.2, 10.sup.-7 cm.sup.2, 10.sup.-1 cm.sup.2, or 10.sup.-10
cm.sup.2. In a preferred embodiment, the regions 12 and 16 are
between about 10.times.10 .mu.m and 500.times.500 .mu.m.
[0239] In some embodiments a single substrate supports more than
about 10 different monomer sequences and preferably more than about
100 different monomer sequences, although in some embodiments more
than about 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, or
10.sup.8 different sequences are provided on a substrate. Of
course, within a region of the substrate in which a monomer
sequence is synthesized, it is preferred that the monomer sequence
be substantially pure. In some embodiments, regions of the
substrate contain polymer sequences which are at least about 1%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,
90%, 95%, 96%, 97%, 98%, or 99% pure.
[0240] According to some embodiments, several sequences are
intentionally provided within a single region so as to provide an
initial screening for biological activity, after which materials
within regions exhibiting significant binding are further
evaluated.
[0241] IV. Details of One Embodiment of a Reactor System
[0242] FIG. 12A schematically illustrates a preferred embodiment of
a reactor system 100 for synthesizing polymers on the prepared
substrate in accordance with one aspect of the invention. The
reactor system includes a body 102 with a cavity 104 on a surface
thereof. In preferred embodiments the cavity 104 is between about
50 and 1000 .mu.m deep with a depth of about 500 .mu.m
preferred.
[0243] The bottom of the cavity is preferably provided with an
array of ridges 106 which extend both into the plane of the Figure
and parallel to the plane of the Figure. The ridges are preferably
about 50 to 200 .mu.m deep and spaced at about 2 to 3 mm. The
purpose of the ridges is to generate turbulent flow for better
mixing. The bottom surface of the cavity is preferably light
absorbing so as to prevent reflection of impinging light.
[0244] A substrate 112 is mounted above the cavity 104. The
substrate is provided along its bottom surface 114 with a
photoremovable protective group such as NVOC with or without an
intervening linker molecule. The substrate is preferably
transparent to a wide spectrum of light, but in some embodiments is
transparent only at a wavelength at which the protective group may
be removed (such as UV in the case of NVOC). The substrate in some
embodiments is a conventional microscope glass slide or cover slip.
The substrate is preferably as thin as possible, while still
providing adequate physical support. Preferably, the substrate is
less than about 1 mm thick, more preferably less than 0.5 mm thick,
more preferably less than 0.1 mm thick, and most preferably less
than 0.05 mm thick. In alternative preferred embodiments, the
substrate is quartz or silicon.
[0245] The substrate and the body serve to seal the cavity except
for an inlet port 108 and an outlet port 110. The body and the
substrate may be mated for sealing in some embodiments with one or
more gaskets. According to a preferred embodiment, the body is
provided with two concentric gaskets and the intervening space is
held at vacuum to ensure mating of the substrate to the
gaskets.
[0246] Fluid is pumped through the inlet port into the cavity by
way of a pump 116 which may be, for example, a model no. B-120-S
made by Eldex Laboratories. Selected fluids are circulated into the
cavity by the pump, through the cavity, and out the outlet for
recirculation or disposal. The reactor may be subjected to
ultrasonic radiation and/or heated to aid in agitation in some
embodiments.
[0247] Above the substrate 112, a lens 120 is provided which may
be, for example, a 2" 100 mm focal length fused silica lens. For
the sake of a compact system, a reflective mirror 122 may be
provided for directing light from a light source 124 onto the
substrate. Light source 124 may be, for example, a Xe(Hg) light
source manufactured by Oriel and having model no. 66024. A second
lens 126 may be provided for the purpose of projecting a mask image
onto the substrate in combination with lens 112. This form of
lithography is referred to herein as projection printing. As will
be apparent from this disclosure, proximity printing and the like
may also be used according to some embodiments.
[0248] Light from the light source is permitted to reach only
selected locations on the substrate as a result of mask 128. Mask
128 may be, for example, a glass slide having etched chrome
thereon. The mask 128 in one embodiment is provided with a grid of
transparent locations and opaque locations. Such masks may be
manufactured by, for example, Photo Sciences, Inc. Light passes
freely through the transparent regions of the mask, but is
reflected from or absorbed by other regions. Therefore, only
selected regions of the substrate are exposed to light.
[0249] As discussed above, light valves (LCD's) may be used as an
alternative to conventional masks to selectively expose regions of
the substrate. Alternatively, fiber optic faceplates such as those
available from Schott Glass, Inc, may be used for the purpose of
contrast enhancement of the mask or as the sole means of
restricting the region to which light is applied. Such faceplates
would be placed directly above or on the substrate in the reactor
shown in FIG. 8A. In still further embodiments, flys-eye lenses,
tapered fiber optic faceplates, or the like, may be used for
contrast enhancement.
[0250] In order to provide for illumination of regions smaller than
a wavelength of light, more elaborate techniques may be utilized.
For example, according to one preferred embodiment, light is
directed at the substrate by way of molecular microcrystals on the
tip of, for example, micropipettes. Such devices are disclosed in
Lieberman et al., "A Light Source Smaller Than the Optical
Wavelength," Science, (1990) 247: 59-61, which is incorporated
herein by reference for all purposes.
[0251] In operation, the substrate is placed on the cavity and
sealed thereto. All operations in the process of preparing the
substrate are carried out in a room lit primarily or entirely by
light of a wavelength outside of the light range at which the
protective group is removed. For example, in the case of NVOC, the
room should be lit with a conventional dark room light which
provides little or no UV light. All operations are preferably
conducted at about room temperature.
[0252] A first, deprotection fluid (without a monomer) is
circulated through the cavity. The solution preferably is of 5 mM
sulfuric acid in dioxane solution which serves to keep exposed
amino groups protonated and decreases their reactivity with
photolysis by-products.
[0253] Absorptive materials such as N,N-diethylamino
2,4-dinitrobenzene, for example, may be included in the
deprotection fluid which serves to absorb light and prevent
reflection and unwanted photolysis.
[0254] The slide is, thereafter, positioned in a light raypath from
the mask such that first locations on the substrate are illuminated
and, therefore, deprotected. In preferred embodiments the substrate
is illuminated for between about 1 and 15 minutes with a preferred
illumination time of about 10 minutes at 10-20 mW/cm.sup.2 with 365
nm light. The slides are neutralized (i.e., brought to a pH of
about 7) after photolysis with, for example, a solution of
di-isopropylethylamine (DIEA) in methylene chloride for about 5
minutes.
[0255] The first monomer is then placed at the first locations on
the substrate. After irradiation, the slide is removed, treated in
bulk, and then reinstalled in the flow cell. Alternatively, a fluid
containing the first monomer, preferably also protected by a
protective group, is circulated through the cavity by way of pump
116. If, for example, it is desired to attach the amino acid Y to
the substrate at the first locations, the amino acid Y (bearing a
protective group on its alpha-nitrogen), along with reagents used
to render the monomer reactive, and/or a carrier, is circulated
from a storage container 118, through the pump, through the cavity,
and back to the inlet of the pump.
[0256] The monomer carrier solution is, in a preferred embodiment,
formed by mixing of a first solution (referred to herein as
solution "A") and a second solution (referred to herein as solution
"B"). Table 2 provides an illustration of a mixture which may be
used for solution A.
2TABLE 2 Representative Monomer Carrier Solution "A" 100 mg NVOC
amino protected amino acid 37 mg HOBT (1-Hydroxybenzotriazole) 250
.mu.l DMF (Dimethylformamide) 86 .mu.l DIEA
(Diisopropylethylamine)
[0257] The composition of solution B is illustrated in Table 3.
Solutions A and B are mixed and allowed to react at room
temperature for about 8 minutes, then diluted with 2 ml of DMF, and
500 .mu.l are applied to the surface of the slide or the solution
is circulated through the reactor system and allowed to react for
about 2 hours at room temperature. The slide is then washed with
DMF, methylene chloride and ethanol.
3TABLE 3 Representative Monomer Carrier Solution "B" 250 .mu.l DMF
111 mg BOP (Benzotriazolyl-n-oxy-tris(dimethylamino)
phosphoniumhexafluoroph- osphate)
[0258] As the solution containing the monomer to be attached is
circulated through the cavity, the amino acid or other monomer will
react at its carboxy terminus with amino groups on the regions of
the substrate which have been deprotected. Of course, while the
invention is illustrated by way of circulation of the monomer
through the cavity, the invention could be practiced by way of
removing the slide from the reactor and submersing it in an
appropriate monomer solution.
[0259] After addition of the first monomer, the solution containing
the first amino acid is then purged from the system. After
circulation of a sufficient amount of the DMF/methylene chloride
such that removal of the amino acid can be assured (e.g., about 50
times the volume of the cavity and carrier lines), the mask or
substrate is repositioned, or a new mask is utilized such that
second regions on the substrate will be exposed to light and the
light 124 is engaged for a second exposure. This will deprotect
second regions on the substrate and the process is repeated until
the desired polymer sequences have been synthesized.
[0260] The entire derivatized substrate is then exposed to a
receptor of interest, preferably labeled with, for example, a
fluorescent marker, by circulation of a solution or suspension of
the receptor through the cavity or by contacting the surface of the
slide in bulk. The receptor will preferentially bind to certain
regions of the substrate which contain complementary sequences.
[0261] Antibodies are typically suspended in what is commonly
referred to as "supercocktail," which may be, for example, a
solution of about 1% BSA (bovine serum albumin), 0.5% Tween in PBS
(phosphate buffered saline) buffer. The antibodies are diluted into
the supercocktail buffer to a final concentration of, for example,
about 0.1 to 4 .mu.g/ml.
[0262] FIG. 12B illustrates an alternative preferred embodiment of
the reactor shown in FIG. 8A. According to this embodiment, the
mask 128 is placed directly in contact with the substrate.
Preferably, the etched portion of the mask is placed face down so
as to reduce the effects of light dispersion. According to this
embodiment, the imaging lenses 120 and 126 are not necessary
because the mask is brought into close proximity with the
substrate.
[0263] For purposes of increasing the signal-to-noise ratio of the
technique, some embodiments of the invention provide for exposure
of the substrate to a first labeled or unlabeled receptor followed
by exposure of a labeled, second receptor (e.g., an antibody) which
binds at multiple sites on the first receptor. If, for example, the
first receptor is an antibody derived from a first species of an
animal, the second receptor is an antibody derived from a second
species directed to epitopes associated with the first species. In
the case of a mouse antibody, for example, fluorescently labeled
goat antibody or antiserum which is antimouse may be used to bind
at multiple sites on the mouse antibody, providing several times
the fluorescence compared to the attachment of a single mouse
antibody at each binding site. This process may be repeated again
with additional antibodies (e.g., goat-mouse-goat, etc.) for
further signal amplification.
[0264] In preferred embodiments an ordered sequence of masks is
utilized. In some embodiments it is possible to use as few as a
single mask to synthesize all of the possible polymers of a given
monomer set.
[0265] If, for example, it is desired to synthesize all 16
dinucleotides from four bases, a 1 cm square synthesis region is
divided conceptually into 16 boxes, each 0.25 cm wide. Denote the
four monomer units by A, B, C, and D. The first reactions are
carried out in four vertical columns, each 0.25 cm wide. The first
mask exposes the left-most column of boxes, where A is coupled. The
second mask exposes the next column, where B is coupled; followed
by a third mask, for the C column; and a final mask that exposes
the right-most column, for D. The first, second, third, and fourth
masks may be a single mask translated to different locations.
[0266] The process is repeated in the horizontal direction for the
second unit of the dimer. This time, the masks allow exposure of
horizontal rows, again 0.25 cm wide. A, B, C, and D are
sequentially coupled using masks that expose horizontal fourths of
the reaction area. The resulting substrate contains all 16
dinucleotides of four bases.
[0267] The eight masks used to synthesize the dinucleotide are
related to one another by translation or rotation. In fact, one
mask can be used in all eight steps if it is suitably rotated and
translated. For example, in the example above, a mask with a single
transparent region could be sequentially used to expose each of the
vertical columns, translated 90.degree., and then sequentially used
to allow exposure of the horizontal rows.
[0268] Tables 4 and 5 provide a simple computer program in Quick
Basic for planning a masking program and a sample output,
respectively, for the synthesis of a polymer chain of three
monomers ("residues") having three different monomers in the first
level, four different monomers in the second level, and five
different monomers in the third level in a striped pattern. The
output of the program is the number of cells, the number of
"stripes" (light regions) on each mask, and the amount of
translation required for each exposure of the mask.
4TABLE 4 Mask Strategy Program DEFINT A-Z DIM b(20), w(20), 1(500)
F$ = "LPT1:"OPEN f$ FOR OUTPUT AS #1 jmax = 3 `Number of residues
b(1) = 3: b(2) = 4: b(3) = 5 `Number of building blocks for res
1,2,3 g = 1: lmax(1) = 1 FOR j = 1 TO jmax: g= g * b(j): NEXT j
w(0) = 0: w(1) = g / b(1) PRINT #1, "MASK2.BAS, " DATE$, TIME$:
PRINT #1, PRINT #1, USING "Number of residues=##"; jamx FOR j = 1
TO jmax PRINT #1, USING " Residue ## ##building blocks"; j; b(j)
NEXT j PRINT #1, " PRINT #1, USING "Number of cells=####"; g: PRINT
#1, FOR j = 2 TO jmax lmax(j) = lmax(j - 1) * b(j - 1) w(j) = w(j -
1)/b(j) NEXT j FOR j - 1 TO jmax PRINT #1, USING "Mask for residue
##"; j: PRINT #1, PRINT #1, USING " Number of stripes=###"; lmax(j)
PRINT #1, USING " Width of each stripe=###"; w(j) FOR 1 = 1 TO
lmax(j) a = 1 + (1 - 1) * w(j - 1) ae = a + w(j) - 1 PRINT #1,
USING " Stripe ## begins at location ### and ends at ###"; 1; a; ae
NEXT 1 PRINT #1, PRINT #1, USING " For each of ## building blocks,
translate mask by ## cell(s)"; b(j); w(j), PRINT #1, : PRINT #1, :
PRINT #1, NEXT j
[0269]
5TABLE 5 Masking Strategy Output Number of residues= 3 Residue 1 3
building blocks Residue 2 4 building blocks Residue 3 5 building
blocks Number of cells= 60 , Mask for residue 1 Number of stripes=
1 Width of each stripe= 20 Stripe 1 begins at location 1 and ends
at 20 For each of 3 building blocks, translate mask by 20 cell(s)
Mask for residue 2 Number of stripes= 3 Width of each stripe= 5
Stripe 1 begins at location 1 and ends at 5 Stripe 2 begins at
location 21 and ends at 25 Stripe 3 begins at location 41 and ends
at 45 For each of 4 building blocks, translate mask by 5 cell(s)
Mask for residue 3 Number of stripes= 12 Width of each stripe= 1
Stripe 1 begins at location 1 and ends at 1 Stripe 2 begins at
location 6 and ends at 6 Stripe 3 begins at location 11 and ends at
11 Stripe 4 begins at location 16 and ends at 16 Stripe 5 begins at
location 21 and ends at 21 Stripe 6 begins at location 26 and ends
at 26 Stripe 7 begins at location 31 and ends at 31 Stripe 8 begins
at location 36 and ends at 36 Stripe 9 begins at location 41 and
ends at 41 Stripe 10 begins at location 46 and ends at 46 Stripe 11
begins at location 51 and ends at 51 Stripe 12 begins at location
56 and ends at 56 For each of 5 building blocks, translate mask by
1 cell(s)
[0270] V. Details of One Embodiment of a Fluorescent Detection
Device
[0271] FIG. 13 illustrates a fluorescent detection device for
detecting fluorescently labeled receptors on a substrate. A
substrate 112 is placed on an x/y translation table 202. In a
preferred embodiment the x/y translation table is a model no.
PM500-A1 manufactured by Newport Corporation. The x/y translation
table is connected to and controlled by an appropriately programmed
digital computer 204 which may be, for example, an appropriately
programmed IBM PC/AT or AT compatible computer. Of course, other
computer systems, special purpose hardware, or the like could
readily be substituted for the AT computer used herein for
illustration. Computer software for the translation and data
collection functions described herein can be provided based on
commercially available software including, for example, "Lab
Windows" licensed by National Instruments, which is incorporated
herein by reference for all purposes.
[0272] The substrate and x/y translation table are placed under a
microscope 206 which includes one or more objectives 208. Light
(about 488 nm) from a laser 210, which in some embodiments is a
model no. 2020-05 argon ion laser manufactured by Spectraphysics,
is directed at the substrate by a dichroic mirror 207 which passes
greater than about 520 nm light but reflects 488 nm light. Dichroic
mirror 207 may be, for example, a model no. FT510 manufactured by
Carl Zeiss. Light reflected from the mirror then enters the
microscope 206 which may be, for example, a model no. Axioscop 20
manufactured by Carl Zeiss. Fluorescein-marked materials on the
substrate will fluoresce>488 nm light, and the fluoresced light
will be collected by the microscope and passed through the mirror.
The fluorescent light from the substrate is then directed through a
wavelength filter 209 and, thereafter through an aperture plate
211. Wavelength filter 209 may be, for example, a model no. OG530
manufactured by Melles Griot and aperture plate 211 may be, for
example, a model no. 477352/477380 manufactured by Carl Zeiss.
[0273] The fluoresced light then enters a photomultiplier tube 212
which in some embodiments is a model no. R943-02 manufactured by
Hamamatsu, the signal is amplified in preamplifier 214 and photons
are counted by photon counter 216. The number of photons is
recorded as a function of the location in the computer 204. Pre-Amp
214 may be, for example, a model no. SR440 manufactured by Stanford
Research Systems and photon counter 216 may be a model no. SR400
manufactured by Stanford Research Systems. The substrate is then
moved to a subsequent location and the process is repeated. In
preferred embodiments the data are acquired every 1 to 100 .mu.m
with a data collection diameter of about 0.8 to 10 .mu.m preferred.
In embodiments with sufficiently high fluorescence, a CCD detector
with broadfield illumination is utilized.
[0274] By counting the number of photons generated in a given area
in response to the laser, it is possible to determine where
fluorescent marked molecules are located on the substrate.
Consequently, for a slide which has a matrix of polypeptides, for
example, synthesized on the surface thereof, it is possible to
determine which of the polypeptides is complementary to a
fluorescently marked receptor.
[0275] According to preferred embodiments, the intensity and
duration of the light applied to the substrate is controlled by
varying the laser power and scan stage rate for improved
signal-to-noise ratio by maximizing fluorescence emission and
minimizing background noise.
[0276] While the detection apparatus has been illustrated primarily
herein with regard to the detection of marked receptors, the
invention will find application in other areas. For example, the
detection apparatus disclosed herein could be used in the fields of
catalysis, DNA or protein gel scanning, and the like.
[0277] VI. Determination of Relative Binding Strength of
Receptors
[0278] The signal-to-noise ratio of the present invention is
sufficiently high that not only can the presence or absence of a
receptor on a ligand be detected, but also the relative binding
affinity of receptors to a variety of sequences can be
determined.
[0279] In practice it is found that a receptor will bind to several
peptide sequences in an array, but will bind much more strongly to
some sequences than others. Strong binding affinity will be
evidenced herein by a strong fluorescent or radiographic signal
since many receptor molecules will bind in a region of a strongly
bound ligand. Conversely, a weak binding affinity will be evidenced
by a weak fluorescent or radiographic signal due to the relatively
small number of receptor molecules which bind in a particular
region of a substrate having a ligand with a weak binding affinity
for the receptor. Consequently, it becomes possible to determine
relative binding avidity (or affinity in the case of univalent
interactions) of a ligand herein by way of the intensity of a
fluorescent or radiographic signal in a region containing that
ligand.
[0280] Semiquantitative data on affinities might also be obtained
by varying washing conditions and concentrations of the receptor.
This would be done by comparison to known ligand receptor pairs,
for example.
[0281] VII. Examples
[0282] The following examples are provided to illustrate the
efficacy of the inventions herein. All operations were conducted at
about ambient temperatures and pressures unless indicated to the
contrary.
[0283] A. Slide Preparation
[0284] Before attachment of reactive groups it is preferred to
clean the substrate which is, in a preferred embodiment a glass
substrate such as a microscope slide or cover slip. According to
one embodiment the slide is soaked in an alkaline bath consisting
of, for example, 1 liter of 95% ethanol with 120 ml of water and
120 grams of sodium hydroxide for 12 hours. The slides are then
washed under running water and allowed to air dry, and rinsed once
with a solution of 95% ethanol.
[0285] The slides are then aminated with, for example,
aminopropyltriethoxysilane for the purpose of attaching amino
groups to the glass surface on linker molecules, although any omega
functionalized silane could also be used for this purpose. In one
embodiment 0.1% aminopropyltriethoxysilane is utilized, although
solutions with concentrations from 10.sup.-7% to 10% may be used,
with about 10.sup.-3% to 2% preferred. A 0.1% mixture is prepared
by adding to 100 ml of a 95% ethanol/5% water mixture, 100
microliters (.mu.l) of aminopropyltriethoxysilane. The mixture is
agitated at about ambient temperature on a rotary shaker for about
5 minutes. 500 .mu.l of this mixture is then applied to the surface
of one side of each cleaned slide. After 4 minutes, the slides are
decanted of this solution and rinsed three times by dipping in, for
example, 100% ethanol.
[0286] After the plates dry, they are placed in a 110-120.degree.
C. vacuum oven for about 20 minutes, and then allowed to cure at
room temperature for about 12 hours in an argon environment. The
slides are then dipped into DMF (dimethylformamide) solution,
followed by a thorough washing with methylene chloride.
[0287] The aminated surface of the slide is then exposed to about
500 .mu.l of, for example, a 30 millimolar (mM) solution of
NVOC-GABA (gamma amino butyric acid) NHS(N-hydroxysuccinimide) in
DMF for attachment of a NVOC-GABA to each of the amino groups.
[0288] The surface is washed with, for example, DMF, methylene
chloride, and ethanol.
[0289] Any unreacted aminopropyl silane on the surface--that is,
those amino groups which have not had the NVOC-GABA attached--are
now capped with acetyl groups (to prevent further reaction) by
exposure to a 1:3 mixture of acetic anhydride in pyridine for 1
hour. Other materials which may perform this residual capping
function include trifluoroacetic anhydride, formicacetic a hydride,
or other reactive acylating agents. Finally, the slides are washed
again with DMF, methylene chloride, and ethanol.
[0290] B. Synthesis of Eight Trimers of "A" and "B"
[0291] FIG. 14 illustrates a possible synthesis of the eight
trimers of the two-monomer set: gly, phe (represented by "A" and
"B," respectively). A glass slide bearing silane groups terminating
in 6-nitro veratryloxycarboxamide (NVOC--NH) residues is prepared
as a substrate. Active esters (pentafluorophenyl, OBt, etc.) of gly
and phe protected at the amino group with NVOC are prepared as
reagents. While not pertinent to this example, if side chain
protecting groups are required for the monomer set, these must not
be photoreactive at the wavelength of light used to protect the
primary chain.
[0292] For a monomer set of size n, n.times.l cycles are required
to synthesize all possible sequences of length l. A cycle consists
of:
[0293] 1. Irradiation through an appropriate mask to expose the
amino groups at the sites where the next residue is to be added,
with appropriate washes to remove the by-products of the
deprotection.
[0294] 2. Addition of a single activated and protected (with the
same photochemically-removable group) monomer, which will react
only at the sites addressed in step 1, with appropriate washes to
remove the excess reagent from the surface.
[0295] The above cycle is repeated for each member of the monomer
set until each location on the surface has been extended by one
residue in one embodiment. In other embodiments, several residues
are sequentially added at one location before moving on to the next
location. Cycle times will generally be limited by the coupling
reaction rate, now as short as 20 min in automated peptide
synthesizers. This step is optionally followed by addition of a
protecting group to stabilize the array for later testing. For some
types of polymers (e.g., peptides), a final deprotection of the
entire surface (removal of photoprotective side chain groups) may
be required.
[0296] More particularly, as shown in FIG. 14A, the glass 20 is
provided with regions 22, 24, 26, 26, 30, 32, 34, and 36. Regions
30, 32, 34, and 36 are masked, as shown in FIG. 14B and the glass
is irradiated and exposed to a reagent containg "A" (e.g., gly),
with the resulting structure shown in FIG. 14C. Thereafter, regions
22, 24, 26, and 28 are masked, the glass is irradiated (as shown in
FIG. 14D) and exposed to a reagent containing "B" (e.g., phe), with
the resulting structure shown in FIG. 14E. The process proceeds,
consecutively masking and exposing the sections as shown until the
structure shown in FIG. 14M, is obtained. The glass is irradiated
and the terminal groups are, optionally, capped by acetylation. As
shown, all possible trimers of gly/phe are obtained.
[0297] In this example, no side chain protective group removal is
necessary. If it is desired, side chain deprotection may be
accomplished by treatment with ethanedithiol and trifluoroacetic
acid.
[0298] In general, the number of steps needed to obtain a
particular polymer chain is defined by:
n.times.l (1)
[0299] where:
[0300] n=the number of monomers in the basis set of monomers,
and
[0301] l=the number of monomer units in a polymer chain.
[0302] Conversely, the synthesized number of sequences of length l
will be:
n.sup.l (2)
[0303] Of course, greater diversity is obtained by using masking
strategies which will also include the synthesis of polymers having
a length of less than 1. If, in the extreme case, all polymers
having a length less than or equal to 1 are synthesized, the number
of polymers synthesized will be:
n.sup.l+n.sup.l-1 + . . . +n.sup.l (3)
[0304] The maximum number of lithographic steps needed will
generally be n for each "layer" of monomers, i.e., the total number
of masks (and, therefore, the number of lithographic steps) needed
will be n.times.l. The size of the transparent mask regions will
vary in accordance with the area of the substrate available for
synthesis and the number of sequences to be formed. In general, the
size of the synthesis areas will be:
size of synthesis areas=(A)/(S)
[0305] where:
[0306] A is the total area available for synthesis; and
[0307] S is the number of sequences desired in the area.
[0308] It will be appreciated by those of skill in the art that the
above method could readily be used to simultaneously produce
thousands or millions of oligomers on a substrate using the
photolithographic techniques disclosed herein. Consequently, the
method results in the ability to practically test large numbers of,
for example, di, tri, tetra, penta, hexa, hepta, octapeptides,
dodecapeptides, or larger polypeptides (or correspondingly,
polynucleotides).
[0309] The above example has illustrated the method by way of a
manual example. It will of course be appreciated that automated or
semi-automated methods could be used. The substrate would be
mounted in a flow cell for automated addition and removal of
reagents, to minimize the volume of reagents needed, and to more
carefully control reaction conditions. Successive masks could be
applied manually or automatically.
[0310] C. Synthesis of a Dimer of an Aminopropyl Group and a
Fluorescent Group
[0311] In synthesizing the dimer of an aminopropyl group and a
fluorescent group, a functionalized durapore membrane was used as a
substrate. The durapore membrane was a polyvinylidine difluoride
with aminopropyl groups. The aminopropyl groups were protected with
the DDZ group by reaction of the carbonyl chloride with the amino
groups, a reaction readily known to those of skill in the art. The
surface bearing these groups was placed in a solution of THF and
contacted with a mask bearing a checkerboard pattern of 1 mm opaque
and transparent regions. The mask was exposed to ultraviolet light
having a wavelength down to at least about 280 nm for about 5
minutes at ambient temperature, although a wide range of exposure
times and temperatures may be appropriate in various embodiments of
the invention. For example, in one embodiment, an exposure time of
between about 1 and 5000 seconds may be used at process
temperatures of between -70 and +50.degree. C.
[0312] In one preferred embodiment, exposure times of between about
1 and 500 seconds at about ambient pressure are used. In some
preferred embodiments, pressure above ambient is used to prevent
evaporation.
[0313] The surface of the membrane was then washed for about 1 hour
with a fluorescent label which included an active ester bound to a
chelate of a lanthanide.
[0314] Wash times will vary over a wide range of values from about
a few minutes to a few hours. These materials fluoresce in the red
and the green visible region. After the reaction with the active
ester in the fluorophore was complete, the locations in which the
fluorophore was bound could be visualized by exposing them to
ultraviolet light and observing the red and the green fluorescence.
It was observed that the derivatized regions of the substrate
closely corresponded to the original pattern of the mask.
[0315] D. Demonstration of Signal Capability
[0316] Signal detection capability was demonstrated using a
low-level standard fluorescent bead kit manufactured by Flow
Cytometry Standarda and having model no. 824. This kit includes 5.8
.mu.m diameter beads, each impregnated with a known number of
fluorescein molecules.
[0317] One of the beads was placed in the illumination field on the
scan stage as shown in FIG. 9 in a field of a laser spot which was
initially shuttered. After being positioned in the illumination
field, the photon detection equipment was turned on. The laser beam
was unblocked and it interacted with the particle bead, which then
fluoresced. Fluorescence curves of beads impregnated with 7,000;
13,000; and 29,000 fluorescein molecules, are shown in FIGS. 11A,
11B, and 11C respectively. On each curve, traces for beads without
fluorescein molecules are also shown. These experiments were
performed with 488 nm excitation, with 100 .mu.W of laser power.
The light was focused through a 40 power 0.75 NA objective.
[0318] The fluorescence intensity in all cases started off at a
high value and then decreased exponentially. The fall-off in
intensity is due to photobleaching of the fluorescein molecules.
The traces of beads without fluorescein molecules are used for
background subtraction. The difference in the initial exponential
decay between labeled and nonlabeled beads is integrated to give
the total number of photon counts, and this number is related to
the number of molecules per bead. Therefore, it is possible to
deduce the number of photons per fluorescein molecule that can be
detected. For the curves illustrated in FIG. 11, this calculation
indicates the radiation of about 40 to 50 photons per fluorescein
molecule are detected.
[0319] E. Determination of the Number of Molecules Per Unit
Area
[0320] Aminopropylated glass microscope slides prepared according
to the methods discussed above were utilized in order to establish
the density of labeling of the slides. The free amino termini of
the slides were reacted with FITC (fluorescein isothiocyanate)
which forms a covalent linkage with the amino group. The slide is
then scanned to count the number of fluorescent photons generated
in a region which, using the estimated 40-50 photons per
fluorescent molecule, enables the calculation of the number of
molecules which are on the surface per unit area.
[0321] A slide with aminopropyl silane on its surface was immersed
in a 1 mM solution of FITC in DMF for 1 hour at about ambient
temperature. After reaction, the slide was washed twice with DMF
and then washed with ethanol, water, and then ethanol again. It was
then dried and stored in the dark until it was ready to be
examined.
[0322] Through the use of curves similar to those shown in FIG. 15,
and by integrating the fluorescent counts under the exponentially
decaying signal, the number of free amino groups on the surface
after derivitization was determined. It was determined that slides
with labeling densities of 1 fluoroscein per
10.sup.3.times.10.sup.3 to .about.2.times.2 nm could be
reproducibly made as the concentration of
aminopropyltriethoxysilane varied from 10.sup.-5% to
10.sup.-1%.
[0323] F. Removal of NVOC and Attachment of A Fluorescent
Marker
[0324] NVOC-GABA groups were attached as described above. The
entire surface of one slide was exposed to light so as to expose a
free amino group at the end of the gamma amino butyric acid. This
slide, and a duplicate which was not exposed, were then exposed to
fluorescein isothiocyanate (FITC).
[0325] FIG. 16A illustrates the slide which was not exposed to
light, but which was exposed to FITC. The units of the x axis are
time and the units of the y axis are counts. The trace contains a
certain amount of background fluorescence. The duplicate slide was
exposed to 350 nm broadband illumination for about 1 minute (12
mW/cm.sup.2, .about.350 nm illumination), washed and reacted with
FITC. The fluorescence curves for this slide are shown in FIG. 16B.
A large increase in the level of fluorescence is observed, which
indicates photolysis has exposed a number of amino groups on the
surface of the slides for attachment of a fluorescent marker.
[0326] G. Use of a Mask in Removal of NVOC
[0327] The next experiment was performed with a 0.1%
aminopropylated slide. Light from a Hg--Xe arc lamp was imaged onto
the substrate through a laser-ablated chrome-on-glass mask in
direct contact with the substrate.
[0328] This slide was illuminated for approximately 5 minutes, with
12 mW of 350 nm broadband light and then reacted with the 1 mM FITC
solution. It was put on the laser detection scanning stage and a
graph was plotted as a two-dimensional representation of position
color-coded for fluorescence intensity. The fluorescence intensity
(in counts) as a function of location is given on the color scale
to the right of FIG. 17A for a mask having 100.times.100 .mu.m
squares.
[0329] The experiment was repeated a number of times through
various masks. The fluorescence pattern for a 50 lam mask is
illustrated in FIG. 17B, for a 20 .mu.m mask in FIG. 17C, and for a
10 .mu.m mask in FIG. 17D. The mask pattern is distinct down to at
least about 10 .mu.m squares using this lithographic technique.
[0330] H. Attachment of YGGFL and Subsequent Exposure to Herz
Antibody and Goat Antimouse
[0331] In order to establish that receptors to a particular
polypeptide sequence would bind to a surface-bound peptide and be
detected, Leu enkephalin was coupled to the surface and recognized
by an antibody. A slide was derivatized with 0.1% amino
propyl-triethoxysilane and protected with NVOC. A 500 .mu.m
checkerboard mask was used to expose the slide in a flow cell using
backside contact printing. The Leu enkephalin sequence
(H.sub.2N-tyrosine, glycine, glycine, phenylalanine,
leucine-CO.sub.2H, otherwise referred to herein as YGGFL) was
attached via its carboxy end to the exposed amino groups on the
surface of the slide. The peptide was added in DMF solution with
the BOP/HOBT/DIEA coupling reagents and recirculated through the
flow cell for 2 hours at room temperature.
[0332] A first antibody, known as the Herz antibody, was applied to
the surface of the slide for 45 minutes at 2 .mu.g/ml in a
supercocktail (containing 1% BSA and 1% ovalbumin also in this
case). A second antibody, goat anti-mouse fluorescein conjugate,
was then added at 2 .mu.g/ml in the supercocktail buffer, and
allowed to incubate for 2 hours.
[0333] The results of this experiment are provided in FIG. 18.
Again, this figure illustrates fluorescence intensity as a function
of position. The fluorescence scale is shown on the right,
according to the color coding. This image was taken at 10 .mu.m
steps. This figure indicates that not only can deprotection be
carried out in a well defined pattern, but also that (1) the method
provides for successful coupling of peptides to the surface of the
substrate, (2) the surface of a bound peptide is available for
binding with an antibody, and (3) that the detection apparatus
capabilities are sufficient to detect binding of a receptor.
[0334] I. Monomer-by-Monomer Formation of YGGFL and Subsequent
Exposure to Labeled Antibody
[0335] Monomer-by-monomer synthesis of YGGFL and GGFL in alternate
squares was performed on a slide in a checkerboard pattern and the
resulting slide was exposed to the Herz antibody. This experiment
and the result thereof are illustrated in FIGS. 19A, 19B, 19C, and
19D.
[0336] In FIG. 19A, a slide is shown which is derivatized with the
aminopropyl group, protected in this case with t-BOC
(t-butoxycarbonyl). The slide was treated with TFA to remove the
t-BOC protecting group. .epsilon.-aminocaproic acid, which was
t-BOC protected at its amino group, was then coupled onto the
aminopropyl groups. The aminocaproic acid serves as a spacer
between the aminopropyl group and the peptide to be synthesized.
The amino end of the spacer was deprotected and coupled to
NVOC-leucine. The entire slide was then illuminated with 12 mW of
325 nm broadband illumination. The slide was then coupled with
NVOC-phenylalanine and washed. The entire slide was again
illuminated, then coupled to NVOC-glycine and washed. The slide was
again illuminated and coupled to NVOC-glycine to form the sequence
shown in the last portion of FIG. 19A.
[0337] As shown in FIG. 19B, alternating regions of the slide were
then illuminated using a projection print using a 500.times.500
.mu.m checkerboard mask; thus, the amino group of glycine was
exposed only in the lighted areas. When the next coupling chemistry
step was carried out, NVOC-tyrosine was added, and it coupled only
at those spots which had received illumination. The entire slide
was then illuminated to remove all the NVOC groups, leaving a
checkerboard of YGGFL in the lighted areas and in the other areas,
GGFL. The Herz antibody (which recognizes the YGGFL, but not GGFL)
was then added, followed by goat anti-mouse fluorescein
conjugate.
[0338] The resulting fluorescence scan is shown in FIG. 19C, and
the color coding for the fluorescence intensity is again given on
the right. Dark areas contain the tetrapeptide GGFL, which is not
recognized by the Herz antibody (and thus there is no binding of
the goat anti-mouse antibody with fluorescein conjugate), and in
the red areas YGGFL is present. The YGGFL pentapeptide is
recognized by the Herz antibody and, therefore, there is antibody
in the lighted regions for the fluorescein-conjugated goat
anti-mouse to recognize.
[0339] Similar patterns are shown for a 50 .mu.m mask used in
direct contact ("proximity print") with the substrate in FIG. 19D.
Note that the pattern is more distinct and the corners of the
checkerboard pattern are touching when the mask is placed in direct
contact with the substrate (which reflects the increase in
resolution using this technique).
[0340] J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL
[0341] A synthesis using a 50 .mu.m checkerboard mask similar to
that shown in FIG. 19 was conducted. However, P was added to the
GGFL sites on the substrate through an additional coupling step. P
was added by exposing protected GGFL to light through a mask, and
subsequence exposure to P in the manner set forth above. Therefore,
half of the regions on the substrate contained YGGFL and the
remaining half contained GGFL.
[0342] The fluorescence plot for this experiment is provided in
FIG. 20. As shown, the regions are again readily discernable. This
experiment demonstrates that antibodies are able to recognize a
specific sequence and that the recognition is not
length-dependent.
[0343] K. Monomer-by-Monomer Synthesis of YGGFL and YPGGFL
[0344] In order to further demonstrate the operability of the
invention, a 50 .mu.m checkerboard pattern of alternating YGGFL and
YPGGFL was synthesized on a substrate using techniques like those
set forth above. The resulting fluorescence plot is provided in
FIG. 21. Again, it is seen that the antibody is clearly able to
recognize the YGGFL sequence and does not bind significantly at the
YPGGFL regions.
[0345] L. Synthesis of an Array or Sixteen Different Amino Acid
Sequences and Estimation of Relative Binding Affinity to Herz
Antibody
[0346] Using techniques similar to those set forth above, an array
of 16 different amino acid sequences (replicated four times) was
synthesized on each of two glass substrates. The sequences were
synthesized by attaching the sequence NVOC-GFL across the entire
surface of the slides. Using a series of masks, two layers of amino
acids were then selectively applied to the substrate. Each region
had dimensions of 0.25 cm.times.0.0625 cm. The first slide
contained amino acid sequences containing only L amino acids while
the second slide contained selected D amino acids. FIGS. 18A and
18B illustrate a map of the various regions on the first and second
slides, respectively. The patterns shown in FIGS. 22A and 22B were
duplicated four times on each slide. The slides were then exposed
to the Herz antibody and fluorescein-labeled goat anti-mouse.
[0347] FIG. 23 is a fluorescence plot of the first slide, which
contained only L amino acids. Red indicates strong binding (149,000
counts or more) while black indicates little or no binding of the
Herz antibody (20,000 counts or less). The bottom right-hand
portion of the slide appears "cut off" because the slide was broken
during processing.
[0348] The sequence YGGFL is clearly most strongly recognized. The
sequences YAGFL and YSGFL also exhibit strong recognition of the
antibody. By contrast, most of the remaining sequences show little
or no binding. The four duplicate portions of the slide are
extremely consistent in the amount of binding shown therein.
[0349] FIG. 24 is a fluorescence plot of the second slide. Again,
strongest binding is exhibited by the YGGFL sequence. Significant
binding is also detected to YaGFL, YsGFL, and YpGFL. The remaining
sequences show less binding with the antibody. Note the low binding
efficiency of the sequence YGGFL.
[0350] Table 6 lists the various sequences tested in order of
relative fluorescence, which provides information regarding
relative binding affinity.
6TABLE 6 Apparent Binding to Herz Ab L-a.a. Set D-a.a. Set YGGFL
YGGFL YAGFL TaGFL YSGFL YsGFL LGGFL YpGFL FGGFL fGGFL YPGFL yGGFL
LAGFL faGFL FAGFL wGGFL WGGFL yaGFL fpGFL waGFL
[0351] VIII. Illustrative Alternative Embodiment
[0352] According to an alternative embodiment of the invention, the
methods provide for attaching to the surface a caged binding member
which in its caged form has a relatively low affinity for other
potentially binding species, such as receptors and specific binding
substances. Such techniques are more fully described in copending
application Ser. No. 404,920, filed Sep. 8, 1989, and incorporated
herein by reference for all purposes.
[0353] According to this alternative embodiment, the invention
provides methods for forming predefined regions on a surface of a
solid support, wherein the predefined regions are capable of
immobilizing receptors. The methods make use of caged binding
members attached to the surface to enable selective activation of
the predefined regions. The caged binding members are liberated to
act as binding members ultimately capable of binding receptors upon
selective activation of the predefined regions. The activated
binding members are then used to immobilize specific molecules such
as receptors on the predefined region of the surface. The above
procedure is repeated at the same or different sites on the surface
so as to provide a surface prepared with a plurality of regions on
the surface containing, for example, the same or different
receptors. When receptors immobilized in this way have a
differential affinity for one or more ligands, screenings and
assays for the ligands can be conducted in the regions of the
surface containing the receptors.
[0354] The alternative embodiment may make use of novel caged
binding members attached to the substrate. Caged (unactivated)
members have a relatively low affinity for receptors of substances
that specifically bind to uncaged binding members when compared
with the corresponding affinities of activated binding members.
Thus, the binding members are protected from reaction until a
suitable source of energy is applied to the regions of the surface
desired to be activated. Upon application of a suitable energy
source, the caging groups labilize, thereby presenting the
activated binding member. A typical energy source will be
light.
[0355] Once the binding members on the surface are activated they
may be attached to a receptor. The receptor chosen may be a
monoclonal antibody, a nucleic acid sequence, a drug receptor, etc.
The receptor will usually, though not always, be prepared so as to
permit attaching it, directly or indirectly, to a binding member.
For example, a specific binding substance having a strong binding
affinity for the binding member and a strong affinity for the
receptor or a conjugate of the receptor may be used to act as a
bridge between binding members and receptors if desired. The method
uses a receptor prepared such that the receptor retains its
activity toward a particular ligand.
[0356] Preferably, the caged binding member attached to the solid
substrate will be a photoactivatable biotin complex, i.e., a biotin
molecule that has been chemically modified with photoactivatable
protecting groups so that it has a significantly reduced binding
affinity for avidin or avidin analogs than does natural biotin. In
a preferred embodiment, the protecting groups localized in a
predefined region of the surface will be removed upon application
of a suitable source of radiation to give binding members, that are
biotin or a functionally analogous compound having substantially
the same binding affinity for avidin or avidin analogs as does
biotin.
[0357] In another preferred embodiment, avidin or an avidin analog
is incubated with activated binding members on the surface until
the avidin binds strongly to the binding members. The avidin so
immobilized on predefined regions of the surface can then be
incubated with a desired receptor or conjugate of a desired
receptor. The receptor will preferably be biotinylated, e.g., a
biotinylated antibody, when avidin is immobilized on the predefined
regions of the surface. Alternatively, a preferred embodiment will
present an avidin/biotinylated receptor complex, which has been
previously prepared, to activated binding members on the
surface.
[0358] IX. Conclusion
[0359] The present inventions provide greatly improved methods and
apparatus for synthesis of polymers on substrates. It is to be
understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reviewing the above description.
By way of example, the invention has been described primarily with
reference to the use of photoremovable protective groups, but it
will be readily recognized by those of skill in the art that
sources of radiation other than light could also be used. For
example, in some embodiments it may be desirable to use protective
groups which are sensitive to electron beam irradiation, x-ray
irradiation, in combination with electron beam lithograph, or x-ray
lithography techniques. Alternatively, the group could be removed
by exposure to an electric current.
[0360] A preferred class of photoremovable protecting groups has
the general formula: 2
[0361] where R.sup.1, R.sup.2, R.sup.3, and R.sup.4 independently
are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen,
hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,
formats, formamido or phosphido group, or adjacent substituents
(i.e., R.sup.1-R.sup.2, R.sup.2-R.sup.3, R.sup.3-R.sup.4) are
substituted oxygen groups that together form a cyclic acetal or
ketal; R.sup.5 is a hydrogen atom, a alkoxyl, alkyl, hydrogen,
halo, aryl, or alkenyl group, and n=0 or 1.
[0362] A preferred protecting group, 6-nitroveratryl (NV), which is
used for protecting the carboxyl terminus of an amino acid or the
hydroxyl group of a nucleotide, for example, is formed when R.sup.2
and R.sup.3 are each a methoxy group, R.sup.1, R.sup.4 and R.sup.5
are each a hydrogen atom, and n=0: 3
[0363] A preferred protecting group, 6-nitroveratryloxycarbonyl
(NVOC), which is used to protect the amino terminus of an amino
acid, for example, is formed when R.sup.2 and R.sup.3 are each a
methoxy group, R.sup.1, R.sup.4 and R.sup.5 are each a hydrogen
atom, and n=1: 4
[0364] Another preferred protecting group, 6-nitropiperonyl (NP),
which is used for protecting the carboxyl terminus of an amino acid
or the hydroxyl group of a nucleotide, for example, is formed when
R.sup.2 and R.sup.3 together form a methylene acetal, R.sup.1,
R.sup.4 and R.sup.5 are each a hydrogen atom, and n=0: 5
[0365] Another preferred protecting group,
6-nitropiperonyloxycarbonyl (NPOC), which is used to protect the
amino terminus of an amino acid, for example, is formed when
R.sup.2 and R.sup.3 together form a methylene acetal, R.sup.1,
R.sup.4 and R.sup.5 are each a hydrogen atom, and n=1: 6
[0366] A most preferred protecting group, methyl-6-nitroveratryl
(MeNV), which is used for protecting the carboxyl terminus of an
amino acid or the hydroxyl group of a nucleotide, for example, is
formed when R.sup.2 and R.sup.3 are each a methoxy group, R.sup.1
and R.sup.4 are each a hydrogen atom, R.sup.5 is a methyl group,
and n=0: 7
[0367] Another most preferred protecting group,
methyl-6-nitroveratryloxyc- arbonyl MeNVOC), which is used to
protect the amino terminus of an amino acid, for example, formed
when R.sup.2 and R.sup.3 are each a methoxy group, R.sup.1 and
R.sup.4 are each a hydrogen tom, R.sup.5 is a methyl group, and
n=1: 8
[0368] Another most preferred protecting group,
methyl-6-nitropiperonyl (MeNP), which is used for protecting the
carboxyl terminus of an amino acid or the hydroxyl group of a
nucleotide, for example, is formed when R.sup.2 and R.sup.3
together form a methylene acetal, R.sup.1 and R.sup.4 are each a
hydrogen atom, R.sup.5 is a methyl group, and n=0: 9
[0369] Another most preferred protecting group,
methyl-6-nitropiperonyloxy- carbonyl (MeNPOC), which is used to
protect the amino terminus of an amino acid, for example, is formed
when R.sup.2 and R.sup.3 together form a methylene acetal, R.sup.1
and R.sup.4 are each a hydrogen atom, R.sup.5 is a methyl group,
and n=1: 10
[0370] A protected amino acid having a photoactivatable oxycarbonyl
protecting group, such NVOC or NPOC or their corresponding methyl
derivatives, MeNVOC or MeNPOC, respectively, on the amino terminus
is formed by acylating the amine of the amino acid with an
activated oxycarbonyl ester of the protecting group. Examples of
activated oxycarbonyl esters of NVOC and MeNVOC have the general
formula: 11
[0371] where X is halogen, mixed anhydride, phenoxy,
p-nitrophenoxy, N-hydroxysuccinimide, and the like.
[0372] A protected amino acid or nucleotide having a
photoactivatable protecting group, such as NV or NP or their
corresponding methyl derivatives, MeNV or MeNP, respectively, on
the carboxy terminus of the amino acid or 5'-hydroxy terminus of
the nucleotide, is formed by acylating the carboxy terminus or
5'-OH with an activated benzyl derivative of the protecting group.
Examples of activated benzyl derivatives of MeNV and MeNP have the
general formula: 12
[0373] where X is halogen, hydroxyl, tosyl, mesyl, trifluormethyl,
diazo, azido, and the like.
[0374] Another method for generating protected monomers is to react
the benzylic alcohol derivative of the protecting group with an
activated ester of the monomer. For example, to protect the
carboxyl terminus of an amino acid, an activated ester of the amino
acid is reacted with the alcohol derivative of the protecting
group, such as 6-nitroveratrol (NVOH). Examples of activated esters
suitable for such uses include halo-formate, mixed anhydride,
imidazoyl formate, acyl halide, and also includes formation of the
activated ester in situ the use of common reagents such as DCC and
the like. See Atherton et al. for other examples of activated
enters.
[0375] A further method for generating protected monomers is to
react the benzylic alcohol derivative of the protecting group with
an activated carbon of the monomer. For example, to protect the
5'-hydroxyl group of a nucleic acid, a derivative having a
5'-activated carbon is reacted with the alcohol derivative of the
protecting group, such as methyl-6-nitropiperonol (MePyROH).
Examples of nucleotides having activating groups attached to the
5'-hydroxyl group have the general formula: 13
[0376] where Y is a halogen atom, a tosyl, mesyl, trifluoromethyl,
azido, or diazo group, and the like.
[0377] Another class of preferred photochemical protecting groups
has the formula: 14
[0378] where R.sup.1, R.sup.2, and R.sup.3 independently are a
hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl,
alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate,
formamido, sulfanates, sulfido or phosphido group, R.sup.4 and
R.sup.5 independently are a hydrogen atom, an alkoxy, alkyl, halo,
aryl, hydrogen, or alkenyl group, and n=0 or 1.
[0379] A preferred protecting group, 1-pyrenylmethyloxycarbonyl
(PyROC), which is used to protect the amino terminus of an amino
acid, for example, is formed when R.sup.1 through R.sup.5 are each
a hydrogen atom and n=1: 15
[0380] Another preferred protecting group, 1-pyrenylmethyl (PyR),
which is used for protecting the carboxy terminus of an amino acid
or the hydroxyl group of a nucleotide, for example, is formed when
R.sup.1 through R.sup.5 are each a hydrogen atom and n=0: 16
[0381] An amino acid having a pyrenylmethyloxycarbonyl protecting
group on its amino terminus is formed by acylation of the free
amine of amino acid with an activated oxycarbonyl ester of the
pyrenyl protecting group. Examples of activated oxycarbonyl esters
of PyROC have the general formula: 17
[0382] where X is halogen, or mixed anhydride, p-nitrophenoxy, or
N-hydroxysuccinimide group, and the like.
[0383] A protected amino acid or nucleotide having a
photoactivatable protecting group, such as PyR, on the carboxy
terminus of the amino acid or 5'-hydroxy terminus of the nucleic
acid, respectively, is formed by acylating the carboxy terminus or
5'-OH with an activated pyrenylmethyl derivative of the protecting
group. Examples of activated pyrenylmethyl derivatives of PyR have
the general formula: 18
[0384] where X is a halogen atom, a hydroxyl, diazo, or azido
group, and the like.
[0385] Another method of generating protected monomers is to react
the pyrenylmethyl alcohol moiety of the protecting group with an
activated ester of the monomer. For example, an activated ester of
an amino acid can be reacted with the alcohol derivative of the
protecting group, such as pyrenylmethyl alcohol (PyROH), to form
the protected derivative of the carboxy terminus of the amino acid.
Examples of activated esters include halo-formate, mixed anhydride,
imidazoyl formate, acyl halide, and also includes formation of the
activated ester in situ and the use of common reagents such as DCC
and the like.
[0386] Clearly, many photosensitive protecting groups are suitable
for use in the present invention.
[0387] In preferred embodiments, the substrate is irradiated to
remove the photoremovable protecting groups and create regions
having free reactive moieties and side products resulting from the
protecting group. The removal rate of the protecting groups depends
on the wavelength and intensity of the incident radiation, as well
as the physical and chemical properties of the protecting group
itself. Preferred protecting groups are removed at a faster rate
and with a lower intensity of radiation. For example, at a given
set of conditions, MeNVOC and MeNPOC are photolytically removed
from the N-terminus of a peptide chain faster than their
unsubstituted parent compounds, NVOC and NPOC, respectively.
[0388] Removal of the protecting group is accomplished by
irradiation to liberate the reactive group and degradation products
derived from the protecting group. Not wishing to be bound by
theory, it is believed that irradiation of an NVOC- and
MeNVOC-protected oligomers occurs by the following reaction
schemes:
[0389]
NVOC-AA.fwdarw.3,4-dimethoxy-6-nitrosobenzaldehyde+CO.sub.2+AA
[0390]
MeNVOC-AA.fwdarw.3,4-dimethoxy-6-nitrosoacetophenone+CO.sub.2+AA
[0391] where AA represents the N-terminus of the amino acid
oligomer.
[0392] Along with the unprotected amino acid, other products are
liberated into solution: carbon dioxide and a
2,3-dimethoxy-6-nitrosophenylcarbonyl compound, which can react
with nucleophilic portions of the oligomer to form unwanted
secondary reactions. In the case of an NVOC-protected amino acid,
the degradation product is a nitrosobenzaldehyde, while the
degradation product for the other is a nitrosophenyl ketone. For
instance, it is believed that the product aldehyde from NVOC
degradation reacts with free amines to form a schiff base (imine)
that affects the remaining polymer synthesis. Preferred
photoremovable protecting groups react slowly or reversibly with
the oligomer on the support.
[0393] Again not wishing to be bound by theory, it is believed that
the product ketone from irradiation of a MeNVOC-protected oligomer
reacts at a slower rate with nucleophiles on the oligomer than the
product aldehyde from irradiation of the same NVOC-protected
oligomer. Although not unambiguously determined, it is believed
that this difference in reaction rate is due to the difference in
general reactivity between aldehyde and ketones towards
nucleophiles due to steric and electronic effects.
[0394] The photoremovable protecting groups of the present
invention are readily removed. For example, the photolysis of
N-protected L-phenylalanine in solution and having different
photoremovable protecting groups was analyzed, and the results are
presented in the following table:
7TABLE Photolysis of Protected L-Phe-OH t.sub.1/2 in seconds
Solvent NBOC NVOC MeNVOC MeNPOC Dioxane 1288 110 24 19 5 mM
H.sub.2SO.sub.4/Dioxane 1575 98 33 22
[0395] The half life, t.sub.1/2, is the time in seconds required to
remove 50% of the starting amount of protecting group. NBOC is the
6-nitrobenzyloxycarbonyl group, NVOC is the
6-nitroveratryloxycarbonyl group, MeNVOC is the
methyl-6-nitroveratryloxycarbonyl group, and MeNPOC is the
methyl-6-nitropiperonyloxycarbonyl group. The photolysis was
carried out in the indicated solvent with 362/364 nm-wavelength
irradiation having an intensity of 10 mW/cm.sup.2, and the
concentration of each protected phenylalanine was 0.10 mM.
[0396] The table shows that deprotection of NVOC-, MeNVOC-, and
MenPOC-protected phenylalanine proceeded faster than the
deprotection of NBOC. Furthermore, it shows that the deprotection
of the two derivatives that are substituted on the benzylic carbon,
MeNVOC and MeNPOC, were photolyzed at the highest rates in both
dioxane and acidified dioxane.
[0397] 1. Use of Photoremovable Groups During Solid-Phase Synthesis
of Peptides
[0398] The formation of peptides on a solid-phase support requires
the stepwise attachment of an amino acid to a substrate-bound
growing chain. In order to prevent unwanted polymerization of the
monomeric amino acid under the reaction conditions, protection of
the amino terminus of the amino acid is required. After the monomer
is coupled to the end of the peptide, the N-terminal protecting
group is removed, and another amino acid is coupled to the chain.
This cycle of coupling and deprotecting is continued for each amino
acid in the peptide sequence. See Merrifield, J. Am. Chem. Soc.
(1963) 85: 2149, and Atherton et al., "Solid Phase Peptide
Synthesis" 1989, IRL Press, London, both incorporated herein by
reference for all purposes. As described above, the use of a
photoremovable protecting group allows removal of selected portions
of the substrate surface, via patterned irradiation, during the
deprotection cycle of the solid phase synthesis. This selectively
allows spatial control of the synthesis--the next amino acid is
coupled only to the irradiated areas.
[0399] In one embodiment, the photoremovable protecting groups of
the present invention are attached to an activated ester of an
amino acid at the amino terminus: 19
[0400] where R is the side chain of a natural or unnatural amino
acid, X is a photoremovable protecting group, and Y is an activated
carboxylic acid derivative. The photoremovable protecting group, X,
is preferably NVOC, NPOC, PyROC, MeNVOC, MeNPOC, and the like as
discussed above. The activated ester, Y, is preferably a reactive
derivative having a high coupling efficiency, such as an acyl
halide, mixed anhydride, N-hydroxysuccinimide ester,
perfluorophenyl ester, or urethane protected acid, and the like.
Other activated esters and reaction conditions are well known (See
Atherton et al.).
[0401] 2. Use of Photoremovable Groups During Solid-Phase Synthesis
of Oligonucleotides
[0402] The formation of oligonucleotides on a solid-phase support
requires the stepwise attachment of a nucleotide to a
substrate-bound growing oligomer. In order to prevent unwanted
polymerization of the monomeric nucleotide under the reaction
conditions, protection of the 5'-hydroxyl group of the nucleotide
is required. After the monomer is coupled to the end of the
oligomer, the 5'-hydroxyl protecting group is removed, and another
nucleotide is coupled to the chain. This cycle of coupling and
deprotecting is continued for each nucleotide in the oligomer
sequence. See Gait, "Oligonucleotide Synthesis: A Practical
Approach" 1984, IRL Press, London, incorporated herein by reference
for all purposes. As described above, the use of a photoremovable
protecting group allows removal, via patterned irradiation, of
selected portions of the substrate surface during the deprotection
cycle of the solid phase synthesis. This selectively allows spatial
control of the synthesis--the next nucleotide is coupled only to
the irradiated areas.
[0403] Oligonucleotide synthesis generally involves coupling an
activated phosphorous derivative on the 3'-hydroxyl group of a
nucleotide with the 5'-hydroxyl group of an oligomer bound to a
solid support. Two major chemical methods exist to perform this
coupling: the phosphate-triester and phosphoamidite methods (See
Gait). Protecting groups of the present invention are suitable for
use in either method.
[0404] In a preferred embodiment, a photoremovable protecting group
is attached to an activated nucleotide on the 5'-hydroxyl group:
20
[0405] where B is the base attached to the sugar ring; R is a
hydrogen atom when the sugar is deoxyribose or R is a hydroxyl
group when the sugar is ribose; P represents an activated
phosphorous group; and X is a photoremovable protecting group. The
photoremovable protecting group, X, is preferably NV, NP, PyR,
MeNV, MeNP, and the like as described above. The activated
phosphorous group, P, is preferably a reactive derivative having a
high coupling efficiency, such as a phosphate-triester,
phosphoamidite or the like. Other activated phosphorous
derivatives, as well as reaction conditions, are well known (See
Gait).
[0406] E. Amino Acid N-Carboxy Anhydrides Protected with a
Photoremovable Group
[0407] During Merrifield peptide synthesis, an activated ester of
one amino acid is coupled with the free amino terminus of a
substrate-bound oligomer. Activated esters of amino acids suitable
for the solid phase synthesis include halo-formate, mixed
anhydride, imidazoyl formate, acyl halide, and also includes
formation of the activated ester in situ and the use of common
reagents such as DCC and the like (See Atherton et al.). A
preferred protected and activated amino acid has the general
formula: 21
[0408] where R is the side chain of the amino acid and X is a
photoremovable protecting group. This compound is a
urethane-protected amino acid having a photoremovable protecting
group attach to the amine. A more preferred activated amino acid is
formed when the photoremovable protecting group has the general
formula: 22
[0409] where R.sup.1, R.sup.2, R.sup.3, and R.sup.4 independently
are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen,
hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,
formate, formamido or phosphido group, or adjacent substituents
(i.e., R.sup.1-R.sup.2, R.sup.2-R.sup.3, R.sup.3-R.sup.4) are
substituted oxygen groups that together form a cyclic acetal or
ketal; and R.sup.5 is a hydrogen atom, a alkoxyl, alky, hydrogen,
halo, aryl, or alkenyl group.
[0410] A preferred activated amino acid is formed when the
photoremovable protecting group is 6-nitroveratryloxycarbonyl. That
is, R.sup.1 and R.sup.4 are each a hydrogen atom, R.sup.2 and
R.sup.3 are each a methoxy group, and R.sup.5 is a hydrogen atom.
Another preferred activated amino acid is formed when the
photoremovable group is 6-nitropiperonyl: R.sup.1 and R.sup.4 are
each a hydrogen atom, R.sup.2 and R.sup.3 together form a methylene
acetal, and R.sup.5 is a hydrogen atom. Other protecting groups are
possible. Another preferred activated ester is formed when the
photoremovable group is methyl-6-nitroveratryl or
methyl-6-nitropiperonyl- .
[0411] Another preferred activated amino acid is formed when the
photoremovable protecting group has the general formula: 23
[0412] where R.sup.1, R.sup.2, and R.sup.3 independently are a
hydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl,
alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate,
formamido, sulfanates, sulfido or phosphido group, and R.sup.4 and
R.sup.5 independently are a hydrogen atom, an alkoxy, alkyl, halo,
aryl, hydrogen, or alkenyl group. The resulting compound is a
urethane-protected amino acid having a pyrenylmethyloxycarbonyl
protecting group attached to the amine. A more preferred embodiment
is formed when R.sup.1 through R.sup.5 are each a hydrogen
atom.
[0413] The urethane-protected amino acids having a photoremovable
protecting group of the present invention are prepared by
condensation of an N-protected amino acid with an acylating agent
such as an acyl halide, anhydride, chloroformate and the like (See
Fuller et al., U.S. Pat. No. 4,946,942 and Fuller et al., J. Amer.
Chem. Soc., (1990) 112: 7414-7416, both herein incorporated by
reference for all purposes).
[0414] Urethane-protected amino acids having photoremovable
protecting groups are generally useful as reagents during
solid-phase peptide synthesis, and because of the spatially
selectivity possible with the photoremovable protecting group, are
especially useful for the spatially addressing peptide synthesis.
These amino acids are difunctional: the urethane group first serves
to activate the carboxy terminus for reaction with the amine bound
to the surface and, once the peptide bond is formed, the
photoremovable protecting group protects the newly formed amino
terminus from further reaction. These amino acids are also highly
reactive to nucleophiles, such as deprotected amines on the surface
of the solid support, and due to this high-reactivity, the
solid-phase peptide coupling times are significantly reduced, and
yields are typically higher.
[0415] 1. Example
[0416] Light activated formation of a thymidine-cytidine dimer was
carried out. A three dimensional representation of a fluorescence
scan showing a checkboard pattern generated by the light-directed
synthesis of a dinucleotide is shown in FIG. 8. 5'-nitroveratryl
thymidine was attached to a synthesis substrate through the 3'
hydroxyl group. The nitroveratryl protecting groups were removed by
illumination through a 500 mm checkerboard mask. The substrate was
then treated with phosphoramidite activated 2'-deoxycytidine. In
order to follow the reaction fluorometrically, the deoxycytidine
had been modified with an FMOC protected aminohexyl linker attached
to the exocyclic amine
(5'-O-dimethoxytrityl-4-N-(6-N-fluorenylmethylcarbamoyl-hexylcarboxy)-2'--
deoxycytidine). After removal of the FMOC protecting group with
base, the regions which contained the dinucleotide were
fluorescently labelled by treatment of the substrate with 1 mM FITC
in DMF for one hour.
[0417] The three-dimensional representation of the fluorescent
intensity data in FIG. 14 clearly reproduces the checkerboard
illumination pattern used during photolysis of the substrate. This
result demonstrates that oligonucleotidesas well as peptides can be
synthesized by the light-directed method.
[0418] C. Binary Masking
[0419] In fact, the means for producing a substrate useful for
these techniques are explained in Pirrung et al. (1992) U.S. Pat.
No. 5,143,854, which is hereby incorporated herein by reference.
However, there are various particular ways to optimize the
synthetic processes. Many of these methods are described in U.S.
Pat. No. 5,489,678.
[0420] Briefly, the binary synthesis strategy refers to an ordered
strategy for parallel synthesis of diverse polymer sequences by
sequential addition of reagents which may be represented by a
reactant matrix, and a switch matrix, the product of which is a
product matrix. A reactant matrix is a 1.times.n matrix of the
building blocks to be added. The switch matrix is all or a subset
of the binary numbers from 1 to n arranged in columns. In preferred
embodiments, a binary strategy is one in which at least two
successive steps illuminate half of a region of interest on the
substrate. In most preferred embodiments, binary synthesis refers
to a synthesis strategy which also factors a previous addition
step. For example, a strategy in which a switch matrix for a
masking strategy halves regions that were previously illuminated,
illuminating about half of the previously illuminated region and
protecting the remaining half (while also protecting about half of
previously protected regions and illuminating about half of
previously protected regions). It will be recognized that binary
rounds may be interspersed with non-binary rounds and that only a
portion of a substrate may be subjected to a binary scheme, but
will still be considered to be a binary masking scheme within the
definition herein. A binary "masking" strategy is a binary
synthesis which uses light to remove protective groups from
materials for addition of other materials such as nucleotides or
amino acids.
[0421] In particular, this procedure provides a simplified and
highly efficient method for saturating all possible sequences of a
defined length polymer. This masking strategy is also particularly
useful in producing all possible oligonucleotide, sequence probes
of a given length.
[0422] D. Applications
[0423] The technology provided by the present invention has very
broad applications. Although described specifically for
polynucleotide sequences, similar sequencing, fingerprinting,
mapping, and screening procedures can be applied to polypeptide,
carbohydrate, or other polymers. In particular, the present
invention may be used to completely sequence a given target
sequence to subunit resolution. This may be for de novo sequencing,
or may be used in conjunction with a second sequencing procedure to
provide independent verification. See, e.g., (1988) Science, 242:
1245. For example, a large polynucleotide sequence defined by
either the Maxam and Gilbert technique or by the Sanger technique
may be verified by using. the present invention.
[0424] In addition, by selection of appropriate probes, a
polynucleotide sequence can be fingerprinted. Fingerprinting is a
less detailed sequence analysis which usually involves the
characterization of a sequence by a combination of defined
features. Sequence fingerprinting is particularly useful because
the repertoire of possible features which can be tested is
virtually infinite. Moreover, the stringency of matching is also
variable depending upon the application. A Southern Blot analysis
may be characterized as a means of simple fingerprint analysis.
[0425] Fingerprinting analysis may be performed to the resolution
of specific nucleotides, or may be used to determine homologies,
most commonly for large segments. In particular, an array of
oligonucleotide probes of virtually any workable size may be
positionally localized on a matrix and used to probe a sequence for
either absolute complementary matching, or homology to the desired
level of stringency using selected hybridization conditions.
[0426] In addition, the present invention provides means for
mapping analysis of a target sequence or sequences. Mapping will
usually involve the sequential ordering of a plurality of various
sequences, or may involve the localization of a particular sequence
within a plurality of sequences. This may be achieved by
immobilizing particular large segments onto the matrix and probing
with a shorter sequence to determine which of the large sequences
contain that smaller sequence. Alternatively, relatively shorter
probes of known or random sequence may be immobilized to the matrix
and a map of various different target sequences may be determined
from overlaps. Principles of such an approach are described in some
detail by Evans et al. (1989) "Physical Mapping of Complex Genomes
by Cosmid Multiplex Analysis," Proc. Natl. Acad. Sci. USA, 86:
5030-5034; Michiels et al. (1987) "Molecular Approaches to Genome
Analysis: A Strategy for the Construction of Ordered Overlap Clone
Libraries," CABIOS, 3: 203-210; Olsen et al. (1986) "Random-Clone
Strategy for Genomic Restriction Mapping in Yeast," Proc. Natl.
Acad. Sci. USA, 83: 7826-7830; Craig, et al. (1990) "Ordering of
Cosmid Clones Covering the Herpes Simplex Virus Type I (HSV-I)
Genome: A Test Case for Fingerprinting by Hybridization," Nuc.
Acids Res., 18: 2653-2660; and Coulson, et al. (1986) "Toward a
Physical Map of the Genome of the Nematode Caenorhabditis elegans,"
Proc. Natl. Acad. Sci. USA, 83: 7821-7825; each of which is hereby
incorporated herein by reference.
[0427] Fingerprinting analysis also provides a means of
identification. In addition to its value in apprehension of
criminals from whom a biological sample, e.g., blood, has been
collected, fingerprinting can ensure personal identification for
other reasons. For example, it may be useful for identification of
bodies in tragedies such as fire, flood, and vehicle crashes. In
other cases the identification may be useful in identification of
persons suffering from amnesia, or of missing persons. Other
forensics applications include establishing the identity of a
person, e.g., military identification "dog tags," or may be used in
identifying the source of particular biological samples.
Fingerprinting technology is described, e.g., in Carrano, et al.
(1989) "A High-Resolution, Fluorescence-Based, Semi-automated
method for DNA Fingerprinting," Genomics, 4: 129-136, which is
hereby incorporated herein by reference. See, e.g., table I, for
nucleic acid applications, and corresponding applications may be
accomplished using polypeptides.
VLSIPS.TM. Project in Nucleic Acids
[0428]
8TABLE I I. Construction of Chips II. Applications A. Sequencing 1.
Primary sequencing 2. Secondary sequencing (sequence checking) 3.
Large scale mapping 4. Fingerprinting B. Duplex/Triplex formation
1. Antisense 2. Sequence specific function modulation (e.g.
promoter inhibition) C. Diagnosis 1. Genetic markers 2. Type
markers a. Blood donors b. Tissue transplants D. Microbiology 1.
Clinical microbiology 2. Food microbiology III. Instrumentation A.
Chip machines B. Detection IV. Software Development A.
Instrumentation software B. Data reduction software C. Sequence
analysis software
[0429] The fingerprinting analysis may be used to perform various
types of genetic screening. For example, a single substrate may be
generated with a plurality of screening probes, allowing for the
simultaneous genetic screening for a large number of genetic
markers. Thus, prenatal or diagnostic screening can be simplified,
economized, and made more generally accessible.
[0430] In addition to the sequencing, fingerprinting, and mapping
applications, the present invention also provides means for
determining specificity of interaction with particular sequences.
Many of these applications were described in U.S. Ser. No.
07/362,901, from which CIP U.S. Ser. No. 07/492,462 issued as U.S.
Pat. No. 5,143,854, and U.S. Ser. No. 07/435,316, from which CEP
U.S. Ser. No. 07/612,671 issued as U.S. Pat. No. 5,252,743.
[0431] E. Detection Methods and Apparatus
[0432] An appropriate detection method applicable to the selected
labeling method can be selected. Suitable labels include
radionucleotides, enzymes, substrates, cofactors, inhibitors,
magnetic particles, heavy metal atoms, and particularly
fluorescers, chemiluminescers, and spectroscopic labels. Patents
teaching the use of such labels include U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241.
[0433] With an appropriate label selected, the detection system
best adapted for high resolution and high sensitivity detection may
be selected. As indicated above, an optically detectable system,
e.g., fluorescence or chemiluminescence would be preferred. Other
detection systems may be adapted to the purpose, e.g., electron
microscopy, scanning electron microscopy (SEM), scanning tunneling
electron microscopy (STEM), infrared microscopy, atomic force
microscopy (AFM), electrical condutance, and image plate
transfer.
[0434] With a detection method selected, an apparatus for scanning
the substrate will be designed. Apparatus, as described in U.S.
Ser. No. 07/362,901, from which CIP U.S. Ser. No. 07/492,462 issued
as U.S. Pat. No. 5,143,854; or U.S. Pat. No. 5,489,678, are
particularly appropriate. Design modifications may also be
incorporated therein.
[0435] F. Data Analysis
[0436] Data is analyzed by processes similar to those described
below in the section describing theoretical analysis. More
efficient algorithms will be mathematically devised, and will
usually be designed to be performed on a computer. Various computer
programs which may more quickly or efficiently make measurement
samples and distinguish signal from noise will also be devised.
See, particularly, U.S. Pat. No. 5,489,678.
[0437] The initial data resulting from the detection system is an
array of data indicative of fluorescent intensity versus location
on the substrate. The data are typically taken over regions
substantially smaller than the area in which synthesis of a given
polymer has taken place. Merely by way of example, if polymers were
synthesized in squares on the substrate having dimensions of 500
microns by 500 microns, the data may be taken over regions having
dimensions of 5 microns by 5 microns. In most preferred
embodiments, the regions over which florescence data are taken
across the substrate are less than about 1/2 the area of the
regions in which individual polymers are synthesized, preferably
less than {fraction (1/10)} the area in which a single polymer is
synthesized, and most preferably less than {fraction (1/100)} the
area in which a single polymer is synthesized. Hence, within any
area in which a given polymer has been synthesized, a large number
of fluorescence data points are collected.
[0438] A plot of number of pixels versus intensity for a scan
should bear a rough resemblance to a bell curve, but spurious data
are observed, particularly at higher intensities. Since it is
desirable to use an average of fluorescent intensity over a given
synthesis region in determining relative binding affinity, these
spurious data will tend to undesirably skew the data.
[0439] Accordingly, in one embodiment of the invention the data are
corrected for removal of these spurious data points, and an average
of the data points is thereafter utilized in determining relative
binding efficiency. In general the data are fitted to a base curve
and measures are used to remove spurious data.
[0440] In an additional analytical tool, various degeneracy
reducing analogues may be incorporated in the hybridization probes.
Various aspects of this strategy are described, e.g., in Macevicz,
S. (1990) PCT publication number WO 90/04652, which is hereby
incorporated herein by reference.
[0441] II. Theoretical Analysis
[0442] The principle of the hybridization sequencing procedure is
based, in part, upon the ability to determine overlaps of short
segments. The VLSIPS.TM. Technology provides the ability to
generate reagents which will saturate the possible short
subsequence recognition possibilities. The principle is most easily
illustrated by using a binary sequence, such as a sequence of zeros
and ones. Once having illustrated the application to a binary
alphabet, the principle may easily be understood to encompass three
letter, four letter, five or more. letter, even 20 letter
alphabets. A theoretical treatment of analysis of subsequence
information, to reconstruction of a target sequence is. provided,
e.g., in Lysov, Yu., et al. (1988) Doklady Akademi. Nauk. SSR, 303:
1508-1511; Khrapko K., et al. (1989) FEBS Letters, 256: 118-122;
Pevzner, P. (1989) J. of Biomolecular Structure and Dynamics, 7:
63-69; and Drmanac, R. et al. (1989) Genomics, 4: 114-128; each of
which is hereby incorporated herein by reference.
[0443] The reagents for recognizing the subsequences will usually
be specific for recognizing a particular polymer subsequence
anywhere within a target polymer. It is preferable that conditions
may be devised which allow absolute discrimination between high
fidelity matching and very low levels of mismatching. The reagent
interaction will preferably exhibit no sensitivity to flanking
sequences, to the subsequence position within the target, or to any
other remote structure within the sequence. For polynucleotide
sequencing, the specific reagents can be oligonucleotide probes;
for polypeptides and carbohydrates, antibodies will be useful
reagents. Antibody reagents should also be useful for other types
of polymers.
[0444] A. Simple n-mer Structure: Theory
[0445] A simple example is presented below of how a sequence of ten
digits comprising zeros and ones would be sequenceable using short
segments of five digits. For example, consider the sample ten digit
sequence:
[0446] 1010011100.
[0447] A VLSIPS.TM. Technology substrate could be constructed, as
discussed elsewhere, which would have reagents attached in a
defined matrix pattern which specifically recognize each of the
possible five digit sequences of ones and zeros. The number of
possible five digit subsequences is 2.sup.5=32. The number of
possible different sequences 10 digits long is 2.sup.10=1,024. The
five contiguous digit subsequences within a ten digit sequence
number six, i.e., positioned at digits 1-5, 2-6, 3-7, 4-8, 5-9, and
6-10. It will be noted that the specific order of the digits in the
sequence is important, and that the order is directional, e.g.,
running left to right versus right to left. The first five digit
sequence contained in the target sequence is 10100. The second is
01001, the third is 10011, the fourth is 00111, the fifth is 01110,
and the sixth is 11100. The VLSIPS.TM.. Technology substrate would
have a matrix pattern of positionally attached reagents which
recognize each of the different 5-mer subsequences. Those reagents
which recognize each of the 6 contained 5-mers will bind the
target, and a label allows the positional determination of where
the sequence specific interaction has occurred. By correlation of
the position in the matrix pattern, the corresponding bound
subsequences can be determined.
[0448] In the above-mentioned sequence, six different 5-mer
sequences would be determined to be present. They would be:
[0449] 10100
[0450] 01001
[0451] 10011
[0452] 00111
[0453] 01110
[0454] 11100
[0455] Any sequence which contains the first five digit sequence,
10100, already narrows the number of possible sequences (e.g., from
1024 possible sequences) which contain it to less than about 192
possible sequences.
[0456] This 192 is derived from the observation that with the
subsequence 10100 at the far left of the sequence, in positions
1-5, there are only 32 possible sequences. Likewise, for that
particular subsequence in positions 2-6, 3-7, 4-8, 5-9, and 6-10.
So, to sum up all of the sequences that could contain 10100, there
are 32 for each position and 6 positions for a total of about 192
possible sequences. However, some of these 10 digit sequences will
have been counted twice. Thus, by virtue of containing the 10100
subsequence, the number of possible 10-mer sequences has been
decreased from 1024 sequences to less than about 192 sequences.
[0457] In this example, not only do we know that the sequence
contains 10100, but we also know that it contains the second five
character sequence, 01001. By virtue of knowing that the sequence
contains 10100, we can look specifically to determine whether the
sequence contains a subsequence of five characters which contains
the four leftmost digits plus a next digit to the left. For
example, we would look for a sequence of X1010, but we find that
there is none. Thus, we know that the 10100 must be at the left end
of the 10-mer. We would also look to see whether the sequence
contains the rightmost four digits-plus a next digit to the right,
e.g., 0100X. We find that the sequence also contains the sequence
01001, and that X is a 1. Thus, we know at least that our target
sequence has an overlap of 0100 and has the left terminal sequence
101001.
[0458] Applying the same procedure to the second 5-mer, we also
know that the sequence must include a sequence of five digits
having the sequence 1001Y where Y must be either 0 or 1. We look
through the fragments and we see that we have a 10011 sequence
within our target, thus Y is also 1. Thus, we would know that our
sequence has a sequence of the first seven being 1010011.
[0459] Moving to the next 5-mer, we know that there must be a
sequence of 0011Z, where Z must be either 0 or 1. We look at the
fragments produced above and see that the target sequence contains
a 00111 subsequence and Z is 1. Thus, we know the sequence must
start with 10100111.
[0460] The next 5-mer must be of the sequence 0111W where W must be
0 or 1. Again, looking up at the fragments produced, we see that
the target sequence contains a 01110 subsequence, and W is a 0.
Thus, our sequence to this point is 101001110. We know that the
last 5-mer must be either 11100 or 11101. Looking above, we see
that it is 11100 and that must be the last of our sequence. Thus,
we have determined that our sequence must have been 1010011100.
[0461] However, it will be recognized from the example above with
the sequences provided therein, that the sequence analysis can
start with any known positive probe subsequence. The determination
may be performed by moving linearly along the sequence checking the
known sequence with a limited number of next positions. Given this
possibility, the sequence may be determined, besides by scanning
all possible oligonucleotide probe positions, by specifically
looking only where the next possible positions would be. This may
increase the complexity of the scanning but may provide a longer
time span dedicated towards scanning and detecting specific
positions of interest relative to other sequence possibilities.
Thus, the scanning apparatus could be set up to work its way along
a sequence from a given contained oligonucleotide to only look at
those positions on the substrate which are expected to have a
positive signal.
[0462] It is seen that given a sequence, it can be deconstructed
into n-mers to produce a set of internal contiguous subsequences.
From any given target sequence, we would be able to determine what
fragments would result. The hybridization sequence method depends,
in part, upon being able to work in the reverse, from a set of
fragments of known sequences to the full sequence. In simple cases,
one is able to start at a single position and work in either or
both directions towards the ends of the sequence as illustrated in
the example.
[0463] The number of possible sequences of a given length increases
very quickly with the length of that sequence. Thus, a 10-mer of
zeros and ones has 1024 possibilities, a 12-mer has 4096. A 20-mer
has over a million possibilities, and a 30-mer has over a billion.
However, a given 30-mer has, at most, 26 different internal 5-mer
sequences. Thus, a 30 character target sequence having over a
million possible sequences can be substantially defined by only 26
different 5-mers. It will be recognized that the probe
oligonucleotides will preferably, but need not necessarily, be of
identical length, and that the probe sequences need not necessarily
be contiguous in that the overlapping subsequences need not differ
by only a single subunit. Moreover, each position of the matrix
pattern need not be homogeneous, but may actually contain a
plurality of probes of known sequence. In addition, although all of
the possible subsequence specifications would be preferred, a less
than full set of sequences specifications could be used. In
particular, although a substantial fraction will preferably be at
least about 70%, it may be less than that. About 20% would be
preferred, more preferably at least about 30% would be desired.
Higher percentages would be especially preferred.
[0464] 2. Example of Four Letter Alphabet
[0465] A four letter alphabet may be conceptualized in at least two
different ways from the two letter alphabet. One way is to consider
the four possible values at each position and to analogize in a
similar fashion to the binary example each of the overlaps. A.
second way is to group the binary digits into groups.
[0466] Using the first means, the overlap comparisons are performed
with a four letter alphabet rather than a two letter alphabet.
Then, in contrast to the binary system with 10 positions where
2.sup.10=1024 possible sequences, in a 4-character alphabet with 10
positions, there will actually be 4.sup.10=1,048,576 possible
sequences. Thus, the complexity of a four character sequence has a
much larger number of possible sequences compared to a two
character sequence. Note, however, that there are still only 6
different internal 5-mers. For simplicity, we shall examine a 5
character string with 3 character subsequences. Instead of only 1
and 0, the characters maybe designated, e.g., A, C, G, and T. Let
us take the sequence GGCTA. The 3-mer subsequences are:
9 GGC GCT CTA
[0467] Given these subsequences, there is one sequence, or at most
only a few sequences which would produce that combination of
subsequences, i.e., GGCTA.
[0468] Alternatively, with a four character universe, the binary
system can be looked at in pairs of digits. The pairs would be 00,
01, 10, and 11. In this manner, the earlier used sequence
1010011100 is looked at as 10,10,01,11,00. Then the first character
of two digits is selected from the possible universe of the four
representations 00, 01, 10, and 11. Then a probe would be in an
even number of digits, e.g., not five digits, but, three pairs of
digits or six digits. A similar comparison is performed and the
possible overlaps determined. The 3-pair subsequences are:
[0469] 10,10,01
[0470] 10,01,11
[0471] 01,11,00
[0472] and the overlap reconstruction produces 10,10,01,11,00.
[0473] The latter of the two conceptual views of the 4 letter
alphabet provides a representation which is similar to what would
be provided in a digital computer. The applicability to a four
nucleotide alphabet is easily seen by assigning, e.g., 00 to A, 01
to C, 10 to G, and 11 to T. And, in fact, if such a correspondence
is used, both examples for the 4 character sequences can be seen to
represent the same target sequence. The applicability of the
hybridization method and its analysis for determining the ultimate
sequence is easily seen if A is the representation of adenine, C is
the representation of cytosine, G is the representation of guanine,
and T is the representation of thymine or uracil.
[0474] 3. Generalization to m-Letter Alphabet
[0475] This reconstruction process may be applied to polymers of
virtually any number of possible characters in the alphabet, and
for virtually any length sequence to be sequenced, though
limitations, as discussed below, will limit its efficiency at
various extremes of length. It will be recognized that the theory
can be applied to a large diversity of systems where sequence is
important.
[0476] For example, the method could be applied to sequencing of a
polypeptide. A polypeptide can have any of twenty natural amino
acid possibilities at each position. A twenty letter alphabet is
amenable to sequencing by this method so long as reagents exist for
recognizing shorter subsequences therein. A preferred reagent for
achieving that goal would be a set of monoclonal antibodies each of
which recognizes a specific three contiguous amino acid
subsequence. A complete set of antibodies which recognize all
possible subsequences of a given length, e.g., 3 amino acids, and
preferably with a uniform affinity, would be 20.sup.3=8000
reagents.
[0477] It will also be recognized that each target sequence which
is recognized by the specific reagents need not have homogeneous
termini. Thus, fragments of the entire target sequence will also be
useful for hybridizing appropriate subsequences. It is, however,
preferable that there not be a significant amount of labeled
homogeneous contaminating extraneous sequences. This constraint
does usually require the purification of the target molecule to be
sequenced, but a specific label technique would dispense with a
purification requirement if the unlabeled extraneous sequences do
not interfere with the labeled sequences.
[0478] In addition, conformational effects of target polypeptide
folding may, in certain embodiments, be negligible if the
polypeptide is fragmented into sufficiently small peptides for if
the interaction is performed under conditions where conformation,
but not specific interaction, is disrupted.
[0479] B. Complications
[0480] Two obvious complications exist with the method of sequence
analysis by hybridization. The first results from a probe of
inappropriate length while the second relates to internally
repeated sequences.
[0481] The first obvious complication is a problem which arises
from an inappropriate length of recognition sequence, which causes
problems with the specificity of recognition. For example, if the
recognized sequence is too short, every sequence which is utilized
will be recognized by every probe sequence. This occurs, e.g., in a
binary system where the probes are each of sequences which occur
relatively frequently, e.g., a two character probe for the binary
system. Each possible two character probe would be expected to
appear 1/4 of the time in every single two character position.
Thus, the above sequence example would be recognized by each of the
00, 10, 01, and 11. Thus, the sequence information is virtually
lost because the resolution is too low and each recognition reagent
specifically binds at multiple sites on the target sequence.
[0482] The number of different probes which bind to a target
depends on the relationship between the probe length and the target
length. At the extreme of short probe length, the just mentioned
problem exists of excessive redundancy and lack of resolution. The
lack of stability in recognition will also be a problem with
extremely short probes. At the extreme of long probe length, each
entire probe sequence is on a different position of a substrate.
However, a problem arises from the number of possible sequences,
which goes up dramatically with the length of the sequence. Also,
the specificity of recognition begins to decrease as the
contribution to binding by any particular subunit may become
sufficiently low that the system fails to distinguish the fidelity
of recognition. Mismatched hybridization may be a problem with the
polynucleotide sequencing applications, though the fingerprinting
and mapping applications may not be so strict in their fidelity
requirements. As indicated above, a thirty position binary sequence
has over a million possible sequences, a number which starts to
become unreasonably large in its required number of different
sequences, even though the target length is still very short.
Preparing a substrate with all sequence possibilities for a long
target may be extremely difficult due to the many different
oligomers which must be synthesized.
[0483] The above example illustrates how a long target sequence may
be reconstructed with a reasonably small number of shorter
subsequences. Since the present day resolution of the regions of
the substrate having defined oligomer probes attached to the
substrate approaches about 10 microns by 10 microns for resolvable
regions, about 10.sup.6, or 1 million, positions can be placed on a
one centimeter square substrate. However, high resolution systems
may have particular disadvantages which may be outweighed using the
lower density substrate matrix pattern. For this reason, a
sufficiently large number of probe sequences can be utilized so
that any given target sequence may be determined by hybridization
to a relatively small number of probes.
[0484] A second complication relates to convergence of sequences to
a single subsequence. This will occur when a particular subsequence
is repeated in the target sequence. This problem can be addressed
in at least two different ways. The first, and simpler way, is to
separate the repeat sequences onto two different targets. Thus,
each single target will not have the repeated sequence and can be
analyzed to its end. This solution, however, complicates the
analysis by requiring that some means for cutting at a site between
the repeats can be located. Typically a careful sequencer would
want to have two intermediate cut points so that the intermediate
region can also be sequenced in both directions across each of the
cut points. This problem is inherent in the hybridization method
for sequencing but can be minimized by using a longer known probe
sequence so that the frequency of probe repeats is decreased.
[0485] Knowing the sequence of flanking sequences of the repeat
will simplify the use of polymerase chain reaction (PCR) or a
similar technique to further definitively determine the sequence
between sequence repeats. Probes can be made to hybridize to those
known sequences adjacent the repeat sequences, thereby producing
new target sequences for analysis. See, e.g., Innis et al. (eds.)
(1990) PCR Protocols: A Guide to Methods and Applications, Academic
Press; and methods for synthesis of oligonucleotide probes, see,
e.g., Gait (1984) Oligonucleotide Synthesis: A Practical Approach,
IRL Press, Oxford.
[0486] Other means for dealing with convergence problems include
using particular longer probes, and using degeneracy reducing
analogues, see, e.g., Macevicz, S. (1990) PCT publication number WO
90/04652, which is hereby incorporated herein by reference. By use
of stretches of the degeneracy reducing analogues with other probes
in particular combinations, the number of probes necessary to fully
saturate the possible oligomer probes is decreased. For example,
with a stretch of 12-mers having the central 4-mer of degenerate
nucleotides, in combination with all of the possible 8-mers, the
collection numbers twice the number of possible 8-mers, e.g.
65,536+65,536=131,072, but the population provides screening
equivalent to all possible 12-mers.
[0487] By way of further explanation, all possible oligonucleotide
8-mers may be depicted in the fashion:
[0488] N1-N-2-N3-N4-N5-N-6-N7-N8,
[0489] in which there are 4.sup.8=65,536 possible 8-mers. As
described in U.S. Pat. No. 5,489,678, automated VLSIPS.TM.
Technology, producing all possible 8-mers requires 4.times.8=32
chemical binary synthesis steps to produce the entire matrix
pattern of 65,536 8-mer possibilities. By incorporating degeneracy
reducing nucleotides, D's, which hybridize nonselectively to any
corresponding complementary nucleotide, new oligonucleotides
12-mers can be made in the fashion:
[0490] N1-N2-N3-N4-D-D-D-D-N5-N6-N7-N8,
[0491] in which there are again, as above, only 4.sup.8=65,536
possible "12-mers," which in reality only have 8 different
nucleotides.
[0492] However, it can be seen that each possible 12-mer probe
could be represented by a group of the two 8-mer types. Moreover,
repeats of less than 12 nucleotides would not converge, or cause
repeat problems in the analysis. Thus, instead of requiring a
collection of probes corresponding to all 12-mers, or
4.sup.12=16,777,216 different 12-mers, the same information can be
derived by making 2 qets of "8-mers" consisting of the typical
8-mer collection of 4.sup.8=65,536 and the "12-mer" set with the
degeneracy reducing analogues, also requiring making 4.sup.8
65,536. The combination of the two sets, requires making
65,536+65,536=131,072 different molecules, but giving the
information of 16,777,216 molecules. Thus, incorporating the
degeneracy reducing analogue decreases the number of molecules
necessary to get 12-mer resolution by a factor of about
128-fold.
[0493] C. Non-Polynucleotide Embodiments
[0494] The above example is directed towards a polynucleotide
embodiment. This application is relatively easily achieved because
the specific reagents will typically be complementary
oligonucleotides, although in certain embodiments other specific
reagents may be desired. For example, there may be circumstances
where other than complementary base pairing will be utilized. The
polynucleotide targets, will usually be single strand, but may be
double or triple stranded in various applications. However, a
triple stranded specific interaction might be sometimes desired, or
a protein or other specific binding molecule may be utilized. For
example, various promoter or DNA sequence specific binding proteins
might be used, including, e.g., restriction enzyme binding domains,
other binding domains, and antibodies. Thus, specific recognition
reagents besides oligonucleotides may be utilized.
[0495] For other polymer targets, the specific reagents will often
be polypeptides. These polypeptides may be protein binding domains
from enzymes or other proteins which display specificity for
binding. Usually an antibody molecule may be used, and monoclonal
antibodies may be particularly desired. Classical methods may be
applied for preparing antibodies, see, e.g., Harlow and Lane (1988)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, New York;
and Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d Ed.) Academic Press, San Diego. Other suitable techniques for
in vitro exposure of lymphocytes to the antigens or selection of
libraries of antibody binding sites are described, e.g., in Huse et
al. (1989) Science, 246: 1275-1281; and Ward et al. (1989) Nature,
341: 544-546, each of which is hereby incorporated herein by
reference. Unusual antibody production methods are also described,
e.g., in Hendricks et al. (1989) BioTechnology, 7: 1271-1274; and
Hiatt et al. (1989) Nature, 342: 76-78, each of which is hereby
incorporated herein by reference. Other molecules which may exhibit
specific binding interaction may be useful for attachment to a
VLSIPS.TM.. Technology substrate by various methods, including the
caged biotin methods, see, e.g., Barrett et al. (1993) U.S. Pat.
No. 5,252,743.
[0496] The antibody specific reagents should be particularly useful
for the polypeptide, carbohydrate, and synthetic polymer
applications. Individual specific reagents might be generated by an
automated process to generate the number of reagents necessary to
advantageously use the high density positional matrix pattern. In
an alternative approach, a plurality of hybridoma cells may be
screened for their ability to bind to a VLSIPS.TM. Technology
matrix possessing the desired sequences whose binding specificity
is desired. Each cell might be individually grown up and its
binding specificity determined by VLSIPS.TM. Technology apparatus
and technology. An alternative strategy would be to expose the same
VLSIPS.TM. Technology matrix to a polyclonal serum of high titer.
By a successively large volume of serum and different animals, each
region of the VLSIPS.TM. Technology substrate would have attached
to it a substantial number of antibody molecules with specificity
of binding. The substrate, with non-covalently bound antibodies
could be derivatized and the antibodies transferred to an adjacent
second substrate in the matrix pattern in which the antibody
molecules had attached to the first matrix. If the sensitivity of
detection of binding interaction is sufficiently high, such a low
efficiency transfer of antibody molecules may produce a
sufficiently high signal to be useful for many purposes, including
the sequencing applications.
[0497] In another embodiment, capillary forces may be used to
transfer the selected reagents to a new matrix, to which the
reagents would be positionally attached in the pattern of the
recognized sequences. Or, the reagents could be transversely
electrophoresed, magnetically transferred, or otherwise transported
to a new substrate in their retained positional pattern.
[0498] III. Polynucleotide Sequencing
[0499] In principle, the making of a substrate having a
positionally defined matrix pattern of all possible
oligonucleotides of a given length involves a conceptually simple
method of synthesizing each and every different possible
oligonucleotide, and affixing them to a definable position.
Oligonucleotide synthesis is presently mechanized and enabled by
current technology, see, e.g.; Pirrung et al. (1992) U.S. Pat. No.
5,143,854; and instruments supplied by Applied Biosystems, Foster
City, Calif.
[0500] A. Preparation of Substrate Matrix
[0501] The production of the collection of specific
oligonucleotides used in polynucleotide sequencing may be produced
in at least two different ways. Present technology certainly allows
production of ten nucleotide oligomers on a solid phase or other
synthesizing system. See, e.g., instrumentation provided by Applied
Biosystems, Foster City, Calif. Although a single oligonucleotide
can be relatively easily made, a large collection of them would
typically require a fairly large amount of time and investment. For
example, there are 4.sup.10=1,048,576 possible ten nucleotide
oligomers. Present technology allows making each and every one of
them in a separate purified form though such might be costly and
laborious.
[0502] Once the desired repertoire of possible oligomer sequences
of a given length have been synthesized, this collection of
reagents may be individually positionally attached to a substrate,
thereby allowing a batchwise hybridization step. Present technology
also would allow the possibility of attaching each and every one of
these 10-mers to a separate specific position on a solid matrix.
This attachment could be automated in any of a number of ways,
particularly through the use of a caged biotin type linking. This
would produce a matrix having each of different possible
10-mers.
[0503] A batchwise hybridization is much preferred because of its
reproducibility and simplicity. An automated process of attaching
various reagents to positionally defined sites on a substrate is
provided in Pirrung et al. (1992) U.S. Pat. Nos. 5,143,854;
5,489,678, and Barrett et al. (1993) U.S. Pat. No. 5,252,743; each
of which is hereby incorporated herein by reference.
[0504] Instead of separate synthesis of each oligonucleotide, these
oligonucleotides are conveniently synthesized in parallel by
sequential synthetic processes on a defined matrix pattern as
provided in Pirrung et al. (1992) U.S. Pat. Nos. 5,143,854; and
5,489,678, which are incorporated herein by reference. Here, the
oligonucleotides are synthesized stepwise on a substrate at
positionally separate and defined positions. Use of photosensitive
blocking reagents allows for defined sequences of synthetic steps
over the surface of a matrix pattern. By use of the binary masking
strategy, the surface of the substrate can be positioned to
generate a desired pattern of regions, each having a defined
sequence oligonucleotide synthesized and immobilized thereto.
[0505] Although the prior art technology can be used to generate
the desired repertoire of oligonucleotide probes, an efficient and
cost effective means would be to use the VLSIPS.TM. Technology
described in Pirrung et al. (1992) U.S. Pat. Nos. 5,143,854, and
5,489,678. In this embodiment, the photosensitive reagents involved
in the production of such a matrix are described below.
[0506] The regions for synthesis may be very small, usually less
than about 100 .mu.m.times.100 .mu.m, more usually less than about
50 .mu.m.times.50 .mu.m. The photolithography technology allows
synthetic regions of less than about 10 .mu.m.times.10 .mu.m, about
3 .mu.m.times.3 .mu.m, or less. The detection also may detect such
sized regions, though larger areas are more easily and reliably
measured.
[0507] At a size of about 30 microns by 30 microns, one million
regions would take about 11 centimeters square or a single wafer of
about 4 centimeters by 4 centimeters. Thus the present technology
provides for making a single matrix of that size having all one
million plus possible oligonucleotides. Region size is sufficiently
small to correspond to densities of at least about 5
regions/cm.sup.2, 20 regions/cm.sup.2, 50 regions/cm.sup.2, 100
regions/cm.sup.2, and greater, including 300 regions/cm.sup.2, 1000
regions/cm.sup.2, 3K regions/cm.sup.2, 100K regions/cm.sup.2, 30K
regions/cm.sup.2, 100K regions/cm.sup.2, 300K regions/cm.sup.2 or
more, even in excess of one million regions/cm.sup.2.
[0508] Although the pattern of the regions which contain specific
sequences is theoretically not important, for practical reasons
certain patterns will be preferred in synthesizing the
oligonucleotides. The application of binary masking algorithms for
generating the pattern of known oligonucleotide probes is described
in related U.S. Pat. No. 5,489,678. By use of these binary masks, a
highly efficient means is provided for producing the substrate with
the desired matrix pattern of different sequences. Although the
binary masking strategy allows for the synthesis of all lengths of
polymers, the strategy may be easily modified to provide only
polymers of a given length. This is achieved by omitting steps
where a subunit is not attached.
[0509] The strategy for generating a specific pattern may take any
of a number of different approaches. These approaches are well
described in related application U.S. Pat. No. Pat. No. 5,489,678
and include a number of binary masking approaches which will not be
exhaustively discussed herein. However, the binary masking and
binary synthesis approaches provide a maximum of diversity with a
minimum number of actual synthetic steps.
[0510] The length of oligonucleotides used in sequencing
applications will be selected on criteria determined to some extent
by the practical limits discussed above. For example, if probes are
made as oligonucleotides, there will be 65,536 possible eight
nucleotide sequences. If a nine subunit oligonucleotide is
selected, there are 262,144 possible permeations of sequences. If a
ten-mer oligonucleotide is selected, there are 1,048,576 possible
permeations of sequences. As the number gets larger, the required
number of positionally defined subunits necessary to saturate the
possibilities also increases. With respect to hybridization
conditions, the length of the matching necessary to confer
stability of the conditions selected can be compensated for. See,
e.g., Kanehisa, M. (1984) Nuc. Acids Res., 12: 203-213, which is
hereby incorporated herein by reference.
[0511] Although not described in detail here, but below for
oligonucleotide probes, the VLSIPS.TM. Technology would typically
use a photosensitive protective group on an oligonucleotide. Sample
oligonucleotides are shown in FIG. 1. In particular, the
photoprotective group on the nucleotide molecules may be selected
from a wide variety of positive light reactive groups preferably
including nitro aromatic compounds such aso-nitro-benzyl
derivatives or benzylsulfonyl. See, e.g., Gait (1984)
Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford,
which is hereby incorporated herein by reference. In a preferred
embodiment, 6-nitro-veratryl oxycarbony (NVOC), 2-nitrobenzyl
oxycarbonyl (NBOC), or alpha,alpha-dimethyl-dimethoxybenzyl
oxycarbonyl (DEZ) is used. Photoremovable protective groups are
described in, e.g., Patchornik (1970) J. Amer. Chem. Soc., 92:
6333-6335; and Amit et al. (1974) J. Organic Chem., 39: 192-196;
each of which is hereby incorporated herein by reference.
[0512] A preferred linker for attaching the oligonucleotide to a
silicon matrix is illustrated in FIG. 2. A more detailed
description is provided below. A photosensitive blocked nucleotide
may be attached to specific locations of unblocked prior cycles of
attachments on the substrate and can be successively built up to
the correct length oligonucleotide probe.
[0513] It should be noted that multiple substrates may be
simultaneously exposed to a single target sequence where each
substrate is a duplicate of one another or where, in combination,
multiple substrates together provide the complete or desired subset
of possible subsequences. This provides the opportunity to overcome
a limitation of the density of positions on a single substrate by
using multiple substrates. In the extreme case, each probe might be
attached to a single bead or substrate and the beads sorted by
whether there is a binding interaction. Those beads which do bind
might be encoded to indicate the subsequence specificity of
reagents attached thereto.
[0514] Then, the target may he bound to the whole collection of
beads and those beads that have appropriate specific reagents on
them will bind to the target. Then a sorting system may be utilized
to sort those beads that actually bind the target from those that
do not. This may be accomplished by presently available cell
sorting devices or a similar apparatus. After the relatively small
number of beads which have bound the target have been collected,
the encoding scheme may be read off to determine the specificity of
the reagent on the bead. An encoding system may include a magnetic
system, a shape encoding system, a color encoding system, or a
combination of any of these, or any other encoding system. Once
again, with the collection of specific interactions that have
occurred, the binding may be analyzed for sequence information,
fingerprint information, or mapping information.
[0515] The parameters of polynucleotide sizes of both the probes
and target sequences are determined by the applications and other
circumstances. The length of the oligonucleotide probes used will
depend in part upon the limitations of the VLSFPS.TM. Technology to
provide the number of desired probes. For example, in an absolute
sequencing application, it is often useful to have virtually all of
the possible oligonucleotides of a given length. As indicated
above, there are 65,536 8-mers, 262,144 9-mers, 1,048,576 10-mers,
4,194,304 11-mers, etc. As the length of the oligomer increases the
number of different probes which must be synthesized also increases
at a rate of a factor of 4 for every additional nucleotide.
Eventually the size of the matrix and the limitations in the
resolution of regions in the matrix will reach the point where an
increase in number of probes becomes disadvantageous. However, this
sequencing procedure requires that the system be able to
distinguish, by appropriate selection of hybridization and washing
conditions, between binding of absolute fidelity and binding of
complementary sequences containing mismatches. On the other hand,
if the fidelity is unnecessary, this discrimination is also
unnecessary and a significantly longer probe may be used.
Significantly longer probes would typically be useful in
fingerprinting or mapping applications.
[0516] The length of the probe is selected for a length that will
allow the probe to bind with specificity to possible targets. The
hybridization conditions are also very important in that they will
determine how closely the homology of complementary binding will be
detected. In fact, a single target may be evaluated at a number of
different conditions to determine its spectrum of specificity for
binding particular probes. This may find use in a number of other
applications besides the polynucleotide sequencing fingerprinting
or mapping. For example, it will be desired to determine the
spectrum of binding affinities and specificities of cell surface
antigens with binding by particular antibodies immobilized on the
substrate surface, particularly under different interaction
conditions. In a related fashion, different regions with reagents
having differing affinities or levels of specificity may allow such
a spectrum to be defined using a single incubation, where various
regions, at a given hybridization condition, show the binding
affinity. For example, fingerprint probes of various lengths, or
with specific defined non-matches may be used. Unnatural
nucleotides or nucleotides exhibiting modified specificity of
complementary binding are described in greater detail in Macevicz
(1990) PCT pub. No. WO 90/04652; and see the section on modified
nucleotides in the Sigma Chemical Company catalogue.
[0517] B. Labeling Target Nucleotide
[0518] The label used to detect the target sequences will be
determined, in part, by the detection methods being applied. Thus,
the labeling method and label used are selected in combination with
the actual detecting systems being used.
[0519] Once a particular label has been selected, appropriate
labeling protocols will be applied, as described below for specific
embodiments. Standard labeling protocols for nucleic acids are
described, e.g., in Sambrook et al.; Kambara, H. et al. (1988)
Biotechnology 6: 816-821; Smith, L. et al. (1985) Nuc. Acids Res.,
13: 2399-2412; for polypeptides, see, e.g., Allen G. (1989)
Sequencing of Proteins and Peptides, Elsevier, N.Y., especially
chapter 5, and Greenstein and Winitz (1961) Chemistry of the Amino
Acids, Wiley and Sons, New York. Carbohydrate labeling is
described, e.g., in Chaplin and Kennedy (1986) Carbohydrate
Analysis: A Practical Approach, IRL Press, Oxford. Labeling of
other polymers will be performed by methods applicable to them as
recognized by a person having ordinary skill in manipulating the
corresponding polymer.
[0520] In some embodiments, the target need not actually be labeled
if a means for detecting where interaction takes place is
available. As described below, for a nucleic acid embodiment, such
may be provided by an intercalating dye which intercalates only
into double stranded segments, e.g., where interaction occurs. See,
e.g., Sheldon et al. U.S. Pat. No. 4,582,789.
[0521] In many uses, the target sequence will be absolutely
homogeneous, both with respect to the total sequence and with
respect to the ends of each molecule. Homogeneity with respect to
sequence is important to avoid ambiguity. It is preferable that the
target sequences of interest not be contaminated with a significant
amount of labeled contaminating sequences. The extent of allowable
contamination will depend on the sensitivity of the detection
system and the inherent signal to noise of the system. Homogeneous
contamination sequences will be particularly disruptive of the
sequencing procedure.
[0522] However, although the target polynucleotide must have a
unique sequence, the target molecules need not have identical ends.
In fact, the homogeneous target molecule preparation may be
randomly sheared to increase the numerical number of molecules.
Since the total information content remains the same, the shearing
results only in a higher number of distinct sequences which may be
labeled and bind to the probe. This fragmentation may give a vastly
superior signal relative to a preparation of the target molecules
having homogeneous ends. The signal for the hybridization is likely
to be dependent on the numerical frequency of the target-probe
interactions. If a sequence is individually found on a larger
number of separate molecules a better signal will result. In fact,
shearing a homogeneous preparation of the target may often be
preferred before the labeling procedure is performed, thereby
producing a large number of labeling groups associated with each
subsequence.
[0523] C. Hybridization Conditions
[0524] The hybridization conditions between probe and target should
be selected such that the specific recognition interaction, i.e.,
hybridization, of the two molecules is both sufficiently specific
and sufficiently stable. See, e.g., Hames and Higgins (1985)
Nucleic Acid Hybridisation: A Practical Approach, IRL Press,
Oxford. These conditions will be dependent both on the specific
sequence and often on the guanine and cytosine (GC) content of the
complementary hybrid strands. The conditions may often be selected
to be universally equally stable independent of the specific
sequences involved. This typically will make use of a reagent such
as an alkylammonium buffer. See, Wood et al. (1985) "Base
Composition-independent Hybridization in Tetramethylammonium
Chloride: A Method for Oligonucleotide Screening of Highly Complex
Gene Libraries," Proc. Natl. Acad. Sci. USA, 82: 1585-1588; and
Krupov et al. (1989) "An Oligonucleotide Hybridization Approach to
DNA Sequencing," FEBS Letters, 256: 118-122; each of which is
hereby incorporated herein by reference. An alklyammonium buffer
tends to minimize differences in hybridization rate and stability
due to GC content. By virtue of the fact that sequences then
hybridize with approximately equal affinity and stability, there is
relatively little bias in strength or kinetics of binding for
particular sequences. Temperature and salt conditions along with
other buffer parameters should be selected such that the kinetics
of renaturation should be essentially independent of the specific
target subsequence or oligonucleotide probe involved. In order to
ensure this, the hybridization reactions will usually be performed
in a single incubation of all the substrate matrices together
exposed to the identical same target probe solution under the same
conditions.
[0525] Alternatively, various substrates may be individually
treated differently. Different substrates may be produced, each
having reagents which bind to target subsequences with
substantially identical stabilities and kinetics of hybridization.
For example, all of the high GC content probes could be synthesized
on a single substrate which is treated accordingly. In this
embodiment, the alkylammonium buffers could be unnecessary. Each
substrate is then treated in a manner such that the collection of
substrates show essentially uniform binding and the hybridization
data of target binding to the individual substrate matrix is
combined with the data from other substrates to derive the
necessary subsequence binding information. The hybridization
conditions will usually be selected to be sufficiently specific
such that the fidelity of base matching will be properly
discriminated. Of course, control hybridizations should be included
to determine the stringency and kinetics of hybridization.
[0526] D. Detection: VLSIPS.TM. Technology Scanning
[0527] The next step of the sequencing process by hybridization
involves labeling of target polynucleotide molecules. A quickly and
easily detectable signal is preferred. The VLSIPS.TM. Technology
apparatus is designed to easily detect a fluorescent label, so
fluorescent tagging of the target sequence is preferred. Other
suitable labels include heavy metal labels, magnetic probes,
chromogenic labels (e.g., phosphorescent labels, dyes, and
fluorophores) spectroscopic labels, enzyme linked labels,
radioactive labels, and labeled binding proteins. Additional labels
are described in U.S. Pat. No. 4,366,241, which is incorporated
herein by reference.
[0528] The detection methods used to determine where hybridization
has taken place will typically depend upon the label selected
above. Thus, for a fluorescent label a fluorescent detection step
will typically be used. Pirrung et al. (1992) U.S. Pat. Nos.
5,143,854 and 5,489,678 describe apparatus and mechanisms for
scanning a substrate matrix using fluorescence detection, but a
similar apparatus is adaptable for other optically detectable
labels.
[0529] The detection method provides a positional localization of
the region where hybridization has taken place. However, the
position is correlated with the specific sequence of the probe
since the probe has specifically been attached or synthesized at a
defined substrate matrix position. Having collected all of the data
indicating the subsequences present in the target sequence, this
data may be aligned by overlap to reconstruct the entire sequence
of the target, as illustrated above.
[0530] It is also possible to dispense with actual labeling if some
means for detecting the positions of interaction between the
sequence specific reagent and the target molecule are available.
This may take the form of an additional reagent which can indicate
the sites either of interaction, or the sites of lack of
interaction, e.g., a negative label. For the nucleic acid
embodiments, locations of double strand interaction may be detected
by the incorporation of intercalating dyes, or other reagents such
as antibody or other reagents that recognize helix formation, see,
e.g., Sheldon, et al. (1986) U.S. Pat. No. 4,582,789, which is
hereby incorporated herein by reference.
[0531] E. Analysis
[0532] Although the reconstruction can be performed manually as
illustrated above, a computer program will typically be used to
perform the overlap analysis. A program may be written and run on
any of a large number of different computer hardware systems. The
variety of operating systems and languages useable will be
recognized by a computer software engineer. Various different
languages may be used, e.g., BASIC; C; PASCAL; etc. A simple flow
chart of data analysis is illustrated in FIG. 4.
[0533] F. Substrate Reuse
[0534] Finally, after a particular sequence has been hybridized and
the pattern of hybridization analyzed, the matrix substrate should
be reusable and readily prepared for exposure to a second or
subsequent target polynucleotides. In order to do so, the hybrid
duplexes are disrupted and the matrix treated in a way which
removes all traces of the original target. The matrix may be
treated with various detergents or solvents to which the substrate,
the oligonucleotide probes, and the linkages to the substrate are
inert. This treatment may include an elevated temperature
treatment, treatment with organic or inorganic solvents,
modifications in pH, and other means for disrupting specific
interaction. Thereafter, a second target may actually be applied to
the recycled matrix and analyzed as before.
[0535] G. Non-Polynucleotide Aspects
[0536] Although the sequencing, fingerprinting, and mapping
functions will make use of the natural sequence recognition
property of complementary nucleotide sequences, the
non-polynucleotide sequences typically require other sequence
recognition reagents. These reagents will take the form, typically,
of proteins exhibiting binding specificity, e.g., enzyme binding
sites or antibody binding sites.
[0537] Enzyme binding sites may be derived from promoter proteins,
restriction enzymes, and the like. See, e.g., Stryer, L. (1988)
Biochemistry, W. H. Freeman, Palo Alto. Antibodies will typically
be produced using standard procedures, see, e.g., Harlow and Lane
(1988) Antibodies; A Laboratory Manual, Cold Spring Harbor Press,
New York; and Goding (1986) Monoclonal Antibodies: Principles and
Practice, (2d Ed.) Academic Press, San Diego.
[0538] Typically, an antigen, or collection of antigens are
presented to an immune system. This may take the form of
synthesized short polymers produced by the VLSIPS.TM. Technology,
or by the other synthetic means, or from isolation of natural
products. For example, antigen for the polypeptides may be made by
the VLSIPS.TM. Technology, by standard peptide synthesis, by
isolation of natural proteins with or without degradation to
shorter segments, or by expression of a collection of short nucleic
acids of random or defined sequences. See, e.g., Tuerk and Gold
(1990) Science, 249: 505-510, for generation of a collection of
randomly mutagenized oligonucleotides useful for expression.
[0539] The antigen or collection is presented to an appropriate
immune system, e.g., to a whole animal as in a standard
immunization protocol, or to a collection of immune cells or
equivalent. In particular, see Ward et al. (1989) Nature, 341:
544-546; and Huse et al. (1989) Science, 246;1275-1281, each of
which is hereby incorporated herein by reference.
[0540] A large diversity of antibodies will be generated, some of
which have specificities for the desired sequences. Antibodies may
be purified having the desired sequence specificities by isolating
the cells producing them. For example, a VLSIPS.TM. Technology
substrate with the desired antigens synthesized thereon may be used
to isolate cells with cell surface reagents which recognize the
antigens. The VLSIPS.TM. Technology substrate may be used as an
affinity reagent to select and recover the appropriate cells.
Antibodies from those cells may be attached to a substrate using
the caged biotin methodology, or by attaching a targeting molecule,
e.g., an oligonucleotide. Alternatively, the supernatants from
antibody producing cells can be easily assayed using a VLSIPS.TM.
Technology substrate to identify the cells producing the
appropriate antibodies.
[0541] Although cells may be isolated, specific antibody molecules
which perform the sequence recognition will also be sufficient.
Preferably populations of antibody with a known specificity can be
isolated. Supernatants from a large population of producing cells
may be passed over a VLSIPS substrate to bind to the desired
antigens attached to the substrate. When a sufficient density of
antibody molecules are attached, they may be removed by an
automated process, preferably as antibody populations exhibiting
specificity of binding.
[0542] In one particular embodiment, a VLSIPS.TM. Technology
substrate, e.g., with a large plurality of fingerprint antigens
attached thereto, is used to isolate antibodies from a supernatant
of a population of cells producing antibodies to the antigens.
Using the substrate as an affinity reagent, the antibodies will
attach to the appropriate positionally defined antigens. The
antibodies may be carefully removed therefrom, preferably by an
automated system which retains their homogeneous specificities. The
isolated antibodies can be attached to a new substrate in a
positionally defined matrix pattern.
[0543] In a further embodiment, these spatially separated
antibodies may be isolated using a specific targeting method for
isolation. In this embodiment, a linker molecule which attaches to
a particular portion of the antibody, preferably away from the
binding site, can be attached to the antibodies. Various reagents
will be used, including staphylococcus protein A or antibodies
which bind to domains remote from the binding site. Alternatively,
the antibodies in the population, before affinity purification, may
be derivatized with an appropriate reagent compatible with new
VLSIPS.TM. Technology synthesis. A preferred reagent is a
nucleotide which can serve as a linker to synthetic VLSIPS.TM.
Technology steps for synthesizing a specific sequence thereon.
Then, by successive VLSIPS.TM. Technology cycles, each of the
antibodies attached to the defined antigen regions can have a
defined oligonucleotide synthesized thereon and corresponding in
area to the region of the substrate having each antigen attached.
These defined oligonucleotides will be useful as targeting reagents
to attach those antibodies possessing the same target sequence
specificity at defined positions on a new substrate, by virtue of
having bound to the antigen region, to a new VLSIPS.TM. Technology
substrate having the complementary target oligonucleotides
positionally located on it. In this fashion, a VLSIPS.TM.
Technology substrate having the desired antigens attached thereto
can be used to generate a second VLSIPS.TM. Technology substrate
with positionally defined reagents which recognize those
antigens.
[0544] The selected antigens will typically be selected to be those
which define particular functionalities or properties, so as to be
useful for fingerprinting and other uses. They will also be useful
for mapping and sequencing embodiments.
[0545] IV. Fingerprinting
[0546] A. General
[0547] Many of the procedures and techniques used in the
polynucleotide sequencing section are also appropriate for
fingerprinting applications. See, e.g., Poustka, et al. (1986) Cold
Spring Harbor Symposia on Quant. Biol., vol. LI, 131-139, Cold
Spring Harbor Press, New York; which is hereby incorporated herein
by reference. The fingerprinting method provided herein is based,
in part, upon the ability to positionally localize a large number
of different specific probes onto a single substrate. This high
density matrix pattern provides the ability to screen for, or
detect, a very large number of different sequences simultaneously.
In fact, depending upon the hybridization conditions,
fingerprinting to the resolution of virtually absolute matching of
sequence is possible thereby approaching an absolute sequencing
embodiment. And the sequencing embodiment is very useful in
identifying the probes useful in further fingerprinting uses. For
example, characteristic features of genetic sequences will be
identified as being diagnostic of the entire sequence. However, in
most embodiments, longer probe and target will be used, and for
which slight mismatching may not need to be resolved.
[0548] B. Preparation of Substrate Matrix
[0549] A collection of specific probes may be produced by either of
the methods described above in the section on sequencing. Specific
oligonucleotide probes of desired lengths may be individually
synthesized on a standard oligonucleotide synthesizer. The length
of these probes is limited only by the ability of the synthesizer
to continue to accurately synthesize a molecule. Oligonucleotides
or sequence fragments may also be isolated from natural sources.
Biological amplification methods may be coupled with synthetic
synthesizing procedures such as, e.g., polymerase chain
reaction.
[0550] In one embodiment, the individually isolated probes may be
attached to the matrix at defined positions. These probe reagents
may be attached by an automated process making use of the caged
biotin methodology described in U.S. Pat. No. 5,252,743, or using
photochemical reagents, see, e.g., Dattagupta et al. (1985) U.S.
Pat. No. 4,542,102 and (1987) U.S. Pat. No. 4,713,326. Each
individually purified reagent can be attached individually at
specific locations on a substrate.
[0551] In another embodiment, the VLSIPS.TM. Technology
synthesizing technique may be used to synthesize the desired probes
at specific positions on a substrate. The probes may be synthesized
by successively adding appropriate monomer subunits, e.g.,
nucleotides, to generate the desired sequences.
[0552] In another embodiment, a relatively short specific
oligonucleotide is used which serves as a targeting reagent for
positionally directing the sequence recognition reagent. For
example, the sequence specific reagents having a separate
additional sequence recognition segment (usually of a different
polymer from the target sequence) can be directed to target
oligonucleotides attached to the substrate. By use of non-natural
targeting reagents, e.g., unusual nucleotide analogues which pair
with other unnatural nucleotide analogues and which do not
interfere with natural nucleotide interactions, the natural and
non-natural portions can coexist on the same molecule without
interfering with their individual functionalities. This can combine
both a synthetic and biological production system analogous to the
technique for targeting monoclonal antibodies to locations on a
VLSIPS.TM. Technology substrate at defined positions. Unnatural
optical isomers of nucleotides may be useful unnatural reagents
subject to similar chemistry, but incapable of interfering with the
natural biological polymers. See also, U.S. Pat. No. 5,547,839
which is hereby incorporated herein by reference.
[0553] After the separate substrate attached reagents are attached
to the targeting segment, the two are crosslinked, thereby
permanently attaching them to the substrate. Suitable crosslinking
reagents are known, see, e.g., Dattagupta et al. (1985) U.S. Pat.
No. 4,542,102 and (1987) "Coupling of nucleic acids to solid
support by photochemical methods," U.S. Pat. No. 4,713,326, each of
which is hereby incorporated herein by reference. Similar linkages
for attachment of proteins to a solid substrate are provided, e.g.,
in Merrifield (1986) Science, 232: 341-347, which is hereby
incorporated herein by reference.
[0554] C. Labeling Target Nucleotides
[0555] The labeling procedures used in the sequencing embodiments
will also be applicable in the fingerprinting embodiments. However,
since the fingerprinting embodiments often will involve relatively
large target molecules and relatively short oligonucleotide probes,
the amount of signal necessary to incorporate into the target
sequence may be less critical than in the sequencing applications.
For example, a relatively long target with a relatively small
number of labels per molecule may be easily amplified or detected
because of the relatively large target molecule size.
[0556] In various embodiments, it may be desired to cleave the
target into smaller segments as in the sequencing embodiments. The
labeling procedures and cleavage techniques described in the
sequencing embodiments would usually also be applicable here.
[0557] D. Hybridization Conditions
[0558] The hybridization conditions used in fingerprinting
embodiments will typically be less critical than for the sequencing
embodiments. The reason is that the amount of mismatching which may
be useful in providing the fingerprinting information would
typically be far greater than that necessary in sequencing uses.
For example, Southern hybridizations do not typically distinguish
between slightly mismatched sequences. Under these circumstances,
important and valuable information may be arrived at with less
stringent hybridization conditions while providing valuable
fingerprinting information. However, since the entire substrate is
typically exposed to the target molecule at one time, the binding
affinity of the probes should usually be of approximately
comparable levels. For this reason, if oligonucleotide probes are
being used, their lengths should be approximately comparable and
will be selected to hybridize under conditions which are common for
most of the probes on the substrate. Much as in a Southern
hybridization, the target and oligonucleotide probes are of lengths
typically greater than about 25 nucleotides. Under appropriate
hybridization conditions, e.g., typically higher salt and lower
temperature, the probes will hybridize irrespective of imperfect
complementarity. In fact, with probes of greater than, e.g., about
fifty nucleotides, the difference in stability of different sized
probes will be relatively minor.
[0559] Typically the fingerprinting is merely for probing
similarity or homology. Thus, the stringency of hybridization can
usually be decreased to fairly low levels. See, e.g., Wetmur and
Davidson (1968) "Kinetics of Renaturation of DNA," J. Mol. Biol.,
31: 349-370; and Kanehisa, M. (1984) Nuc. Acids Res., 12:
203-213.
[0560] E. Detection: VLSIPS.TM. Technology Scanning
[0561] Detection methods will be selected which are appropriate for
the selected label. The scanning device need not necessarily be
digitized or placed into a specific digital database, though such
would most likely be done. For example, the analysis in
fingerprinting could be photographic. Where a standardized
fingerprint substrate matrix is used, the pattern of hybridizations
may be spatially unique and may be compared photographically. In
this manner, each sample may have a characteristic pattern of
interactions and the likelihood of identical patterns will
preferably be such low frequency that the fingerprint pattern
indeed becomes a characteristic pattern virtually as unique as an
individual's fingertip fingerprint. With a standardized substrate,
every individual could be, in theory, uniquely identifiable on the
basis of the pattern of hybridizing to the substrate.
[0562] Of course, the VLSIPS.TM. Technology scanning apparatus may
also be useful to generate a digitized version of the fingerprint
pattern. In this way, the identification pattern can be provided in
a linear string of digits. This sequence could also be used for a
standardized identification system providing significant useful
medical transferability of specific data. In one embodiment, the
probes used are selected to be of sufficiently high resolution to
measure the antigens of the major histocompatibility complex. It
might even be possible to provide transplantation matching data in
a linear stream of data. The fingerprinting data may provide a
condensed version, or summary, of the linear genetic data, or any
other information data base.
[0563] F. Analysis
[0564] The analysis of the fingerprint will often be much simpler
than a total sequence determination. However, there may be
particular types of analysis which will be substantially simplified
by a selected group of probes. For example, probes which exhibit
particular populational heterogeneity may be selected. In this way,
analysis may be simplified and practical utility enhanced merely by
careful selection of the specific probes and a careful matrix
layout of those probes.
[0565] G. Substrate Reuse
[0566] As with the sequencing application, the fingerprinting
usages may also take advantage of the reusability of the substrate.
In this way, the interactions can be disrupted, the substrate
treated, and the renewed substrate is equivalent to an unused
substrate.
[0567] H. Non-Polynucleotide Aspects
[0568] Besides polynucleotide applications, the fingerprinting
analysis may be applied to other polymers, especially polypeptides,
carbohydrates, and other polymers, both organic and inorganic.
Besides using the fingerprinting method for analyzing a particular
polymer, the fingerprinting method may be used to characterize
various samples. For example, a cell or population of cells may be
tested for their expression of specific antigens or their mRNA
sequence intent. For example, a T-cell may be classified by virtue
of its combination of expressed surface antigens. With specific
reagents which interact with these antigens, a cell or a population
of cells or a lysed cell may be exposed to a VLSIPS.TM. Technology
substrate. The biological sample may be classified or characterized
by analyzing the pattern of specific interaction. This may be
applicable to a cell or tissue type, to the messenger RNA
population expressed by a cell to the genetic content of a cell, or
to virtually any sample which can be classified and/or identified
by its combination of specific molecular properties.
[0569] The ability to generate a high density means for screening
the presence or absence of specific interactions allows for the
possibility of screening for, if not saturating, all of a very
large number of possible interactions. This is very powerful in
providing the means for testing the combinations of molecular
properties which can define a class of samples. For example, a
species of organism may be characterized by its DNA sequences,
e.g., a genetic fingerprint. By using a fingerprinting method, it
may be determined that all members of that species are sufficiently
similar in specific sequences that they can be easily identified as
being within a particular group. Thus, newly defined classes may be
resolved by their similarity in fingerprint patterns.
Alternatively, a non-member of that group will fail to share those
many identifying characteristics. However, since the technology
allows testing of a very large number of specific interactions, it
also provides the ability to more finely distinguish between
closely related different cells or samples. This will have
important applications in diagnosing viral, bacterial, and other
pathological on nonpathological infections.
[0570] In particular, cell classification may be defined by any of
a nether of different properties. For example, a cell class may be
defined by its DNA sequences contained therein. This allows species
identification for parasitic or other infections. For example, the
human cell is presumably genetically distinguishable from a monkey
cell, but different human cells will share many genetic markers. At
higher resolution, each individual human genome will exhibit unique
sequences that can define it as a single individual.
[0571] Likewise, a developmental stage of a cell type may be
definable by its pattern of expression of messenger RNA. For
example, in particular stages of cells, high levels of ribosomal
RNA are found whereas relatively low levels of other. types of
messenger RNAs may be found. The high resolution distinguishability
provided by this fingerprinting method allows the distinction
between cells which have relatively minor differences in its
expressed mRNA population. Where a pattern is shown to be
characteristic of a stage, a stage may be defined by that
particular pattern of messenger RNA expression.
[0572] In a similar manner, the antigenic determinants found on a
protein may very well define the cell class. For example,
immunological T-cells are distinguishable from B-cells because, in
part, the cell surface antigens on the cell types are
distinguishable. Different T-cell subclasses can be also
distinguished from one another by whether they contain particular
T-cell antigens. The present invention provides the possibility for
high resolution testing of many different interactions
simultaneously, and the definition of new cell types will be
possible.
[0573] The high resolution VLSIPS.TM. Technology substrate may also
be used as a very powerful diagnostic tool to test the combination
of presence, of a plurality of different assays from a biological
sample. For example, a cancerous condition may be indicated by a
combination of various different properties found in the blood. For
example, a cancerous condition may be indicated by a combination of
expression of various soluble antigens found in the blood along
with a high number of various cellular antigens found on
lymphocytes and/or particular cell degradation products. With a
substrate as provided herein, a large number of different features
can be simultaneously performed on a biological sample. In fact,
the high resolution of the test will allow more complete
characterization of parameters which define particular diseases.
Thus, the power of diagnostic tests may be limited by the extent of
statistical correlation with a particular condition rather than
with the number of antigens or interactions which are tested. The
present invention provides the means to generate this large
universe of possible reagents and the ability to actually
accumulate that correlative data.
[0574] In another embodiment, a substrate as provided herein may be
used for genetic screening. This would allow for simultaneous
screening of thousands of genetic markers. As the density of the
matrix is increased, many more molecules can be simultaneously
tested. Genetic screening then becomes a simpler method as the
present invention provides the ability to screen for thousands,
tens of thousands, and hundreds of thousands, even millions of
different possible genetic features. However, the number of high
correlation genetic markers for conditions numbers only in the
hundreds. Again, the possibility for screening a large number of
sequences provides the opportunity for generating the data which
can provide correlation between sequences and specific conditions
or susceptibility. The present invention provides the means to
generate extremely valuable correlations useful for the genetic
detection of the causative mutation leading to medical conditions.
In still another embodiment, the present invention would be
applicable to distinguishing two individuals having identical
genetic compositions. The antibody population within an individual
is dependent both on genetic and historical factors. Each
individual experiences a unique exposure to various infectious
agents, and the combined antibody expression is partly determined
thereby. Thus, individuals may also be fingerprinted by their
immunological content, either of actively expressed antibodies, or
their immunological memory. Similar soils of immunological and
environmental histories may be useful for fingerprinting, perhaps
in combination with other screening properties. In particular, the
present invention may be useful for screening allergic reactions or
susceptibilities, and a simple IgE specificity may be useful in
determining a spectrum of allergies.
[0575] With the definition of new classes of cells, a cell sorter
will be used to purify them. Moreover, new markers for defining
that class of cells will be identified. For example, where the
class is defined by its RNA content, cells may he screened by
antisense probes which detect the presence or absence of specific
sequences therein. Alternatively, cell lysates may provide
information useful in correlating intracellular properties with
extracellular markers which indicate functional differences. Using
standard cell sorter technology with a fluorescence or labeled
antisense probe which recognizes the internal presence of the
specific sequences of interest, the cell sorter will be able to
isolate a relatively homogeneous population of cells possessing the
particular marker. Using successive probes the sorting process
should be able to select for cells having a combination of a large
number of different markers.
[0576] In a non-polynucleotide embodiment, cells may be defined by
the presence of other markers. The markers may be carbohydrates,
proteins, or other molecules. Thus, a substrate having particular
specific reagents, e.g., antibodies, attached to it should be able
to identify cells having particular patterns of marker expression.
Of course, combinations of these made be utilized and a cell class
may be defined by a combination of its expressed mRNA, its
carbohydrate expression, its antigens, and other properties. This
fingerprinting should be useful in determining the physiological
state of a cell or population of cells.
[0577] Having defined a cell type whose function or properties are
defined by the reagents attachable to a VLSIPS substrate, such as
cellular antigens, these structural manifestations of function may
be used to sort cells to generate a relatively homogeneous
population of that class of cells. Standard cell sorter technology
may be applied to purify such a population, see, e.g., Dangl, J.
and Herzenberg (1982) "Selection of hybridomas and hybridoma
variants using the fluorescence activated cell sorter," J.
Immunological Methods, 52: 1-14; and Becton Dickinson, Fluorescence
Activated Cell Sorter Division, San Jose, Calif., and Coulter
Diagnostics, Hialeah, Fla.
[0578] With the fingerprinting method as an identification means
arises from mosaicism problems in an organism. A mosaic organism is
one whose genetic content in different cells is significantly
different. Various clonal populations should have similar genetic
fingerprints, though different clonal populations may have
different genetic contents. See, for example, Suzuki et al. An
Introduction to Genetic Analysis (4th Ed.), Freeman and Co., New
York, which is hereby incorporated herein by reference. However,
this problem should be a relatively rare problem and could be more
carefully evaluated with greater experience using the
fingerprinting methods.
[0579] V. Mapping
[0580] A. General
[0581] The use of the present invention for mapping parallels its
use for fingerprinting and sequencing. Where a polymer is a linear
molecule, the mapping provides the ability to locate particular
segments along the length of the polymer. Branched polymers can be
treated as a series of individual linear polymers. The mapping
provides the ability to locate, in a relative sense, the order of
various subsequences. This may be achieved using at least two
different approaches.
[0582] The first approach is to take the large sequence and
fragment it at specific points. The fragments are then ordered and
attached to a solid substrate. For example, the clones resulting
from a chromosome walking process may be individually attached to
the substrate by methods, e.g., caged biotin techniques, indicated
earlier. Segments of unknown map position will be exposed to the
substrate and will hybridize to the segment which contains that
particular sequence. This procedure allows the rapid determination
of a number of different labeled segments, each mapping requiring
only a single hybridization step once the substrate is generated.
The substrate may be regenerated by removal of the interaction, and
the next mapping segment applied.
[0583] In an alternative method, a plurality of subsequences can be
attached to a substrate. Various short probes may be applied to
determine which segments may contain particular overlaps. The
theoretical basis and a description of this mapping procedure is
contained in, e.g., Evans et al. 1989 "Physical Mapping of Complex
Genomes by Cosmid Multiplex Analysis," Proc. Natl. Acad. Sci. USA,
86: 5030-5034, and other references cited above in the Section
labeled "Overall Description." Using this approach, the details of
the mapping embodiment are very similar to those used in the
fingerprinting embodiment.
[0584] B. Preparation of Substrate Matrix
[0585] The substrate may be generated in either of the methods
generally applicable in the sequencing and fingerprinting
embodiments. The substrate may be made either synthetically, or by
attaching otherwise purified probes or sequences to the matrix. The
probes. or sequences may be derived either from synthetic or
biological means. As indicated above, the solid phase substrate
synthetic methods may be utilized to generate a matrix with
positionally defined sequences. In the mapping embodiment, the
importance of saturation of all possible subsequences of a
preselected length is far less important than in the sequencing
embodiment, but the length of the probes used may be desired to be
much longer. The processes for making a substrate which has longer
oligonucleotide probes should not be significantly different from
those described for the sequencing embodiments, but the
optimization parameters may be modified to comply with the mapping
needs.
[0586] C. Labeling
[0587] The labeling methods will be similar to those applicable in
sequencing and fingerprinting embodiments. Again, it may be
desirable to fragment the target sequences.
[0588] D. Hybridization/Specific Interaction
[0589] The specificity of interaction between the targets and probe
would typically be closer to those used for fingerprinting
embodiments, where homology is more important than absolute
distinguishability of high fidelity complementary hybridization.
Usually, the hybridization conditions will be such that merely
homologous segments will interact and provide a positive signal.
Much like the fingerprinting embodiment, it may be useful to
measure the extent of homology by successive incubations at higher
stringency conditions. Or, a plurality of different probes, each
having various levels of homology may be used. In either way, the
spectrum of homologies can be measured.
[0590] Where non-nucleic acid hybridization is involved, the
specific interactions may also be compared in a fingerprint-like
manner. The specific reagents may have less specificity, e.g.,
monoclonal antibodies which recognize a broader spectrum of
sequences may be utilized relative to a sequencing embodiment.
Again, the specificity of interaction may be measured under various
conditions of increasing stringency to determine the spectrum of
matching across the specific probes selected, or a number of
different stringency reagents may be included to indicate the
binding affinity.
[0591] E. Detection
[0592] The detection methods used in the mapping procedure will be
virtually identical to those used in the fingerprinting embodiment.
The detection methods will be selected in combination with the
labeling methods.
[0593] F. Analysis
[0594] The analysis of the data in a mapping embodiment will
typically be somewhat different from that in fingerprinting. The
fingerprinting embodiment will test for the presence or absence of
specific or homologous segments. However, in the mapping
embodiment, the existence of an interaction is coupled with some
indication of the location of the interaction. The interaction is
mapped in some manner to the physical polymer sequence. Some means
for determining the relative positions of different probes, is
performed. This may be achieved by synthesis of the substrate in
pattern, or may result from analysis of sequences after they have
been attached to the substrate.
[0595] For example, the probes may be randomly positioned at
various locations on the substrate. However, the relative positions
of the various reagents in the original polymer may be determined
by using short fragments, e.g., individually, as target molecules
which determine the proximity of different probes. By an automated
system of testing each different short fragment of the original
polymer, coupled with proper analysis, it will be possible to
determine which probes are adjacent one another on the original
target sequence and correlate that with positions on the matrix. In
this way, the matrix is useful for determining the relative
locations of various new segments in the original target molecule.
This sort of analysis is described in Evans, and the related
references described above.
[0596] G. Substrate Reuse
[0597] The substrate should be reusable in the manner described in
the fingerprinting section. The substrate is renewed by removal of
the specific interactions and is washed and prepared for successive
cycles of exposure to new target sequences.
[0598] H. Non-Polynucleotide Aspects
[0599] The mapping procedure may be used on other molecules than
polynucleotides. Although hybridization is one type of specific
interaction which is clearly useful for use in this mapping
embodiment, antibody reagents may also be very useful. In the same
way that polypeptide sequencing or other polymers may be sequenced
by the reagents and techniques described in the sequencing section
and fingerprinting section, the mapping embodiment may also be used
similarly.
[0600] In another form of mapping, as described above in the
fingerprinting section, the developmental map of a cell or
biological system may be measured using fingerprinting type
technology. Thus, the mapping may be along a temporal dimension
rather than along a polymer dimension. The mapping or
fingerprinting embodiments may also be used in determining the
genetic rearrangements which may be genetically important, as in
lymphocyte and B-cell development. In another example, various
rearrangements or chromosomal dislocations may be tested by either
the fingerprinting or mapping methods. These techniques are similar
in many respects and the fingerprinting and mapping embodiments may
overlap in many respects.
[0601] VI. Additional Screening and Applications
[0602] A. Specific Interactions
[0603] As originally indicated in the parent filing of VLSIPS.TM.
Technology, the production of a high density plurality of spatially
segregated polymers provides the ability to generate a very large
universe or repertoire of individually and distinct sequence
possibilities. As indicated above, particular oligonucleotides may
be synthesized in automated fashion at specific locations on a
matrix. In fact, these oligonucleotides may be used to direct other
molecules to specific locations by linking specific
oligonucleotides to other reagents which are in batch exposed to
the matrix and hybridized in a complementary fashion to only those
locations where the complementary oligonucleotide has been
synthesized on the matrix. This allows for spatially attaching a
plurality of different reagents onto the matrix instead of
individually attaching each separate reagent at each specific
location. Although the caged biotin method allows automated
attachment, the speed of the caged biotin attachment process is
relatively slow and requires a separate reaction for each reagent
being attached. By use of the oligonucleotide method, the
specificity of position can be done in an automated and parallel
fashion. As each reagent is produced, instead of directly attaching
each reagent at each desired position, the reagent may be attached
to a specific desired complementary oligonucleotide which will
ultimately be specifically directed toward locations on the matrix
having a complementary oligonucleotide attached thereat.
[0604] In addition, the technology allows screening for specificity
of interaction with particular reagents. For example, the
oligonucleotide sequence specificity of binding of a potential
reagent may be tested by presenting to the reagent all of the
possible subsequences available for binding. Although secondary or
higher order sequence specific features might not be easily
screenable using this technology, it does provide a convenient,
simple, quick, and thorough screen of interactions between a
reagent and its target recognition sequences. See, e.g., Pfeifer et
al. (1989) Science, 246: 810-812.
[0605] For example, the interaction of a promoter protein with its
target binding sequence may be tested for many different, or all,
possible binding sequences. By testing the strength of interactions
under various different conditions, the interaction of the promoter
protein with each of the different potential binding sites may be
analyzed. The spectrum of strength of interactions with each
different potential binding site may provide significant insight
into the types of features which are important in determining
specificity.
[0606] An additional example of a sequence specific interaction
between reagents is the testing of binding of a double stranded
nucleic acid structure with a single stranded oligonucleotide.
Often, a triple stranded structure is produced which has
significant aspects of sequence specificity. Testing of such
interactions with either sequences comprising only natural
nucleotides, or perhaps the testing of nucleotide analogs may be
very important in screening for particularly useful diagnostic or
therapeutic reagents. See, e.g., Haner and Dervan (1990)
Biochemistry, 29: 9761-6765, and references therein.
[0607] B. Sequence Comparisons
[0608] Once a gene is sequenced, the present invention provides a
means to compare alleles or related sequences to locate and
identify differences from the control sequence. This would be
extremely useful in further analysis of genetic variability at a
specific gene locus.
[0609] C. Categorizations
[0610] As indicated above in the fingerprinting and mapping
embodiments, the present invention is also useful in defining
specific stages in the temporal sequence of cells, e.g.,
development, and the resulting tissues within an organism. For
example, the developmental stage of a cell, or population of cells,
can be dependent upon the expression of particular messenger RNAs
or cellular antigens. The screening procedures provided allow for
high resolution definition of new classes of cells. In addition,
the temporal development of particular cells will be characterized
by the presence or expression of various mRNAs. Means to
simultaneously screen a plurality or very large number of different
sequences are provided. The combination of different markers made
available dramatically increases the ability to distinguish fairly
closely related cell types. Other markers may be combined with
markers and methods made available herein to define new
classifications of biological samples, e.g., based upon new
combinations of markers.
[0611] The presence or absence of particular marker sequences will
be used to define temporal developmental stages. Once the stages
are defined, fairly simple methods can be applied to actually
purify those particular cells. For example, antisense probes or
recognition reagents may be used with a cell sorter to select those
cells containing or expressing the critical markers. Alternatively,
the expression of those sequences may result in specific antigens
which may also be used in defining cell classes and sorting those
cells away from others. In this way, for example, it should be
possible to select a class of omnipotent immune system cells which
are able to completely regenerate a human immune system. Based upon
the cellular classes defined by the parameters made available by
this technology, purified classes of cells having identifiable
differences, structural or functional, are made available.
[0612] In an alternative embodiment, a plurality of antigens or
specific binding proteins attached to the substrate may be used to
define particular cell types. For example, subclasses of T-cells
are defined, in part, by the combination of expressed cell surface
antigens. The present invention allows for the simultaneous
screening of a large plurality of different antigens together.
Thus, higher resolution classification of different T-cell
subclasses becomes possible and, with the definitions and
functional differences which correlate with those antigenic or
other parameters, the ability to purify those cell types becomes
available. This is applicable not only to T-cells, lymphocyte
cells, or even to freely circulating cells. Many of the cells for
which this would be most useful will be immobile cells found in
particular tissues or organs. Tumor cells will be diagnosed or
detected using these fingerprinting techniques. Coupled with a
temporal change in structure, developmental classes may also be
selected and defined using these technologies. The present
invention also provides the ability not only to define new classes
of cells based upon functional or structural differences, but it
also provides the ability to select or purify populations of cells
which share these particular properties. Standard cell sorting
procedures using antibody markers may be used to detect
extracellular features. Intracellular features would also be
detectable by introducing the label reagents into the cell. In
particular, antisense DNA or RNA molecules may be introduced into a
cell to detect RNA sequences therein. See, eg., Weintraub (1990)
Scientific American, 262: 40-46.
[0613] D. Statistical Correlations
[0614] In an additional embodiment, the present invention also
allows for the high resolution correlation of medical conditions
with various different markers. For example, the presently
available technology, when applied to amniocentesis or other
genetic screening methods, typically screens for tens of different
markers at most. The present invention allows simultaneous
screening for tens, hundreds, thousands, tens of thousands,
hundreds of thousands, and even millions of different genetic
sequences. Thus, applying the fingerprinting methods of the present
invention to a sufficiently large population allows detailed
statistical analysis to be made, thereby correlating particular
medical conditions with particular markers, typically antigenic or
genetic. Tumor specific antigens will be identified using the
present invention.
[0615] Various medical conditions may be correlated against an
enormous data base of the sequences within an individual. Genetic
propensities and correlations then become available and high
resolution genetic predictability and correlation become much more
easily performed. With the enormous data base, the reliability of
the predictions is also better tested. Particular markers which are
partially diagnostic of particular medical conditions or medical
susceptibilities will be identified and provide direction in
further studies and more careful analysis of the markers involved.
Of course, as indicated above in the sequencing embodiment, the
present invention will find much use in intense sequencing
projects. For example, sequencing of the entire human genome in the
human genome project will be greatly simplified and enabled by the
present invention.
[0616] VI. Formation of Substrate
[0617] The substrate is provided with a pattern of specific
reagents which are positionally localized on the surface of the
substrate. This matrix of positions is defined by the automated
system which produces the substrate. The instrument will typically
be one similar to that described in Pirrung et al. (1992) U.S. Pat.
No. 5,143,854, and U.S. Pat. No. 5,489,678. The instrumentation
described therein is directly applicable to the applications used
here. In particular, the apparatus comprises a substrate, typically
a silicon containing substrate, on which positions on the surface
may be defined by a coordinate system of positions. These positions
can be individually addressed or detected by the VLSIPS.TM.
Technology apparatus.
[0618] Typically, the VLSIPS.TM. Technology apparatus uses optical
methods used in semiconductor fabrication applications. In this
way, masks may be used to photo-activate positions for attachment
or synthesis of specific sequences on the substrate. These
manipulations may be automated by the types of apparatus described
in Pirrung et al. (1992) U.S. Pat. Nos. 5,143,854 and
5,489,678.
[0619] Selectively removable protecting groups allow creation of
well defined areas of substrate surface having differing
reactivities. Preferably, the protecting groups are selectively
removed from the surface by applying a specific activator, such as
electromagnetic radiation of a specific wavelength and intensity.
More preferably, the specific activator exposes selected areas of
surface to remove the protecting groups in the exposed areas.
[0620] Protecting groups of the present invention are used in
conjunction with solid phase oligomer syntheses, such as peptide
syntheses using natural or unnatural amino acids, nucleotide
syntheses using deoxyribonucleic and ribonucleic acids,
oligosaccharide syntheses, and the like. In addition to protecting
the substrate surface from unwanted reaction, the protecting groups
block a reactive end of the monomer to prevent self-polymerization.
For instance, attachment of a protecting group to the amino
terminus of an activated amino acid, such as the
N-hydroxysuccinimide-activated ester of the amino acid prevents the
amino terminus of one monomer from reacting with the activated
ester portion of another during peptide synthesis.
[0621] Alternatively, the protecting group may be attached to the
carboxyl group of an amino acid to prevent reaction at this site.
Most protecting groups can be attached to either the amino or the
carboxyl group of an amino acid, and the nature of the chemical
synthesis will dictate which reactive group will require a
protecting group. Analogously, attachment of a protecting group to
the 5'-hydroxyl group of a nucleoside during synthesis using for
example, phosphate-triester coupling chemistry, prevents the
5'-hydroxyl of one nucleoside from reacting with the 3'-activated
phosphate-triester of another.
[0622] Regardless of the specific use, protecting groups are
employed to protect a moiety on a molecule from reacting with
another reagent. Protecting groups of the present invention have
the following characteristics: they prevent selected reagents from
modifying the group to which they are attached; they are stable
(that is, they remain attached) to the synthesis reaction
conditions; they are removable under conditions that do not
adversely affect the remaining structure; and once removed, do not
react appreciably with the surface or surface-bound oligomer. The
selection of a suitable protecting group will depend, of course, on
the chemical nature of the monomer unit and oligomer, as well as
the specific reagents they are to protect against.
[0623] In a preferred embodiment, the protecting groups will be
photoactivatable. The properties and uses of photoreactive
protecting compounds have been reviewed. See, McCray et al., Ann.
Rev. of Biophys. and Biophys. Chem., (1989) 18: 239-270, which is
incorporated herein by reference. Preferably, the photosensitive
protecting groups will be removable by radiation in the ultraviolet
(UV) or visible portion of the electromagnetic spectrum. More
preferably, the protecting groups will be removable by radiation in
the near UV or visible portion of the spectrum. In some
embodiments, however, activation may be performed by other methods
such as localized heating, electron beam lithography, laser
pumping, oxidation or reduction with microelectrodes, and the like.
Sulfonyl compounds are suitable reactive groups for electron beam
lithography. Oxidative or reductive removal is accomplished by
exposure of the protecting group to an electric current source,
preferably using microelectrodes directed to the predefined regions
of the surface which are desired for activation. A more detailed
description of these protective groups is provided in U.S. Pat. No.
5,489,678, which is hereby incorporated herein by reference.
[0624] The density of reagents attached to a silicon substrate may
be varied by standard procedures. The surface area for attachment
of reagents may be increased by modifying the silicon surface. For
example, a matte surface may be machined or etched on the substrate
to provide more sites for attachment of the particular reagents.
Another way to increase the density of reagent binding sites is to
increase the derivitization density of the silicon. Standard
procedures for achieving this are described, below.
[0625] One method to control the derivatization density is to
highly derivatize the substrate with photochemical groups at high
density. The substrate is then photolyzed for various predetermined
times, which photoactivate the groups at a measurable rate, and
react them with a capping reagent. By this method, the density of
linker groups may be modulated by using a desired time and
intensity of photoactivation.
[0626] In many applications, the number of different sequences
which may be provided may be limited by the density and the size of
the substrate on which the matrix pattern is generated. In
situations where the density is insufficiently high to allow the
screening of the desired number of sequences, multiple substrates
may be used to increase the number of sequences tested. Thus, the
number of sequences tested may be increased by using a plurality of
different substrates. Because the VLSIPS.TM. Technology apparatus
is almost fully automated, increasing the number of substrates does
not lead to a significant increase in the number of manipulations
which must be performed by humans. This again leads to greater
reproducibility and speed in the handling of these multiple
substrates.
[0627] A. Instrumentation
[0628] The concept of using, VLSIPS.TM. Technology generally allows
a pattern or a matrix of reagents to be generated. The procedure
for making the pattern is performed by any of a number of different
methods. An apparatus and instrumentation useful for generating a
high density VLSIPS.TM. Technology substrate is described in detail
in Pirrung et al. (1992) U.S. Pat. Nos. 5,143,854 and
5,489,678.
[0629] B. Binary Masking
[0630] The details of the binary masking are described in an
accompanying application filed simultaneously with this, U.S. Pat.
No. 5,489,678, whose specification is incorporated herein by
reference.
[0631] For example, the binary masking technique allows for
producing a plurality of sequences based on the selection of either
of two possibilities at any particular location. By a series of
binary masking steps, the binary decision may be the determination,
on a particular synthetic cycle, whether or not to add any
particular one of the possible subunits. By treating various
regions of the matrix pattern in parallel, the binary masking
strategy provides the ability to carry out spatially addressable
parallel synthesis.
[0632] C. Synthetic Methods
[0633] The synthetic methods in making a substrate are described in
the parent application, which issued as U.S. Pat. No. 5,143,854.
The construction of the matrix pattern oil the substrate will
typically be generated by the use of photo-sensitive reagents. By
use of photolithographic optical methods, particular segments of
the substrate an be irradiated with light to activate or deactivate
blocking, agents, e.g., to protect or deprotect particular chemical
groups. By an appropriate sequence of photo-exposure steps at
appropriate times with appropriate masks and with appropriate
reagents, the substrates can have known polymers synthesized at
positionally defined regions on the substrate. Methods for
synthesizing various substrates are described in Pirrung et al.
(1992) U.S. Pat. Nos. 5,143,854 and 5,489,678. By a sequential
series of these photo-exposure and reaction manipulations, a
defined matrix pattern of known sequences may be generated, and is
typically referred to as a VLSIPS.TM. Teleology substrate. In the
nucleic acid synthesis embodiment, nucleosides used in the
synthesis of DNA by photolytic methods will typically be one of the
two forms shown below: 24
[0634] In I, the photolabile group at the 5' position is
abbreviated NV (nitroveratryl) and in II, the group is abbreviated
NVOC (nitroveratryl oxycarbonyl). Although not shown in FIG. C the
bases (adenine, cytosine, and guanine) contain exocyclic NH.sub.2
groups which must be protected during DNA synthesis, Thymine
contains no exocyclic NH.sub.2 and therefore requires no
protection. The standard protecting groups for these amines are
shown below: 25
[0635] Guanine (G).
[0636] Other amides of the general formula: 26
[0637] where R may be alkyl or aryl have been used.
[0638] Another type of protecting group FMOC (9-fluorenyl
methoxycarbonyl) is currently being used to protect the exocyclic
amines of the three bases: 27
[0639] The advantage of the FMOC group is that it is removed under
mild conditions (dilute organic bases) and can be used for all
three bases. The amide protecting groups require more harsh
conditions to be removed (N.sub.3/MeOH with heat).
[0640] Nucleosides used as 5'-OH probes, useful in verifying
correct VLSIPS.TM. Technology synthetic function, include, for
example the following: 28
[0641] These compounds are used to detect where on a substrate
photolysis has occurred by the attachment of either III or V to the
newly generated 5'-OH. In the case of III, after the phosphate
attachment is made, the substrate is treated with a dilute base to
remove the FMOC group. The resulting amine can be reacted with FITC
and the substrate examined by fluorescence microscopy. This
indicates the proper generation of a 5'-OH. In the case of compound
IV, after the phosphate attachment is made, the substrate is
treated with FITC labeled streptavidin and the substrate again may
be examined by fluorescence microscopy. Other probes, although not
nucleoside based, have included the following: 29
[0642] The method of attachment of the first nucleoside to the
surface of the substrate depends on the functionality of the groups
at the substrate surface. If the surface is amine functionalized,
an amide bond is made (see example below). 30
[0643] If the surface is hydroxy functionalized, a phosphate bond
is made (see example below): 31
[0644] In both cases, the thymidine example is illustrated, but any
one of the four phosphoramidite activated nucleosides can be used
in the first step.
[0645] Photolysis of the photolabile group NV or NVOC on the
5'-positions of the nucleosides is carried out at 362 nm with an
intensity of 14 mW/cm.sup.2 for 10 minutes with the substrate side
(side containing the photolabile group) immersed in dioxane. After
the coupling of the next nucleoside is complete, the photolysis is
repeated followed by another coupling until the desired oligomer is
obtained.
[0646] One of the most common 3'-O-protecting groups is the ester,
in particular the acetate: 32
[0647] The groups can be removed by mild base treatment 0.1 N
NaOH/MeOH or K.sub.2CO.sub.3/H.sub.2O/MeOH.
[0648] Another group used most often is the silyl ether: 33
[0649] These groups can be removed by neutral conditions using 1 M
tetra-n-butylammonium fluoride in THF or under acid conditions.
[0650] With respect to photodeprotection, the nitroveratryl group
could also be used to protect the 3'-position. 34
[0651] Here, light (photolysis) would be used to remove these
protecting groups. A variety of ethers can also be used in the
protection of the 3'-O-position: 35
[0652] Removal of these groups usually involves acid or catalytic
methods.
[0653] Note that corresponding linkages and photoblocked amino
acids are described in detail in U.S. Pat. No. 5,489,678, which is
hereby incorporated herein by reference.
[0654] Although the specificity of interactions at particular
locations will usually be homogeneous due to a homogeneous polymer
being synthesized at each defined location, for certain purposes,
it may be useful to have mixed polymers with a commensurate mixed
collection of interactions occurring at specific defined locations,
or degeneracy reducing analogues, which have been discussed above
and show broad specificity in binding. Then, a positive interaction
signal may result from any of a number of sequences contained
therein.
[0655] As an alternative method of generating a matrix pattern on a
substrate, preformed polymers may be individually attached at
particular sites on the substrate. This may be performed by
individually attaching reagents one at a time to specific positions
on the matrix, a process which may be automated. See, e.g., U.S.
Ser. No. 07/435,316, from which CIP U.S. Ser. No. 07/612,671 issued
as U.S. Pat. No. 5,252,743. Another way of generating a
positionally defined matrix pattern on a substrate is to have
individually specific reagents which interact with each specific
position on the substrate. For example, oligonucleotides may be
synthesized at defined locations on the substrate. Then the
substrate would have on its surface a plurality of regions having
homogeneous oligonucleotides attached at each position.
[0656] In particular, at least four different substrate preparation
procedures are available for treating a substrate surface. They are
the standard VLSIPS.TM. Technology method, polymeric substrates,
Durapore.TM., and synthetic beads or fibers. The treatment labeled
"standard VLSIPS.TM. Technology" method is described in U.S. Pat.
No. 5,489,678, and involves applying amino-propyltriethoxysilane to
a glass surface.
[0657] The polymeric substrate approach involves either of two ways
of generating a polymeric substrate. The first uses a high
concentration of aminopropyltriethoxysilane (2-20%) in an aqueous
ethanol solution (95%). This allows the silane compound to
polymerize both in solution and on the substrate surface, which
provides a high density of amines on the surface of the glass. This
density is contrasted with the standard VLSIPS.TM. Technology
method. This polymeric method allows for the deposition on the
substrate surface of a monolayer due to the anhydrous method used
with the aforementioned silane.
[0658] The second polymeric method involves either the coating or
covalent binding of an appropriate acrylic acid polymer onto the
substrate surface. In particular, e.g., in DNA synthesis, a monomer
such as a hydroxypropylacrylate is used to generate a high density
of hydroxyl groups on the substrate surface, allowing for the
formation of phosphate bonds. An example of such a compound is
shown: 36
[0659] The method using a Durapore.TM. membrane (Millipore)
consists of a polyvinylidine difluoride coating with crosslinked
polyhydroxylpropyl acrylate [PVDF-HPA]: 37
[0660] Here the building up of, e.g., a DNA oligomer, can be
started immediately since phosphate bonds to the surface can be
accomplished in the first step with no need for modification. A
nucleotide dimer (5'-C-T-3') has been successfully made on this
substrate. A nucleotide dimer (5'-C-T-3') has been successfully
made on this substrate.
[0661] The fourth method utilizes synthetic beads or fibers. This
would use another substrate, such as a teflon copolymer graft bead
or fiber, which is covalently coated with an organic layer
(hydrophilic) terminating in hydroxyl sites (commercially available
from Molecular Biosystems, Inc.) This would offer the same
advantage as the Durapore.TM. membrane, allowing for immediate
phosphate linkages, but would give additional contour by the
3-dimensional growth of oligomers.
[0662] A matrix pattern of new reagents may be targeted to each
specific oligonucleotide position by attaching a complementary
oligonucleotide to which the substrate bound form is complementary.
For instance, a number of regions may have homogeneous
oligonucleotides synthesized at various locations. Oligonucleotide
sequences complementary to each of these can be individually
generated and linked to a particular specific reagents. Often these
specific reagents will be antibodies. As each of these is specific
for finding its complementary oligonucleotide, each of the specific
reagents will bind through the oligonucleotide to the appropriate
matrix position. A single step having a combination of different
specific reagents being attached specifically to a particular
oligonucleotide will thereby bind to its complement at the defined
matrix position. The oligonucleotides will typically then be
covalently attached, using, e.g., an acridine dye, for
photocrosslinking. Psoralen is a commonly used acridine dye for
photocrosslinking purposes, see, e.g., Song et al. (1979)
Photochem. Photobiol., 29: 1177-1197; Cimino et al. (1985) Ann.
Rev. Biochem, 54: 115-1193; Parsons (1980) Photochem. Photobiol.
32: 813-821; and Dattagupta et al. (1985) U.S. Pat. No. 4,542,102,
and (1987) U.S. Pat. No. 4,713,326; each of which is hereby
incorporated herein by reference. This method allows a single
attachment manipulation to attach all of the specific reagents to
the matrix at defined positions and results in the specific
reagents being homogeneously located at defined positions. In many
embodiments, the specific reagents will be antibodies.
[0663] In an alternative embodiment, antibody molecules may be used
to specifically direct binding to defined positions on a substrate.
The VLSIPS Technology may be used to generate specific epitopes at
each position on the substrate. Antibody molecules having
specificity of interaction may be used to attach oligonucleotides,
thereby avoiding the interference of internal polynucleotide
sequences from binding to the substrate complementary
oligonucleotides. In fact, the specificity of interaction for
positional targeting may be achieved by use of nucleotide analogues
which do not interact with the natural nucleotides. For example,
other synthetic nucleotides have been made which undergo base
pairing, thereby providing the specificity of targeting, but the
synthetic nucleotides also do not interact with the natural
biological nucleotides. Thus, synthetic oligonucleotides would be
useful for attachment to biological nucleotides and specific
targeting. Moreover, the VLSIPS synthetic processes would be useful
in generating the VLSIPS substrate, and standard oligonucleotide
synthesis could be applied, with minor modifications, to produce
the complementary sequences which would be attached to other
specific reagents.
[0664] D. Surface Immobilization
[0665] 1. Caged Biotin
[0666] An alternative method of attaching reagents in a
positionally defined matrix pattern is to use a caged biotin
system. See Barrett et al. (1993) U.S. Pat. No. 5,252,743, which is
hereby incorporated herein by reference, for additional details on
the chemistry and application of caged biotin embodiments. In
short, the caged biotin has a photosensitive blocking moiety which
prevents the combination of avidin to biotin. At positions where
the photo-lithographic process has removed the blocking group, high
affinity biotin sites are generated. Thus, by a sequential series
of photolithographic deblocking steps interspersed with exposure of
those regions to appropriate biotin containing reagents, only those
locations where the deblocking takes place will form an
avidin-biotin interaction. Because the avidin-biotin binding is
very tight, this will usually be virtually irreversible
binding.
[0667] 2. Crosslinked Interactions
[0668] The surface immobilization may also take place by photo
crosslinking of defined oligonucleotides linked to specific
reagents. After hybridization of the complementary
oligonucleotides, the oligonucleotides may be crosslinked by a
reagent by psoralen or another similar type of acridine dye. Other
useful cross linking reagents are described in Dattagupta et al.
(1995) U.S. Pat. No. 4,542,102, and (1987) U.S. Pat. No.
4,713,326.
[0669] In another embodiment, colony or phage plaque transfer of
biological polymers may be transferred directly onto a silicon
substrate. For example, a colony plate may be transferred onto a
substrate having a generic oligonucleotide sequence which
hybridizes to another generic complementary sequence contained on
all of the vectors into which inserts are cloned. This will
specifically only bind those molecules which are actually contained
in the vectors containing the desired complementary sequence. This
immobilization allows for producing a matrix onto which a sequence
specific reagent can bind, or for other purposes. In a further
embodiment, a plurality of different vectors each having a specific
oligonucleotide attached to the vector may be specifically attached
to particular regions on a matrix having a complementary
oligonucleotide attached thereto.
[0670] VIII. Hybridization/Specific Interaction
[0671] A. General
[0672] As discussed previously in the VLSIPS.TM. Technology parent
applications, the VLSIPS.TM. Technology substrates may be used for
screening for specific interactions with sequence specific targets
or probes.
[0673] In addition, the availability of substrates having the
entire repertoire of possible sequences of a defined length opens
up the possibility of sequencing by hybridization. This sequence
may be de novo determination of an unknown sequence, particularly
of nucleic acid, verification of a sequence determined by another
method, or an investigation of changes in a previously sequenced
gene, locating and identifying specific changes. For example, often
Maxam and Gilbert sequencing techniques are applied to sequences
which have been determined by Sanger and Coulson. Each of those
sequencing technologies have problems with resolving particular,
types of sequences. Sequencing by hybridization may serve as a
third and independent method for verifying other sequencing
techniques. See, e.g., (1988) Science, 242: 1245.
[0674] In addition, the ability to provide a large repertoire of
particular sequences allows use of short subsequence and
hybridization as a means to fingerprint a sample. This may be used
in a nucleic acid, as well as other polymer embodiments. For
example, fingerprinting to a high degree of specificity of sequence
matching may be used for identifying highly similar samples, e.g.,
those exhibiting high homology to the selected probes. This may
provide a means for determining classifications of particular
sequences. This should allow determination of whether particular
genomes of bacteria, phage, or even higher cells might be related
to one another.
[0675] In addition, fingerprinting may be used to identify an
individual source of biological sample. See, e.g., Lander, E.
(1989) Nature, 339: 501-505, and references therein. For example, a
DNA fingerprint may be used to determine whether a genetic sample
arose from another individual. This would be particularly useful in
various sorts of forensic tests to determine, e.g., paternity or
sources of blood samples. Significant detail on the particulars of
genetic fingerprinting for identification purposes are described
in, e.g., Morris et al. (1989) "Biostatistical evolution of
evidence from continuous allele frequency distribution DNA probes
in reference to disputed paternity of identity," J. Forensic
Science, 34: 1311-1317; and Neufeld et al. (1990) Scientific
American, 262: 46-53; each of which is hereby incorporated herein
by reference.
[0676] In another embodiment, a fingerprinting-like procedure may
be used for classifying cell types by analyzing a pattern of
specific nucleic acids present in the cell. A series of antibodies
may be used to identify cell markers, e.g., proteins, usually on
the cell surface, but intracellular markers may also be used.
Antigens which are extracellularly expressed are preferred so cell
lysis is unnecessary in the screening, but intracellular markers
may also be useful. The markers will usually be proteins, but may
be nucleic acids, lipids, metabolites, carbohydrates, or other
cellular components. See, e.g., Winkelgren, I. (1990) Science News,
136: 234-237, which indicates extracellular DNA may be common, and
suggesting that such might be characteristic of cell types, stage,
or physiology. This may also be useful in defining the temporal
stage of development of cells, e.g., stem cells or other cells
which undergo temporal changes in development. For example, the
stage of a cell, or group of cells, may be tested or defined by
isolating a sample of mRNA from the population and testing to see
what sequences are present in messenger populations. Direct
samples, or amplified samples, may be used. Where particular mRNA
or other nucleic acid sequences may be characteristic of or shown
to be characteristic of particular developmental stages,
physiological states, or other conditions, this fingerprinting
method may define them. Similar sorts of fingerprinting may be used
for determining T-cell classes or perhaps even to generate
classification schemes for such proteins as major
histocompatibility complex antigens. Thus, the ability to make
these substrates allows both the generation of reagents which will
be used for defining subclasses or classes of cells or other
biological materials, but also provides the mechanisms for
selecting those cells which may be found in defined population
groups.
[0677] In addition to cell classification defined by such a
combination of properties, typically expression of extracellular
antigens, the present invention also provides the means for
isolating homogeneous population of cells. Once the antigenic
determinants which define a cell class have been identified, these
antigens may be used in a sequential selection process to isolate
only those cells which exhibit the combination of defining
structural properties.
[0678] The present invention may also be used for mapping sequences
within a larger segment. This may be performed by at least two
methods, particularly in reference to nucleic acids. Often,
enormous segments of DNA are subcloned into a large plurality of
subsequences. Ordering these subsequences may be important in
determining the overlaps of sequences upon nucleotide
determinations. Mapping may be performed by immobilizing
particularly large segments onto a matrix using the VLSIPS.TM.
Technology. Alternatively, sequences may be ordered by virtue of
subsequences shared by overlapping segments. See, e.g., Craig et
al. (1990) Nuc. Acids Res., 18: 2653-2660; Michiels et al. (1987)
CABIOS, 3: 203-210; and Olson et al. (1986) Proc. Natl. Acad. Sci.
USA, 83: 7826-7830.
[0679] B. Important Parameters
[0680] The extent of specific interaction between reagents
immobilized to the VLSIPS.TM. Technology substrate and another
sequence specific reagent may be modified by the conditions of the
interaction. Sequencing embodiments typically require high fidelity
hybridization and the ability to discriminate perfect matching from
imperfect matching. Fingerprinting and mapping embodiments may be
performed using less stringent conditions, depending upon the
circumstances.
[0681] For example, the specificity of antibody/antigen interaction
may depend upon such parameters as pH, salt concentration, ionic
composition, solvent composition, detergent composition and
concentration, and chaotropic agent concentration. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Press, New York. By careful control of these parameters, the
affinity of binding may be mapped across different sequences.
[0682] In a nucleic acid hybridization embodiment, the specificity
and kinetics of hybridization have been described in detail by,
e.g., Wetmur and Davidson (1968) J. Mol. Biol., 31: 349-370,
Britten and Kohne (1968) Science, 161: 529-530, and Kanehisa,
(1984) Nuc. Acids Res., 12: 203-213, each of which is hereby
incorporated herein by reference. Parameters which are well known
to affect specificity and kinetics of reaction include salt
conditions, ionic composition of the solvent, hybridization
temperature, length of oligonucleotide matching sequences, guanine
and cytosine (GC) content, presence of hybridization accelerators,
pH, specific bases found in the matching sequences, solvent
conditions, and addition of organic solvents.
[0683] In particular, the salt conditions required for driving
highly mismatched sequences to completion typically include a high
salt concentration. The typical salt used is sodium chloride
(NaCl), however, other ionic salts may he utilized, e.g., KCl.
Depending on the desired stringency hybridization, the salt
concentration will often be less than about 3 molar, more often
less than 2.5 molar, usually less than about 2 molar, and more
usually less than about 1.5 molar. For applications directed
towards higher stringency matching, the salt concentrations would
typically be lower. Ordinary high stringency conditions will
utilize salt concentration of less than about 1 molar, more often
less then about 750 millimolar, usually less than about 500
millimolar, and may be as low as about 250 or 150 millimolar.
[0684] The kinetics of hybridization and the stringency of
hybridization both depend upon the temperature at which the
hybridization is performed and the temperature at which the washing
steps are performed. Temperatures at which steps for low stringency
hybridization are desired would typically be lower temperatures,
e.g., ordinarily at least about 15.degree. C., more ordinarily at
least about 20.degree. C., usually at least about 25.degree. C.,
and more usually at least about 30.degree. C. For those
applications requiring high stringency hybridization, or fidelity
of hybridization and sequence matching, temperatures at which
hybridization and washing steps are performed would typically be
high. For example, temperatures in excess of about 35.degree. C.
would often be used, more often in excess of about 40.degree. C.,
usually at least about 45.degree. C., and occasionally even
temperatures as high as about 50.degree. C. or 60.degree. C. or
more. Of course, the hybridization of oligonucleotides may be
disrupted by even higher temperatures. Thus, for stripping of
targets from substrates, as discussed below, temperatures as high
as 80.degree. C., or even higher may be used.
[0685] The base composition of the specific oligonucleotides
involved in hybridization affects the temperature of melting, and
the stability of hybridization as discussed in the above
references. However, the bias of GC rich sequences to hybridize
faster and retain stability at higher temperatures can be
compensated for by the inclusion in the hybridization incubation or
wash steps of various buffers. Sample buffers which accomplish this
result include the triethly-and trimethyl ammonium buffers. See,
e.g., Wood et al. (1987) Proc. Natl. Acad. Sci. USA, 82: 1585-1588,
and Khrapko, K. et al. (1989) FEBS Letters, 256: 118-122.
[0686] The rate of hybridization can also be affected by the
inclusion of particular hybridization accelerators. These
hybridization accelerators include the volume exclusion agents
characterized by dextran sulfate, or polyethylene glycol (PEG).
Dextran sulfate is typically included at a concentration of between
1% and 40% by weight. The actual concentration selected depends
upon the application, but typically a faster hybridization is
desired in which the concentration is optimized for the system in
question. Dextran sulfate is often included at a concentration of
between 0.5% and 2% by weight or dextran sulfate at a concentration
between about 0.5% and 5%. Alternatively, proteins which accelerate
hybridization may be added, e.g., the recA protein found in E. coli
or other homologous proteins.
[0687] With respect to those embodiments where specific reagents
are not oligonucleotides, the conditions of specific interaction
would depend on the affinity of binding between the specific
reagent and its target. Typically parameters which would be of
particular importance would be pH, salt concentration anion and
cation compositions, buffer concentration, organic solvent
inclusion, detergent concentration, and inclusion of such reagents
such as chaotropic agents. In particular, the affinity of binding
may be tested over a variety of conditions by multiple washes and
repeat scans or by using reagents with differences in binding
affinity to determine which reagents bind or do not bind under the
selected binding and washing conditions. The spectrum of binding
affinities may provide an additional dimension of information which
may be very useful in identification purposes and mapping.
[0688] Of course, the specific hybridization conditions will be
selected to correspond to a discriminatory condition which provides
a positive signal where desired but fails to show a positive signal
at affinities where interaction is not desired. This may be
determined by a number of titration steps or with a number of
controls which will be run during the hybridization and/or washing
steps to determine at what point the hybridization conditions have
reached the stage of desired specificity.
[0689] IX. Detection Methods
[0690] Methods for detection depend upon the label selected. The
criteria for selecting an appropriate label are discussed below,
however, a fluorescent label is preferred because of its extreme
sensitivity and simplicity. Standard labeling procedures are used
to determine the positions where interactions between a sequence
and a reagent take place. For example, if a target sequence is
labeled and exposed to a matrix of different probes, only those
locations where probes do interact with the target will exhibit any
signal. Alternatively, other methods may be used to scan the matrix
to determine where interaction takes place. Of course, the spectrum
of interactions may be determined in a temporal manner by repeated
scans of interactions which occur at each of a multiplicity of
conditions. However, instead of testing each individual interaction
separately, a multiplicity of sequence interactions may be
simultaneously determined on a matrix.
[0691] A. Labeling Techniques
[0692] The target polynucleotide may be labeled by any of a number
of convenient detectable markers. A fluorescent label is preferred
because it provides a very strong signal with low background. It is
also optically detectable at high resolution and sensitivity
through a quick scanning procedure. Other potential labeling
moieties include radioisotopes, chemiluminescent compounds, labeled
binding proteins, heavy metal atoms, spectroscopic markers,
magnetic labels, and linked enzymes.
[0693] Another method for labeling may bypass any label of the
target sequence. The target may be exposed to the probes, and a
double strand hybrid is formed at those positions only. Addition of
a double strand specific reagent will detect where hybridization
takes place. An intercalative dye such as ethidium bromide may be
used as long as the probes themselves do not fold back on
themselves to a significant extent forming hairpin loops. See,
e.g., Sheldon et al. (1986) U.S. Pat. No. 4,582,789. However, the
length of the hairpin loops in short oligonucleotide probes would
typically be insufficient to form a stable duplex.
[0694] In another embodiment, different targets may be
simultaneously sequenced where each target has a different label.
For instance, one target could have a green fluorescent label and a
second target could have a red fluorescent label. The scanning step
will distinguish sites of binding of the red label from those
binding the green fluorescent label. Each sequence can be analyzed
independently from one another.
[0695] Suitable chromogens will include molecules and compounds
which absorb light in a distinctive range of wavelengths so that a
color may be observed, or emit light when irradiated with radiation
of a particular wave length or wave length range, e.g.,
fluorescers. Biliproteins, e.g., phycoerythrin, may also serve as
labels.
[0696] A wide variety of suitable dyes are available, being
primarily chosen to provide an intense color with minimal
absorption by their surroundings. Illustrative dye types include
quinoline dyes, triarylmethane dyes, acridine dyes, alizarine dyes,
phthaleins, insect dyes, azo dyes, anthraquinoid dyes, cyanine
dyes, phenazathionium dyes, and phenazoxonium dyes.
[0697] A wide variety of fluorescers may be employed either by
themselves or in conjunction with quencher molecules. Fluorescers
of interest fall into a variety of categories having certain
primary functionalities. These primary functionalities include 1-
and 2-aminonaphthalene, p,p'-diaminostilbenes, pyrenes, quaternary
phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone
imines, anthracenes, oxacarbocyanine, merocyanine,
3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl
benzene, 1,2-benzophenazin, retinol, bis-3-aminopyridinium salts,
hellebrigenin, tetracycline, sterophenol,
benzimidzaolylphenylamine, 2-oxo-3-chromen, indole, xanthen,
7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin,
porphyrins, triarylmethanes and flavin. Individual fluorescent
compounds which have functionalities for linking or which can be
modified to incorporate such functionalities include, e.g., dansyl
chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol;
rhodamineisothiocyanate; N-phenyl-1-amino-8-sulfonatonaphthalene;
N-phenyl-2-amino-6-sulfonatonaph- thalene;
4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid;
pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;
N-phenyl, N-methyl-2-aminonaphthalene-6-sulfonate; ethidium
bromide; stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl
phosphatidylethanolamine; N,N'-dioctadecyl oxacarbocyanine;
N,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)butyrate;
d-3-aminodesoxy-equilenin; 12-(9'-anthroyl)stearate;
2-methylanthracene; 9-vinylanthracene;
2,2'-(vinylene-p-phenylene)bisbenzoxazole;
p-bis[2-(4-methyl-5-phenyl-oxa- zolyl)]benzene;
6-dimethylamino-1,2-benzophenazin; retinol; bis(3'-aminopyridinium)
1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;
chlorotetracycline; N-(7-dimethylamino-4-methyl-2-oxo-3--
chromenyl)maleimide; N-[p-(2-benzimidazolyl)-phenyl]maleimide;
N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin;
4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin;
rose bengal; and 2,4-diphenyl-3(2H)-furanone.
[0698] Desirably, fluorescers should absorb light above about 300
nm, preferably about 350 nm, and more preferably above about 400
nm, usually emitting at wavelengths greater than about 10 nm higher
than the wavelength of the light absorbed. It should be noted that
the absorption and emission characteristics of the bound dye may
differ from the unbound dye. Therefore, when referring to the
various wavelength ranges and characteristics of the dyes, it is
intended to indicate the dyes as employed and not the dye which is
unconjugated and characterized in an arbitrary solvent.
[0699] Fluorescers are generally preferred because by irradiating a
fluorescer with light, one can obtain a plurality of emissions.
Thus, a single label can provide for a plurality of measurable
events.
[0700] Detectable signal may also be provided by chemiluminescent
and bioluminescent sources. Chemiluminescent sources include a
compound which becomes electronically excited by a chemical
reaction and may then emit light which serves as the detectible
signal or donates energy to a fluorescent acceptor. A diverse
number of families of compounds have been found to provide
chemiluminescence under a variety of conditions. One family of
compounds is 2,3-dihydro-1,-4-phthalazinedione. The most popular
compound is luminol, which is the 5-amino compound. Other members
of the family include the 5-amino-6,7,8-trimethoxy- and the
dimethylamino[ca]benz analog. These compounds can be made to
luminesce with alkaline hydrogen peroxide or calcium hypochlorite
and base. Another family of compounds is the
2,4,5-triphenylimidazoles, with lophine as the common name for the
parent product. Chemiluminescent analogs include para-dimethylamino
and -methoxy substituents. Chemiluminescence may also be obtained
with oxalates, usually oxalyl active esters, e.g., p-nitrophenyl
and a peroxide, e.g., hydrogen peroxide, under basic conditions.
Alternatively, luciferins may be used in conjunction with
luciferase or lucigenins to provide bioluminescence.
[0701] Spin labels are provided by reporter molecules with an
unpaired electron spin which can be detected by electron spin
resonance (ESR) spectroscopy. Exemplary spin labels include organic
free radicals, transitional metal complexes, particularly vanadium,
copper, iron, and manganese, and the like. Exemplary spin labels
include nitroxide free radicals.
[0702] B. Scanning System
[0703] With the automated detection apparatus, the correlation of
specific positional labeling is converted to the presence on the
target of sequences for which the reagents have specificity of
interaction. Thus, the positional information is directly converted
to a database indicating what sequence interactions have occurred.
For example, in a nucleic acid hybridization application, the
sequences which have interacted between the substrate matrix and
the target molecule can be directly listed from the positional
information. The detection system used is described in Pirrung et
al. (1992) U.S. Pat. Nos. 5,143,854; and 5,489,678. Although the
detection described therein is a fluorescence detector, the
detector may be replaced by a spectroscopic or other detector. The
scanning system may make use of a moving detector relative to a
fixed substrate, a fixed detector with a moving substrate, or a
combination. Alternatively, mirrors or other apparatus can be used
to transfer the signal directly to the detector. See, e.g., U.S.
Pat. No. 5,489,678, which is hereby incorporated herein by
reference.
[0704] The detection method will typically also incorporate some
signal processing to determine whether the signal at a particular
matrix position is a true positive or may, be a spurious signal.
For example, a signal from a region which has actual positive
signal may tend to spread over and provide a positive signal in an
adjacent region which actually should not have one. This may occur,
e.g., where the scanning system is not properly discriminating with
sufficiently high resolution in its pixel density to separate the
two regions. Thus, the signal over the spatial region may be
evaluated pixel by pixel to determine the locations and the actual
extent of positive signal. A true positive signal should, in
theory, show a uniform signal at each pixel location. Thus,
processing by plotting number of pixels with actual signal
intensity should have a clearly uniform signal intensity. Regions
where the signal intensities show a fairly wide dispersion may be
particularly suspect and the scanning system may be programmed to
more carefully scan those positions.
[0705] In another embodiment, as the sequence of a target is
determined at a particular location, the overlap for the sequence
would necessarily have a known sequence. Thus, the system can
compare the possibilities for the next adjacent position and look
at these in comparison with each other. Typically, only one of the
possible adjacent sequences should give a positive signal and the
system might be programmed to compare each of these possibilities
and select that one which gives a strong positive. In this way, the
system can also simultaneously provide some means of measuring the
reliability of the determination by indicating what the average
signal to background ratio actually is.
[0706] More sophisticated signal processing techniques can be
applied to the initial determination of whether a positive signal
exists or not. See, e.g., U.S. Pat. No. 5,489,678.
[0707] From a listing of those sequences which interact, data
analysis may be performed on a series of sequences. For example, in
a nucleic acid sequence application, each of the sequences may be
analyzed for their overlap regions and the original target sequence
may be reconstructed from the collection of specific subsequences
obtained therein. Other sorts of analyses for different
applications may also be performed, and because the scanning system
directly interfaces with a computer the information need not be
transferred manually. This provides for the ability to handle large
amounts of data with very little human intervention. This, of
course, provides significant advantages over manual manipulations.
Increased throughput and reproducibility is thereby provided by the
automation of a vast majority of steps in any of these
applications.
[0708] XI. Data Analysis
[0709] A. General
[0710] Data analysis will typically involve aligning the proper
sequences with their overlaps to determine the target sequence.
Although the target "sequence" may not specifically correspond to
any specific molecule, especially where the target sequence is
broken and fragmented in the sequencing process, the sequence
corresponds to a contiguous sequence of the subfragments.
[0711] The data analysis can be performed by a computer using an
appropriate program. See, e.g., Drmanac, R. et al. (1989) Genomics,
4: 114-128; and a commercially available analysis program available
from the Genetic Engineering Center, P.O. Box 794, 11000 Belgrade,
Yugoslavia. Although the specific manipulations necessary to
reassemble the target sequence from fragments may take many forms,
one embodiment uses a sorting program to sort all of the
subsequences using a defined hierarchy. The hierarchy need not
necessarily correspond to any physical hierarchy, but provides a
means to determine, in order, which subfragments have actually been
found in the target sequence. In this manner, overlaps can be
checked and found directly rather than having to search throughout
the entire set after each selection process. For example, where the
oligonucleotide probes are 10-mers, the first 9 positions can be
sorted. A particular subsequence can be selected as in the
examples, to determine where the process starts. As analogous to
the theoretical example provided above, the sorting procedure
provides the ability to immediately find the position of the
subsequence which contains the first 9 positions and can compare
whether there exists more than 1 subsequence during the first 9
positions. In fact, the computer can easily generate all of the
possible target sequences which contain given combination of
subsequences. Typically there will be only one, but in various
situations, there will be more.
[0712] An exemplary flow chart for a sequencing program is provided
in FIG. 4. In general terms, the program provides for automated
scanning of the substrate to determine the positions of probe and
target interaction. Simple processing of the intensity of the
signal may be incorporated to filter out clearly spurious signals.
The positions with positive interaction are correlated with the
sequence specificity of specific matrix positions, to generate the
set of matching subsequences. This information is further
correlated with other target sequence information, e.g.,
restriction fragment analysis. The sequences are then aligned using
overlap data, thereby leading to possible corresponding target
sequences which will, optimally, correspond to a single target
sequence.
[0713] B. Hardware
[0714] A variety of computer systems may be used to run a
sequencing program. The program may be written to provide both the
detecting and scanning steps together and will typically be
dedicated to a particular scanning apparatus. However, the
components and functional steps may be separated and the scanning
system may provide an output, e.g., through tape or an electronic
connection into a separate computer which separately runs the
sequencing analysis program. The computer may be any of a number of
machines provided by standard computer manufacturers, e.g., IBM
compatible machines, Apple.TM. machines, VAX machines, and others,
which may often use a UNIX.TM. operating system. Of course, the
hardware used to run the analysis program will typically determine
what programming language would be used.
[0715] C. Software
[0716] Software would be easily developed by a person of ordinary
skill in the programming art, following the flow chart provided, or
based upon the input provided and the desired result.
[0717] Of course, an exemplary embodiment is a polynucleotide
sequence system. However, the theoretical and mathematical
manipulations necessary for data analysis of other linear
molecules, such as polypeptides, carbohydrates, and various other
polymers are conceptually similar. Simple branching polymers will
usually also be sequencable using similar technology. However,
where there is branching, it may be desired that additional
recognition reagents be used to determine the nature and location
of branches. This can easily be provided by use of appropriate
specific reagents which would be generated by methods similar to
those used to produce specific reagents for linear polymers.
[0718] XII. Substrate Reuse
[0719] Where a substrate is made with specific reagents that are
relatively insensitive to the handling and processing steps
involved in a single cycle of use, the substrate may often be
reused. The target molecules are usually stripped off of the solid
phase specific recognition molecules. Of course, it is preferred
that the manipulations and conditions be selected as to be mild and
to not affect the substrate. For example, if a substrate is acid
labile, a neutral pH would be preferred in all handling steps.
Similar sensitivities would be carefully respected where recycling
is desired.
[0720] A. Removal of Label
[0721] Typically for a recycling, the previously attached specific
interaction would be disrupted and removed. This will typically
involve exposing the substrate to conditions under which the
interaction between probe and target is disrupted. Alternatively,
it may be exposed to conditions where the target is destroyed. For
example, where the probes are oligonucleotides and the target is a
polynucleotide, a heating and low salt wash will often be
sufficient to disrupt the interactions. Additional reagents may be
added such as detergents, and organic or inorganic solvents which
disrupt the interaction between the specific reagents and target.
In an embodiment where the specific reagents are antibodies, the
substrate may be exposed to a gentle detergent which will denature
the specific binding between the antibody and its target. The
conditions are selected to avoid severe disruption or destruction
of the structure of the antibody and to maintain the specificity of
the antibody binding site. Conditions with specific pH, detergent
concentration, salt concentration, ionic concentration, and other
parameters may be selected which disrupt the specific
interactions.
[0722] B. Storage and Peservation
[0723] As indicated above, the matrix will typically be maintained
under conditions where the matrix itself and the linkages and
specific reagents are preserved. Various specific reservatives may
be added which prevent degradation. For example, if the reagents
are acid or base labile, a neutral pH buffer will typically be
added. It is also desired to avoid destruction of the matrix by
growth of organisms which may destroy organic reagents attached
thereto. For this reason, a preservative such as cyanide or azide
may be added. However, the chemical preservative should also be
selected to preserve the chemical nature of the linkages and other
components of the substrate. Typically, a detergent may also be
included.
[0724] C. Processes to Avoid Degradation of Oligomers
[0725] In particular, a substrate comprising a large number of
oligomers will be treated in a fashion which is known to maintain
the quality and integrity of oligonucleotides. These include
storing the substrate in a carefully controlled environment under
conditions of lower temperature, cation depletion (EDTA and EGTA),
sterile conditions, and inert argon or nitrogen atmosphere.
[0726] XIII. Integrated Sequencing Strategy
[0727] A. Initial Mapping Strategy
[0728] As indicated above, although the VLSIPS.TM. Technology may
be applied to sequencing embodiments, it is often useful to
integrate other concepts to simplify the sequencing. For example,
nucleic acids may be easily sequenced by careful selection of the
vectors and hosts used for amplifying and generating the specific
target sequences. For example, it may be desired to use specific
vectors which have been designed to interact most efficiently with
the VLSIPS.TM. Technology substrate. This is also important in
fingerprinting and mapping strategies. For example, vectors may be
carefully selected having particular complementary sequences which
are designed to attach to a genetic or specific oligomer on the
substrate. This is also applicable to situations where it is
desired to target particular sequences to specific locations on the
matrix.
[0729] In one embodiment, unnatural oligomers may be used to target
natural probes to specific locations on the VLSIPS.TM. Technology
substrate. In addition, particular probes may be generated for the
mapping embodiment which are designed to have specific combinations
of characteristics. For example, the construction of a mapping
substrate may depend upon use of another automated apparatus which
takes clones isolated from a chromosome walk and attaches them
individually or in bulk to the VLSIPS.TM. Technology substrate.
[0730] In another embodiment, a variety of specific vectors having
known and particular "targeting" sequences adjacent to the cloning
sites may be individually used to clone a selected probe, and the
isolated probe will then be targetable to a site on the VLSIPS.TM.
Technology substrate with a sequence complementary to the "target"
sequence.
[0731] B. Selection of Smaller Clones
[0732] In the fingerprinting and mapping embodiments, the selection
of probes may be very important. Significant mathematical analysis
may be applied to determine which specific sequences should be used
as those probes. Of course, for fingerprinting use, these sequences
would be most desired that show significant heterogeneity across
the human population. Selection of the specific sequences which
would most favorably be utilized will tend to be single copy
sequences within the genome.
[0733] Various hybridization selection procedures may be applied to
select sequences which tend not to be repeated within a genome, and
thus would tend to be conserved across individuals. For example,
hybridization selections may be made for non-repetitive and single
copy sequences. See, e.g., Britten and Kohne (1968) "Repeated
Sequences in DNA," Science, 161: 529-540. On the other hand, it may
be desired under certain circumstances to use repeated sequences.
For example, where a fingerprint may be used to identify or
distinguish different species, or where repetitive sequences may be
diagnostic of specific species, repetitive sequences may be desired
for inclusion in the fingerprinting probes. In either case, the
sequencing capability will greatly assist in the selection of
appropriate sequences to be used as probes.
[0734] Also as indicated above, various means for constructing an
appropriate substrate may involve either mechanical or automated
procedures. The standard VLSIPS.TM. Technology automated procedure
involves synthesizing oligonucleotides or short polymers directly
on the substrate. In various other embodiments, it is possible to
attach separately synthesized reagents onto the matrix in an
ordered array. Other circumstances may lend themselves to transfer
a pattern from a petri plate onto a solid substrate. Also, there
are methods for site specifically directing collections of reagents
to specific locations using unnatural nucleotides or equivalent
sorts of targeting molecules.
[0735] While a brute force manual transfer process may be utilized
sequentially for attaching various samples to successive positions,
instrumentation for automating such procedures may also be devised.
The automated system for performing such would preferably be
relatively easily designed and conceptually easily understood.
[0736] XIV. Commercial Applications
[0737] A. Sequencing
[0738] As indicated above, sequencing may be performed either de
novo or as a verification of another sequencing method. The present
hybridization technology provides the ability to sequence nucleic
acids and polynucleotides de novo, or as a means to verify either
the Maxam and Gilbert chemical sequencing technique or Sanger and
Coulson dideoxy-sequencing techniques. The hybridization method is
useful to verify sequencing determined by any other sequencing
technique and to closely compare two similar sequences, e.g., to
identify and locate sequence differences.
[0739] Besides polynucleotide sequencing, the present invention
also provides means for sequencing other polymers. This includes
polypeptides, carbohydrates, synthetic organic polymers, and other
polymers. Again, the sequencing may be either verification or de
novo.
[0740] Of course, sequencing can be very important in many
different sorts of environments. For example, it will be useful in
determining the genetic sequence of particular markers in various
individuals. In addition, polymers may be used as markers or for
information containing molecules to encode information. For
example, a short polynucleotide sequence may be included in large
bulk production samples indicating the manufacturer, date, and
location of manufacture of a product. For example, various drugs
may be encoded with this information with a small number of
molecules in a batch. For example, a pill may have somewhere from
10 to 100 to 1,000 or more very short and small molecules encoding
this information. When necessary, this information may be decoded
from a sample of the material using a polymerase chain reaction
(PCR) or other amplification method. This encoding system may be
used to provide the origin of large bulky samples without
significantly affecting the properties of those samples. For
example, chemical samples may also be encoded by this method
thereby providing means for identifying the source and
manufacturing details of lots. The origin of bulk hydrocarbon
samples may be encoded. Production lots of organic compounds such
as benzene or plastics may be encoded with a short molecule
polymer. Food stuffs may also be encoded using similar marking
molecules. Even toxic waste samples can be encoded determining the
source or origin. In this way, proper disposal can be traced or
more easily enforced.
[0741] Similar sorts of encoding may be provided by
fingerprinting-type analysis. Whether the resolution is absolute or
less so, the concept of coding information on molecules such as
nucleic acids, which can be amplified and later decoded, may be a
very useful and important application.
[0742] This technology also provides the ability to include markers
for origins of biological materials. For example, a patented animal
line may be transformed with a particular unnatural sequence which
can be traced back to its origin. With a selection of multiple
markers, the likelihood could be negligible that a combination of
markers would have independently arisen from a source other than
the patented or specifically protected source. This technique may
provide a means for tracing the actual origin of particular
biological materials. Bacteria, plants, and animals will be subject
to marking by such encoding sequences.
[0743] B. Fingerprinting
[0744] As indicated above, fingerprinting technology may also be
used for data encryption. Moreover, fingerprinting allows for
significant identification of particular individuals. Where the
fingerprinting technology is standardized, and used for
identification of large numbers of people, related equipment and
peripheral processing will be developed to accompany the underlying
technology. For example, specific equipment may be developed for
automatically taking a biological sample and generating or
amplifying the information molecules within the sample to be used
in fingerprinting analysis. Moreover, the fingerprinting substrate
may be mass produced using particular types of automatic equipment.
Synthetic equipment may produce the entire matrix simultaneously by
stepwise synthetic methods as provided by the VLSIPS.TM.
technology. The attachment of specific probes onto a substrate may
also be automated, e.g., making use of the caged biotin technology.
See, e.g., Barrett et al. (1993) U.S. Pat. No. 5,252,743. As
indicated above, there are automated methods for actually
generating the matrix and substrate with distinct sequence reagents
positionally located at each of the matrix positions. Where such
reagents are, e.g., unnatural amino acids, a targeting function may
be utilized which does not interfere with a natural nucleotide
functionality.
[0745] In addition, peripheral processing may be important and may
be dedicated to this specific application. Thus, automated
equipment for producing the substrates may be designed, or
particular systems which take in a biological sample and output
either a computer readout or an encoded instrument, e.g., a card or
document which indicates the information and can provide that
information to others. An identification having a short magnetic
strip with a few million bits may be used to provide individual
identification and important medical information useful in a
medical emergency.
[0746] In fact, data banks may be set up to correlate all of this
information of fingerprinting with medical information. This may
allow for the determination of correlations between various medical
problems and specific DNA sequences. By collating large populations
of medical records with genetic information, genetic propensities
and genetic susceptibilities to particular medical conditions may
be developed. Moreover, with standardization of substrates, the
micro encoding data may be also standardized to reproduce the
information from a centralized data bank or on an encoding device
carried on an individual person. On the other hand, if the
fingerprinting procedure is sufficiently quick and routine, every
hospital may routinely perform a fingerprinting operation and from
that determine many important medical parameters for an
individual.
[0747] In particular industries, the VLSIPS.TM. Technology
sequencing, fingerprinting, or mapping technology will be
particularly appropriate. As mentioned above, agricultural
livestock suppliers may be able to encode and determine whether
their particular strains are being used by others. By incorporating
particular markers into their genetic stocks, the markers will
indicate origin of genetic material. This is applicable to seed
producers, livestock producers, and other suppliers of medical or
agricultural biological materials.
[0748] This may also be useful in identifying individual animals or
plants. For example, these markers may be useful in determining
whether certain fish return to their original breeding grounds,
whether sea turtles always return to their original birthplaces, or
to determine the migration patterns and viability of populations of
particular endangered species. It would also provide means for
tracking the sources of particular animal products. For example, it
might be useful for determining the origins of controlled animal
substances such as elephant ivory or particular bird populations
whose importation or exportation is controlled.
[0749] As indicated above, polymers may be used to encode important
information on source and batch and supplier. This is described in
greater detail, e.g., "Applications of PCR to industrial problems,"
(1990) in Chemical and Engineering News 68: 145, which is hereby
incorporated herein by reference. In fact, the synthetic method can
be applied to the storage of enormous amounts of information. Small
substrates may encode enormous amounts of information, and its
recovery will make use of the inherent replication capacity. For
example, on regions of 10 .mu.m.times.10 .mu.m, 1 cm.sup.2 has
10.sup.6 regions. In theory, the entire human genome could be
attached in 1000 nucleotide segments on a 3 cm.sup.2 surface.
Genomes of endangered species may be stored on these
substrates.
[0750] Fingerprinting may also be used for genetic tracing or for
identifying individuals for forensic science purposes.
[0751] See, e.g., Morris, J. et al. (1989) "Biostatistical
Evaluation of Evidence From Continuous Allele Frequency
Distribution DNA Probes in Reference to Disputed Paternity and
Identity," J. Forensic Science, 34: 1311-1317, and references
provided therein; each of which is hereby incorporated herein by
reference.
[0752] In addition, the high resolution fingerprinting allows the
distinguishability to high resolution of particular samples. As
indicated above, new cell classifications may be defined based on
combinations of a large number of properties. Similar applications
will be found in distinguishing different species of animals or
plants. In fact, microbial identification may become dependent on
characterization of the genetic content. Tumors or other cells
exhibiting abnormal physiology will be detectable by use of the
present invention. Also, knowing the genetic fingerprint of a
microorganism may provide very useful information on how to treat
an infection by such organism.
[0753] Modifications of the fingerprint embodiments may be used to
diagnose the condition of the organism. For example, a blood sample
is presently used for diagnosing any of a number of different
physiological conditions. A multi-dimensional fingerprinting method
made available by the present invention could become a routine
means for diagnosing an enormous number of physiological features
simultaneously. This may revolutionize the practice of medicine in
providing information on an enormous number of parameters together
at one time. In another way, the genetic predisposition may also
revolutionize the practice of medicine providing a physician with
the ability to predict the likelihood of particular medical
conditions arising at any particular moment. It also provides the
ability to apply preventive medicine.
[0754] The present invention might also find application in use for
screening new drugs and new reagents which may be very important in
medical diagnosis or other applications. For example, a description
of generating a population of monoclonal antibodies with defined
specificities may be very useful for producing various drugs or
diagnostic reagents.
[0755] Also available are kits with the reagents useful for
performing sequencing, fingerprinting, and mapping procedures. The
kits will have various compartments with the desired necessary
reagents, e.g., substrate, labeling reagents for target samples,
buffers, and other useful accompanying products.
[0756] C. Mapping
[0757] The present invention also provides the means for mapping
sequences within enormous stretches of sequence. For example,
nucleotide sequences may be mapped within enormous chromosome size
sequence maps. For example, it would be possible to map a
chromosomal location within the chromosome which contains hundreds
of millions of nucleotide base pairs. In addition, the mapping and
fingerprinting embodiments allow for testing of chromosomal
translocations, one of the standard problems for which
amniocentesis is performed.
[0758] Thus, the present invention provides a powerful tool and the
means for performing sequencing, fingerprinting, and mapping
functions on polymers. Although most easily and directly applicable
to polynucleotides, polypeptides, carbohydrates, and other sorts of
molecules can be advantageously utilized using the present
technology.
[0759] The present invention will be better understood by reference
to the following illustrative examples. The following examples are
offered by way of illustration and not by way of limitation.
Experimental
[0760] I. Sequencing
[0761] A. polynucleotide
[0762] B. polypeptide
[0763] C. short peptide
[0764] 1. Herz antibody indentification
[0765] II. Fingerprinting
[0766] A. polynucleotide fingerprint
[0767] B. peptide fingerprint
[0768] C. cell classification scheme
[0769] D. temporal development scheme
[0770] 1. developmental antigens
[0771] 2. developmental mRNA expression
[0772] E. diagnostic test
[0773] 1. viral identification
[0774] 2. bacterial identification
[0775] 3. other microbiological identifications
[0776] 4. allergy test (immobilized antigens)
[0777] F. individual (animal/plant) identification
[0778] 1. genetic
[0779] 2. immunological
[0780] G. genetic screen
[0781] 1. test alleles with markers
[0782] 2. aminocentesis
[0783] III. Mapping
[0784] A. positionally located clones (caged biotin)
[0785] 1. short probes, long targets
[0786] 2. long targets, short probes
[0787] B. positionally defined clones
[0788] IV. Conclusion
[0789] Relevant applications whose techniques are incorporated
herein by reference are Pirrung, et al., U.S. Ser. No. 07/362,901,
from which CIP Ser. No. 07/492,462 issued as U.S. Pat. No.
5,143,854, U.S. Ser. No. 07/435,316, from which CIP Ser. No.
07/612,671 issued as U.S. Pat. Nos. 5,252,743, and 5,489,678.
[0790] Also, additional relevant techniques are described, e.g., in
Sambrook, J., et al. (1989) Molecular Cloning: a Laboratory Manual,
2d Ed., vols 1-3, Cold Spring Harbor Press, New York; Greenstein
and Winitz (1961) Chemistry of the Amino Acids, Wiley and Sons, New
York; Bodzansky, M. (1988) Peptide Chemistry: a Practical Textbook,
Springer-Verlag, New York; Harlow and Lane (1988) Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, New York; Glover, D.
(ed.) (1987) DNA Cloning: A Practical Approach, vols 1-3, IRL
Press, Oxford; Bishop and Rawlings (1987) Nucleic Acid and Protein
Sequence Analysis: A Practical Approach, IRL Press, Oxford; Hames
and Higgins (1985) Nucleic Acid Hybridisation: A Practical
Approach, IRL Press, Oxford; Wu et al. (1989) Recombinant DNA
Methodoloy, Academic Press, San Diego; Goding (1986) Monoclonal
Antibodies: Principles and Practice, (2d ed.), Academic Press, San
Diego; Finegold and Barron (1986) Bailey and Scott's Diagnostic
Microbiology, (7th ed.), Mosby Co., St. Louis; Collins et al.
(1989) Microbiological Methods, (6th ed.), Butterworth, London;
Chaplin and Kennedy (1986) Carbohydrate Analysis: A Practical
Approach, IRL Press, Oxford; Van Dyke (ed.) (1985) Bioluminescence
and Chemiluminescence: Instruments and Applications, vol 1, CRC
Press, Boca Rotan; and Ausubel et al. (ed.) (1990) Current
Protocols in Molecular Bioloy, Greene Publishing and
Wiley-Interscience, New York; each of which is hereby incorporated
herein by reference.
[0791] The following examples are provided to illustrate the
efficacy of the inventions herein. All operations were conducted at
about ambient temperatures and pressures unless indicated to the
contrary.
[0792] I. Sequencing
[0793] A. Polynucleotide
[0794] 1. HPLC of the Photolysis of
5'-O-nitroveratryl-thymidine
[0795] In order to determine the time for photolysis of
5'-O-nitroveratryl thymidine to thymidine a 100 .mu.M solution of
NV-Thym-OH(5'-O-nitroverat- ryl thymidine) in dioxane was made and
about 200 .mu.l aliquots were irradiated (in a quartz cuvette 1
cm.times.2 mm) at 362.3 nm for 20 sec, 40 sec, 60 sec, 2 min, 5
min, 10 min, 15 min, and 20 min. The resulting irradiated mixtures
were then analyzed by HPLC using a Varian MicroPak SP column
(C.sub.8 analytical) at a flow rate of 1 ml/min and a solvent
system of 40% CH.sub.3CN and 60% water. Thymidine has a retention
time of 1.2 min and NVO-Thym-OH has a retention time of 2.1 min. It
was seen that after 10 min of exposure the deprotection was
complete.
[0796] 2. Preparation and Detection of Thymidine-Cytidine Dimer
(FITC) The Reaction is Illustrated: 38
[0797] To an aminopropylated glass slide (standard VLSIPS.TM.
Technology) was added a mixture of the following:
[0798] 12.2 mg of NVO-Thym-CO.sub.2H (IX)
[0799] 3.4 mg of HOBT (N-hydroxybenztriazole)
[0800] 8.8 .mu.l DIEA (Diisopropylethylamine)
[0801] 11.1 mg BOP reagent
[0802] 2.5 ml DMF
[0803] After 2 h coupling time (standard VLSIPS) the plate was
washed, acetylated with acetic anhydride/pyridine, washed, dried,
and photolyzed in dioxane at 362 nm at 14 mW/cm.sup.2 for 10 min
using a 500 .mu.m checkerboard mask. The slide was then taken and
treated with a mixture of the following:
[0804] 107 mg of FMOC-amine modified C (III)
[0805] 21 mg of tetrazole
[0806] 1 ml anhydrous CH.sub.3CN
[0807] After being treated for approximately 8 min, the slide was
washed off with CH.sub.3CN, dried, and oxidized with
I.sub.2/H.sub.2O/THF/lutidi- ne for 1 min. The slide was again
washed, dried, and treated for 30 min with a 20% solution of DBU in
DMF. After thorough rinsing of the slide, it was next exposed to a
FITC solution (1 mM fluorescein isothiocyanate [FITC] in DMF) for
50 min, then washed, dried, and examined by fluorescence
microscopy. This reaction is illustrated: 3940
[0808] 3. Preparation and Detection of Thymidine-Cytidine Dimer
(Biotin)
[0809] An aminopropyl glass slide, was soaked in a solution of
ethylene oxide (20% in DMF) to generate a hydroxylated surface. The
slide was added to a mixture of the following:
[0810] 32 mg of NVO-T-OCED (X)
[0811] 11 mg of tetrazole
[0812] 0.5 ml of anhydrous CH.sub.3CN
[0813] After 8 min the plate was then rinsed with acetonitrile,
then oxidized with I.sub.2/H.sub.2O/THF/lutidine for 1 min, washed
and dried. The slide was then exposed to a 1:3 mixture of acetic
anhydride:pyridine for 1 h, then washed and dried. The substrate
was then photolyzed in dioxane at 362 nm at 14 mW/cm.sup.2 for 10
min using a 500 .mu.m checkerboard mask, dried, and then treated
with a mixture of the following:
[0814] 65 mg of biotin modified C (IV)
[0815] 11 mg of tetrazole
[0816] 0.5 ml anhydrous CH.sub.3CN
[0817] After 8 min the slide was washed with CH.sub.3CN then
oxidized with 12/H.sub.2O/THF/lutidine for 1 min, washed, and then
dried. The slide was then soaked for 30 min in a PBS/0.05% Tween 20
buffer and the solution then shaken off. The slide was next treated
with FITC-labeled streptavidin at 10 .mu.g/ml in the same buffer
system for 30 min. After this time the streptavidin-buffer system
was rinsed off with fresh PBS/0.05% Tween 20 buffer and then the
slide was finally agitated in distilled water for about 1/2 h.
After drying, the slide was examined by fluorescence microscopy
(see FIG. 2 and FIG. 3).
[0818] 4. Substrate Preparation
[0819] Before attachment of reactive groups it is preferred to
clean the substrate which is, in a preferred embodiment, a glass
substrate such as a microscope slide or cover slip. A roughened
surface will be useable but a plastic or other solid substrate is
also appropriate. According to one embodiment the slide is soaked
in an alkaline bath consisting of, e.g., 1 liter of 95% ethanol
with 120 ml of water and 120 grams of sodium hydroxide for 12
hours. The slides are washed with a buffer and under running water,
allowed to air dry, and rinsed with a solution of 95% ethanol.
[0820] The slides are then aminated with, e.g.,
aminopropyltriethoxysilane for the purpose of attaching amino
groups to the glass surface on linker molecules, although other
omega functionalized silanes could also be used for this purpose.
In one embodiment 0.1% aminopropyltriethoxysilane is utilized,
although solutions with concentrations from 10.sup.-7% to 10% may
be used, with about 10.sup.-3% to 2% preferred. A 0.1% mixture is
prepared by adding to 100 ml of a 95% ethanol/5% water mixture, 100
microliters (.mu.l) of aminopropyltriethoxysilane. The mixture is
agitated at about ambient temperature on a rotary shaker for an
appropriate amount of time, e.g., about 5 minutes. 500 .mu.l of
this mixture is then applied to the surface of one side of each
cleaned slide. After 4 minutes or more, the slides are decanted of
this solution and thoroughly rinsed three times or more by dipping
in 100% ethanol.
[0821] After the slides dry, they are heated in a 110-120.degree.
C. vacuum oven for about 20 minutes, and then allowed to cure at
room temperature for about 12 hours in an argon environment. The
slides are then dipped into DMF (dimethylformamide) solution,
followed by a thorough washing with methylene chloride.
[0822] 5. Linker Attachment, Blocking of Free Sites
[0823] The aminated surface of the slide is then exposed to about
500 .mu.l of, for example, a 30 millimolar (mM) solution of
NVOC-nucleotide-NHS (N-hydroxysuccinimide) in DMF for attachment of
a NVOC-nucleotide to each of the amino groups. See, e.g., SIGMA
Chemical Company for various nucleotide derivatives. The surface is
washed with, for example, DMF, methylene chloride, and ethanol.
[0824] Any unreacted aminopropyl silane on the surface, i.e., those
amino groups which have not had the NVOC-nucleotide attached, are
now capped with acetyl groups (to prevent further reaction) by
exposure to a 1:3 mixture of acetic anhydride in pyridine for 1
hour. Other materials which may perform this residual capping
function include trifluoroacetic anhydride, formicacetic anhydride,
or other reactive acylating agents. Finally, the slides are washed
again with DMF, methylene chloride, and ethanol.
[0825] 6. Synthesis of Eight Trimers of C and T
[0826] FIG. 4 illustrates a possible synthesis of the eight trimers
of the two-monomer set: cytosine and thymine (represented by C and
T, respectively). A glass slide bearing silane groups terminating
in 6-nitroveratryloxycarboxamide (NVOC--NH) residues is prepared as
a substrate. Active esters (pentafluorophenyl, OBt, etc.) of
cytosine and thymine protected at the 5' hydroxyl group with NVOC
are prepared as reagents. While not pertinent to this example, if
side chain protecting groups are required for the monomer set,
these must not be photoreactive at the wavelength of light used to
protect the primary chain.
[0827] For a monomer set of size n, n.times.l cycles are required
to synthesize all possible sequences of length l. A cycle consists
of:
[0828] 1. Irradiation through an appropriate mask to expose the
5'-OH groups at the sites where the next residue is to be added,
with appropriate washes to remove the by-products of the
deprotection.
[0829] 2. Addition of a single activated and protected (with the
same photochemically-removable group) monomer, which will react
only at the sites addressed in step 1, with appropriate washes to
remove the excess reagent from the surface.
[0830] The above cycle is repeated for each member of the monomer
set until each location on the surface has been extended by one
residue in one embodiment. In other embodiments, several residues
are sequentially added at one location before moving on to the next
location. Cycle times will generally be limited by the coupling
reaction rate, now as short as about 10 min in automated
oligonucleotide synthesizers. This step is optionally followed by
addition of.
[0831] Of course, greater diversity is obtained by using masking
strategies which will also include the synthesis of polymers having
a length of less than 1. If, in the extreme case, all polymers
having a length less than or equal to 1 are synthesized, the number
of polymers synthesized will be:
n.sup.l+n.sup.l-1 + . . . +n.sup.l. (3)
[0832] The maximum number of lithographic steps needed will
generally be n for each "layer" of monomers, i.e., the total number
of masks (and, therefore, the number of lithographic steps) needed
will be n.times.l. The size of the transparent mask regions will
vary in accordance with the area of the substrate available for
synthesis and the number of sequences to be formed. In general, the
size of the synthesis areas will be:
size of synthesis areas=(A)/(S)
[0833] where:
[0834] A is the total area available for synthesis; and
[0835] S is the number of sequences desired in the area.
[0836] It will be appreciated by those of skill in the art that the
above method could readily be used to simultaneously produce
thousands or millions of oligomers on a substrate using the
photolithographic techniques disclosed herein. Consequently, the
method results in the ability to practically test large numbers of,
for example, di, tri, tetra, penta, hexa, hepta, octa, nona, deca,
even dodecanucleotides, or larger polynucleotides (or
correspondingly, polypeptides).
[0837] The above example has illustrated the method by way of a
manual example. It will of course be appreciated that automated or
semi-automated methods could be used. The substrate would be
mounted in a flow cell for automated addition and removal of
reagents, to minimize the volume of reagents needed, and to more
carefully control reaction conditions. Successive masks will be
applicable manually or automatically. See, e.g., Pirrung et al.
(1992) U.S. Pat. Nos. 5,143,854 and 5,489,678.
[0838] 7. Labeling of Target
[0839] The target oligonucleotide can be labeled using standard
procedures referred to above. As discussed, for certain situations,
a reagent which recognizes interaction, e.g., ethidium bromide, may
be provided in the detection step. Alternatively, fluorescence
labeling techniques may be applied, see, e.g., Smith, et al. (1986)
Nature, 321: 674-679; and Prober, et al. (1987) Science, 238:
336-341. The techniques described therein will be followed with
minimal modifications as appropriate for the label selected.
[0840] 8. Dimers of A, C, G, and T
[0841] The described technique may be applied, with photosensitive
blocked nucleotides corresponding to adenine, cytosine, guanine,
and thymine, to make combinations of polynucleotides consisting of
each of the four different nucleotides. All 16 possible dimers
would be made using a minor modification of the described
method.
[0842] 9. 10-mers of A, C, G, and T
[0843] The described technique for making dimers of A, C, G, and T
may be further extended to make longer oligonucleotides. The
automated system described, e.g., in Pirrung et al. (1992) U.S.
Pat. Nos. 5,143,854, and 5,489,678, can be adapted to make all
possible 10-mers composed of the 4 nucleotides A, C, G, and T. The
photosensitive, blocked nucleotide analogues have been described
above, and would be readily adaptable to longer
oligonucleotides.
[0844] 10. Specific Recognition Hybridization to 10-Mers
[0845] The described hybridization conditions are directly
applicable to the sequence specific recognition reagents attached
to the substrate, produced as described immediately above. The
10-mers have an inherent property of hybridizing to a complementary
sequence. For optimum discrimination between full matching and some
mismatch, the conditions of hybridization should be carefully
selected, as described above. Careful control of the conditions,
and titration of parameters should be performed to determine the
optimum collective conditions.
[0846] 11. Hybridization
[0847] Hybridization conditions are described in detail, e.g., in
Hames and Higgins (1985) Nucleic Acid Hybridisation: A Practical
Approach; and the considerations for selecting particular
conditions are described, e.g., in Wetmur and Davidson, (1988) J.
Mol. Biol., 31: 349-370, and Wood et al. (1985) Proc. Natl. Acad.
Sci. USA, 82: 1585-1588. As described above, conditions are desired
which can distinguish matching along the entire length of the probe
from where there is one or more mismatched bases. The length of
incubation and conditions will be similar, in many respects, to the
hybridization conditions used in Southern blot transfers.
Typically, the GC bias may be minimized by the introduction of
appropriate concentrations of the alkylammonium buffers, as
described above.
[0848] Titration of the temperature and other parameters is desired
to determine the optimum conditions for specificity and
distinguishability of absolutely matched hybridization from
mismatched hybridization.
[0849] A fluorescently labeled target or set of targets are
generated, as described in Prober, et al. (1987) Science, 238:
336-341, or Smith, et al. (1986) Nature, 321: 674-679. Preferably,
the target or targets are of the same length as, or slightly
longer, than the oligonucleotide probes attached to the substrate
and they will have known sequences. Thus, only a few of the probes
hybridize perfectly with the target, and which particular ones did
would be known.
[0850] The substrate and probes are incubated under appropriate
conditions for a sufficient period of time to allow hybridization
to completion. The time is measured to determine when the
probe-target hybridizations have reached completion. A salt buffer
which minimizes GC bias is preferred, incorporating, e.g., buffer,
such as tetramethyl ammonium or tetraethyl ammonium ion at between
about 2.4 and 3.0 M. See Wood, et al. (1985) Proc. Nat'l Acad. Sci.
USA, 82: 1585-1588.
[0851] This time is typically at least about 30 min, and may be as
long as about 1-5 days. Typically very long matches will hybridize
more quickly, very short matches will hybridize less quickly,
depending upon relative target and probe concentrations. The
hybridization will be performed under conditions where the reagents
are stable for that time duration.
[0852] Upon maximal hybridization, the conditions for washing are
titrated. Three parameters initially titrated are time,
temperature, and cation concentration of the wash step. The matrix
is scanned at various times to determine the conditions at which
the distinguishability between true perfect hybrid and mismatched
hybrid is optimized. These conditions will be preferred in the
sequencing embodiments.
[0853] 12. Positional Detection of Specific Interaction
[0854] As indicated above, the detection of specific interactions
may be performed by detecting the positions where the labeled
target sequences are attached. Where the label is a fluorescent
label, the apparatus described, e.g., in Pirrung et al. (1992) U.S.
Pat. No. 5,143,854, may be advantageously applied. In particular,
the synthetic processes described above will result in a matrix
pattern of specific sequences attached to the substrate, and a
known pattern of interactions can be converted to corresponding
sequences.
[0855] In an alternative embodiment, a separate reagent which
differentially interacts with the probe and interacted
probe/targets can indicate where interaction occurs or does not
occur. A single-strand specific reagent will indicate where no
interaction has taken place, while a double-strand specific reagent
will indicate where interaction has taken place. An intercalating
dye, e.g., ethidium bromide, may be used to indicate the positions
of specific interaction.
[0856] 13. Analysis
[0857] Conversion of the positional data into sequence specificity
will provide the set of subsequences whose analysis by overlap
segments, may be performed, as described above. Analysis is
provided by the methodology described above, or using, e.g.,
software available from the Genetic Engineering Center, P.O. Box
794, 11000 Belgrade, Yugoslavia (Yugoslav group). See, also,
Macevicz, PCT publication no. WO 90/04652, which is hereby
incorporated herein by reference.
[0858] B. Polypeptide
[0859] The description of the preparation of short peptides on a
substrate incorporates by reference sections in Pirrung et al.
(1992) U.S. Pat. No. 5,143,854, and described below.
[0860] 1. Slide Preparation
[0861] Preparation of the substrate follows that described above
for nucleotides.
[0862] 2. Linker Attachment, Blocking of Free Sites
[0863] The aminated surface of the slide is exposed to about 500
ill of, e.g., a 30 millimolar (mM) solution of NVOC-GABA (gamma
amino butyric acid) NHS(N-hydroxysuccinimide) in DMF for attachment
of a NVOC-GABA to each of the amino groups. The surface is washed
with, for example, DMF, methylene chloride, and ethanol. See U.S.
Pat. No. 5,489,678 for details on amino acid chemistry.
[0864] Any unreacted aminopropyl silane on the surface, i.e., those
amino groups which have not had the NVOC-GABA attached, are now
capped with acetyl groups (to prevent further reaction) by exposure
to a 1:3 mixture of acetic anhydride in pyridine for 1 hour. Other
materials which may perform this residual capping function include
trifluoroacetic anhydride, formicacetic anhydride, or other
reactive acylating agents. Finally, the slides are washed again
with DMF, methylene chloride, and ethanol.
[0865] 3. Synthesis of 8 Trimers of "A" and "B"
[0866] See Pirrung et al. (1992) U.S. Pat. No. 5,143,854 which
describes the preparation of glycine and phenylalanine trimers. The
technique is similar to the method described above for making
trimers of C and T, but substituting photosensitive blocked glycine
for the C derivative and photosensitive blocked phenylalanine for
the T derivative trimers of C and T, but substituting
photosensitive blocked glycine for the C derivative and
photosensitive blocked phenylalamine for the T derivative.
[0867] 4. Synthesis of a Dimer of an Aminopropyl Group and a
Fluorescent Group
[0868] In synthesizing the dimer of an aminopropyl group and a
fluorescent group, a functionalized Durapore.TM. membrane was used
as a substrate. The Durapore.TM. membrane was a polyvinylidine
difluoride with aminopropyl groups. The aminopropyl groups were
protected with the DDZ group by reaction of the carbonyl chloride
with the amino groups, a reaction readily known to those of skill
in the art. The surface bearing these groups was placed in a
solution of THF and contacted with a mask bearing a checkerboard
pattern of 1 mm opaque and transparent regions. The mask was
exposed to ultraviolet light having a wavelength down to at least
about 280 nm for about 5 minutes at ambient temperature, although a
wide range of exposure times and temperatures may be appropriate in
various embodiments of the invention. For example, in one
embodiment, an exposure time of between about 1 and 5000 seconds
may be used at process temperatures of between -70 and +50.degree.
C.
[0869] In one preferred embodiment, exposure times of between about
1 and 500 seconds at about ambient pressure are used. In some
preferred embodiments, pressure above ambient is used to prevent
evaporation.
[0870] The surface of the membrane was then washed for about 1 hour
with a fluorescent label which included an active ester bound to a
chelate of a lanthanide. Wash times will vary over a wide range of
values from about a few minutes to a few hours. These materials
fluoresce in the red and the green visible region. After the
reaction with the active ester in the fluorophore was complete, the
locations in which the fluorophore was bound could be visualized by
exposing them to ultraviolet light and observing the red and the
green fluorescence. It was observed that the derivatized regions of
the substrate closely corresponded to the original pattern of the
mask.
[0871] 5. Demonstration of Signal Capability
[0872] Signal detection capability was demonstrated using a
low-level standard fluorescent bead kit manufactured by Flow
Cytometry Standards and having model no. 824. This kit includes 5.8
.mu.m diameter beads, each impregnated with a known number of
fluorescein molecules.
[0873] One of the beads was placed in the illumination field on the
scan stage in a field of a laser spot which was initially
shuttered. After being positioned in the illumination field, the
photon detection equipment was turned on. The laser beam was
unblocked and it interacted with the particle bead, which then
fluoresced. Fluorescence curves of beads impregnated with 7,000 and
29,000 fluorescein molecules, arc shown in FIGS. 11A and 11B,
respectively of Pirrung et al. (1992) U.S. Pat. No. 5,143,854. On
each curve, traces for beads without fluorescein molecules are also
shown. These experiments were performed with 488 nm excitation,
with 100 .mu.W of laser power. The light was focused through a 40
power 0.75 NA objective.
[0874] The fluorescence intensity in all cases started off at a
high value and then decreased exponentially. The fall-off in
intensity is due to photobleaching of the fluorescein molecules.
The traces of beads without fluorescein molecules are used for
background subtraction. The difference in the initial exponential
decay between labeled and nonlabeled beads is integrated to give
the total number of photon counts, and this number is related to
the number of molecules per bead. Therefore, it is possible to
deduce the number of photons per fluorescein molecule that can be
detected. This calculation indicates the radiation of about 40 to
50 photons per fluorescein molecule are detected.
[0875] 6. Determination of the Number of Molecules Per Unit
Area
[0876] Aminopropylated glass microscope slides prepared according
to the methods discussed above were utilized in order to establish
the density of labeling of the slides. The free amino termini of
the slides were reacted with FITC (fluorescein isothiocyanate)
which forms a covalent linkage with the amino group. The slide is
then scanned to count the number of fluorescent photons generated
in a region which, using the estimated 40-50 photons per
fluorescent molecule, enables the calculation of the number of
molecules which are on the surface per unit area.
[0877] A slide with aminopropyl silane on its surface was immersed
in a 1 mM solution of FITC in DMF for 1 hour at about ambient
temperature. After reaction, the slide was washed twice with DMF
and then washed with ethanol, water, and then ethanol again. It was
then dried and stored in the dark until it was ready to be
examined.
[0878] Through the use of curves similar to those shown in FIG. 11
of Pirrung et al. (1992) U.S. Pat. No. 5,143,854, and by
integrating the fluorescent counts under the exponentially decaying
signal, the number of free amino groups on the surface after
derivitization was determined. It was determined that slides with
labeling densities of 1 fluorescein per 10.sup.3.times.10.sup.3 to
.about.2.times.2 nm could be reproducibly made as the concentration
of aminopropyltriethoxysilane varied from 10.sup.-5% to
10.sup.-1%.
[0879] 7. Removal of NVOC and Attachment of a Flourescent
Marker
[0880] FIG. 12A of Pirrung et al. (1992) U.S. Pat. No. 5,143,854
illustrates the slide which was not exposed to light, but which was
exposed to FITC. The units of the x axis are time and the units of
the y axis are counts. The trace contains a certain amount of
background fluorescence. The duplicate slide was exposed to 350 mm
broadband illumination for about 1 minute (12 mW/cm.sup.2, about
350 nm illumination), washed and reacted with FITC. A large
increase in the level of fluorescence is observed, which indicates
photolysis has exposed a number of amino groups on the surface of
the slides for attachment of a fluorescent marker.
[0881] 8. Use of a Mask in Removal of NVOC
[0882] The next experiment was performed with a 0.1%
aminopropylated slide. Light from a Hg--Xe arc lamp was imaged onto
the substrate through a laser-ablated chrome-on-glass mask in
direct contact with the substrate.
[0883] This slide was illuminated for approximately 5 minutes, with
12 mW of 350 nm broadband light and then reacted with the 1 mM FITC
solution. It was put on the laser detection scanning stage and a
graph was plotted as a two-dimensional representation of position
color-coded for fluorescence intensity. The experiment was repeated
a number of times through various masks. The fluorescence patterns
for a 100.times.100 .mu.m mask, a 50 .mu.m mask, a 20 .mu.m mask,
and a 10 .mu.m mask indicate that the mask pattern is distinct down
to at least about 10 .mu.m squares using this lithographic
technique.
[0884] 9. Attachment of YGGFL and Subsequent Exposure to Herz
Antibody and Goat Anti-Mouse Antibody
[0885] In order to establish that receptors to a particular
polypeptide sequence would bind to a surface-bound peptide and be
detected, Leu enkephalin was coupled to the surface and recognized
by an antibody. A slide was derivatized with 0.1% amino
propyl-triethoxysilane and protected with NVOC. A 500 .mu.m
checkerboard mask was used to expose the slide in a flow cell using
backside contact printing. The Leu enkephalin sequence
(H.sub.2N-tyrosine, glycine, glycine, phenylalanine, leucine-COOH,
otherwise referred to herein as YGGFL) was attached via its carboxy
end to the exposed amino groups on the surface of the slide. The
peptide was added in DMF solution with the BOP/HOBT/DIEA coupling
reagents and recirculated through the flow cell for 2 hours at room
temperature.
[0886] A first antibody, known as the Herz antibody, was applied to
the surface of the slide for 45 minutes at 2 .mu.g/ml in a
supercocktail (containing 1% BSA and 1% ovalbumin also in this
case). A second antibody, goat anti-mouse fluorescein conjugate,
was then added at 2 .mu.g/ml in the supercocktail buffer, and
allowed to incubate for 2 hours.
[0887] The results of this experiment were plotted as fluorescence
intensity as a function of position. This image was taken at 10
.mu.m steps and showed that not only can deprotection be carried
out in a well defined pattern, but also that (1) the method
provided for successful coupling of peptides to the surface of the
substrate, (2) the surface of a bound peptide was available for
binding with an antibody, and (3) the detection apparatus
capabilities were sufficient to detect binding of a receptor.
Moreover, the Herz antibody is a sequence specific reagent which
may be used advantageously as a sequence specific recognition
reagent. It may be used, if specificity is high, for sequencing
purposes, and, at least, for fingerprinting and mapping uses.
[0888] 10. Monomer-by-Monomer Formation of YGGFL and Subsequent
Exposure to Labeled Antibody
[0889] Monomer-by-monomer synthesis of YGGFL and GGFL in alternate
squares was performed on a slide in a checkerboard pattern and the
resulting slide was exposed to the Herz antibody.
[0890] A slide is derivatized with the aminopropyl group, protected
in this case with t-BOC (t-butoxycarbonyl). The slide was treated
with TFA to remove the t-BOC protecting group E-aminocaproic acid,
which was t-BOC protected at its amino group, was then coupled onto
the aminopropyl groups. The aminocaproic acid serves as a spacer
between the aminopropyl group and the peptide to be synthesized.
The amino end of the spacer was deprotected and coupled to
NVOC-leucine. The entire slide was then illuminated with 12 mW of
325 nm broadband illumination. The slide was then coupled with
NVOC-phenylalanine and washed. The entire slide was again
illuminated, then coupled to NVOC-glycine and washed. The slide was
again illuminated and coupled to NVOC-glycine to form the sequence
shown in the last portion of FIG. 13A of Pirrung et al. (1992) U.S.
Pat. No. 5,143,854.
[0891] Alternating regions of the slide were then illuminated using
a projection print using a 500.times.500 .mu.m checkerboard mask;
thus, the amino group of glycine was exposed only in the lighted
areas. When the next coupling chemistry step was carried out,
NVOC-tyrosine was added, and it coupled only at those spots which
had received illumination. The entire slide was then illuminated to
remove all the NVOC groups, leaving a checkerboard of YGGFL in the
lighted areas and in the other areas, GGFL. The Herz antibody
(which recognizes the YGGFL, but not GGFL) was then added, followed
by goat anti-mouse fluorescein conjugate.
[0892] The resulting fluorescence scan showed dark areas containing
the tetrapeptide GGFL, which is not recognized by the Herz antibody
(and thus there is no binding of the goat anti-mouse-antibody with
fluorescein conjugate), and red areas in which YGGFL was present.
The YGGFL pentapeptide is recognized by the Herz antibody and,
therefore, there is antibody in the lighted regions for the
fluorescein-conjugated goat anti-mouse to recognize.
[0893] Similar patterns for a 50 .mu.m mask used in direct contact
("proximity print") with the substrate provided a pattern which was
more distinct and the corners of the checkerboard pattern were
touching as a result of the mask being placed in direct contact
with the substrate (which reflects the increase in resolution using
this technique).
[0894] 11. Monomer-by-Monomer Synthesis of YGGFL and PGGFL
[0895] A synthesis using a 50 .mu.m checkerboard mask was
conducted. However, P was added to the GGFL sites on the substrate
through an additional coupling step. P was added by exposing
protected GGFL to light through a mask, and subsequence exposure to
P in the manner set forth above. Therefore, half of the regions on
the substrate contained YGGFL and the remaining half contained
PGGFL.
[0896] The fluorescence plot for this experiment showed the regions
are again readily discernable between those in which binding did
and did not occur. This experiment demonstrated that antibodies are
able to recognize a specific sequence and that the recognition is
not length-dependent.
[0897] 12. Monomer-by-Monomer Synthesis of YGGFL and YPGGFL
[0898] In order to further demonstrate the operability of the
invention, a 50 .mu.m checkerboard pattern of alternating YGGFL and
YPGGFL was synthesized on a substrate using techniques like those
set forth above. The resulting fluorescence plot showed that the
antibody was clearly able, to recognize the YGGFL sequence and did
not bind significantly at the YPGGFL regions.
[0899] 13. Synthesis of an Array of Sixteen Different Amino Acid
Sequences and Estimation of Relative Binding Affinity to Herz
Antibody
[0900] Using techniques similar to those set forth above, an array
of 16 different amino acid sequences (replicated four times) was
synthesized on each of two glass substrates. The sequences were
synthesized by attaching the sequence NVOC-GFL across the entire
surface of the-slides. Using a series of masks, two layers of amino
acids were then selectively applied to the substrate. Each region
had dimensions of 0.25 cm.times.0.0625 cm. The first slide
contained amino acid sequences containing only L-amino acids while
the second slide contained selected D-amino acids. Various regions
on the first and second slides, were duplicated four times on each
slide. The slides were then exposed to the Herz antibody and
fluorescein-labeled goat anti-mouse antibodies.
[0901] A fluorescence plot of the first slide, which contained only
L-amino acids showed red areas (indicating strong binding, i.e.,
149,000 counts or more) and black areas (indicating little or no
binding of the Herz antibody, i.e., 20,000 counts or less). The
sequence YGGFL was clearly most strongly recognized. The sequences
YAGFL and YSGFL also exhibited strong recognition of the antibody.
By contrast, most of the remaining sequences showed little or no
binding. The four duplicate portions of the slide were extremely
consistent in the amount of binding shown therein.
[0902] A fluorescence plot of the D-amino acid slide indicated that
strongest binding was exhibited by the YGGFL sequence. Significant
binding was also detected to YaGFL, YsGFL, and YpGFL. The remaining
sequences showed less binding with the antibody. Low binding
efficiency of the sequence yGGFL was observed.
[0903] Table 6 lists the various sequences tested in order of
relative fluorescence, which provides information regarding
relative binding affinity.
10TABLE 6 Apparent Binding to Herz Ab L-a.a. Set D-a.a. Set YGGFL
YGGFL YAGFL YaGFL YSGFL YsGFL LGGFL YpGFL FGGFL fGGFL YPGFL yGGFL
LAGFL faGFL FAGFL wGGFL WGGFL yaGFL fpGFL waGFL
[0904] 14. Illustrative Alternative Embodiment
[0905] According to an alternative embodiment of the invention, the
methods provide for attaching to the surface a caged binding member
which, in its caged form, has a relatively low affinity for other
potentially binding species, such as receptors and specific binding
substances. Such techniques are more fully described in copending
application Ser. No. 404,920, filed Sep. 8, 1989, and incorporated
herein by reference for all purposes. See also U.S. Ser. No.
07/435,316, from which CIP U.S. Ser. No. 07/612,671 issued as U.S.
Pat. No. 5,252,743, each of which is hereby incorporated herein by
reference.
[0906] According to this alternative embodiment, the invention
provides methods for forming predefined regions on a surface of a
solid support, wherein the predefined regions are capable of
immobilizing receptors. The methods make use of caged binding
members attached to the surface to enable selective activation of
the predefined regions. The caged binding members are liberated to
act as binding members ultimately capable of binding receptors upon
selective activation of the predefined regions. The activated
binding members are then used to immobilize specific molecules such
as receptors on the predefined region of the surface. The above
procedure is repeated at the same or different sites on the surface
so as to provide a surface prepared with a plurality of regions on
the surface containing, for example, the same or different
receptors. When receptors immobilized in this way have a
differential affinity for one or more ligands, screenings and
assays for the ligands can be conducted in the regions of the
surface containing the receptors.
[0907] The alternative embodiment may make use of novel caged
binding members attached to the substrate. Caged (unactivated)
members have a relatively low affinity for receptors of substances
that specifically bind to uncaged binding members when compared
with the corresponding affinities of activated binding members.
Thus, the binding members are protected from reaction until a
suitable source of energy is applied to the regions of the surface
desired to be activated. Upon application of a suitable energy
source, the caging groups labilize, thereby presenting the
activated binding member. A typical energy source will be
light.
[0908] Once the binding members on the surface are activated they
may be attached to a receptor. The receptor chosen may be a
monoclonal antibody, a nucleic acid sequence, a drug receptor, etc.
The receptor will usually, though not always, be prepared so as to
permit attaching it, directly or indirectly, to a binding member.
For example, a specific binding substance having a strong binding
affinity for the binding member and a strong affinity for the
receptor or a conjugate of the receptor may be used to act as a
bridge between binding members and receptors if desired. The method
uses a receptor prepared such that the receptor retains its
activity toward a particular ligand.
[0909] Preferably, the caged binding member attached to the solid
substrate will be a photoactivatable biotin complex, i.e., a biotin
molecule that has been chemically modified with photoactivatable
protecting groups so that it has a significantly reduced binding
affinity for avidin or avidin analogs than does natural biotin. In
a preferred embodiment, the protecting groups localized in a
predefined region of the surface will be removed upon application
of a suitable source of radiation to give binding members, that is
biotin or a functionally analogous compound having substantially
the same binding affinity for avidin or avidin analogs as does
biotin. surface will be removed upon application of a suitable
source of radiation to give binding members, that is biotin or a
functionally analogous compound having substantially the same
binding affinity for avidin or avidin analogs as does biotin.
[0910] In another preferred embodiment, avidin or an avidin analog
is incubated with activated binding members on the surface until
the avidin binds strongly to the binding members. The avidin so
immobilized on predefined regions of the surface can then be
incubated with a desired receptor or conjugate of a desired
receptor. The receptor will preferably be biotinylated, e.g., a
biotinylated antibody, when avidin is immobilized on the predefined
regions of the surface. Alternatively, a preferred embodiment will
present an avidin/biotinylated receptor complex, which has been
previously prepared, to activated binding members on the
surface.
[0911] II. Fingerprinting
[0912] The above section on generation of reagents for sequencing
provides specific reagents useful for fingerprinting applications.
Fingerprinting embodiments may be applied towards polynucleotide
fingerprinting, polypeptide fingerprinting, cell and tissue
classification, cell and tissue temporal development stage
classification, diagnostic tests, forensic uses for individual
identification, classification of organisms, and genetic screening
of individuals. Mapping applications are also described below.
[0913] A. Polynucleotide Fingerprint
[0914] Polynucleotide fingerprinting may use reagents similar to
those described above for probing a sequence for the presence of
specific subsequences found therein. Typically, the subsequences
used for fingerprinting will be longer than the sequences used in
oligonucleotide sequencing. In particular, specific long segments
may be used to determine the similarity of different samples of
nucleic acids. They may also be used to fingerprint whether
specific combinations of information are provided therein.
Particular probe sequences are selected and attached in a
positional manner to a substrate. The means for attachment may be
either using a caged biotin method described, e.g., in Barrett et
al. (1993) U.S. Pat. No. 5,252,743, or by another method using
targeting molecules. For example, a short polypeptide of specific
sequence may be attached to an oligonucleotide and targeted to
specific positions on a substrate having antibodies attached
thereto, the antibodies exhibiting specificity for binding to those
short peptide sequences. In another embodiment, an unnatural
nucleotide or similar complementary binding molecule may be
attached to the fingerprinting probe and the probe thereby directed
towards complementary sequences on a VLSIPS.TM. technology
substrate. Typically, unnatural nucleotides would be preferred,
e.g., unnatural optical isomers, which would not interfere with
natural nucleotide interactions.
[0915] Having produced a substrate with particular fingerprint
probes attached thereto at positionally defined regions, the
substrate may be used in a manner quite similar to the sequencing
embodiment to provide information as to whether the fingerprint
probes are detecting the corresponding sequence in a target
sequence. This will often provide information similar to a Southern
blot hybridization.
[0916] B. Polypeptide Fingerprint
[0917] A polypeptide fingerprint may be performed using antibodies
which recognize specific antigens on the polypeptide. For example,
monoclonal antibodies which recognize specific sequences or
antigens on a polypeptide may be used to determine whether those
epitopes are found on a particular protein. For example, particular
patterns of epitopes would be found on various types of proteins.
This will lead to the discovery that specific epitopes, or
antigenic determinants, which are characteristic of, e.g., beta
sheet segments, will be identified as will particular different
types of domains in various protein types. Thus, a screening method
may be devised which can classify polypeptides, either native or
denatured, into various new classes defined by the epitopes
existing thereon.
[0918] In addition, once the substrate is generated in the manners
described above, a target peptide is exposed to the substrate. The
target may be either native or denatured, though the conditions
used to denature the polypeptide may interfere with the specific
interaction between the polypeptide and the recognition reagent.
This method is not dependent on the fact that the polypeptide is a
single chain, thus protein complexes may also be fingerprinted
using this methodology. Structures such as multi-subunit proteins,
associations of proteins, ribosomes, nucleosomes, and other small
cellular structures may also be fingerprinted and classified
according to the presence of specific recognizable features
thereon.
[0919] Peptide fingerprinting may be useful, for example, in
correlating with particular physiological conditions or
developmental stages of a cell or organism. Thus, a biological
sample may be fingerprinted to determine the presence in that
sample of a plurality of different polypeptides which are each
individually fingerprinted. In an alternative embodiment, a
polypeptide itself is not fingerprinted but a biological sample is
fingerprinted searching for specific epitopes, e.g., polypeptide,
carbohydrate, nucleic acid, or any of a number of other specific
recognizable structural features.
[0920] The conditions for the interactions using antibodies is
described, e.g., in Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Press, New York. The conditions should
be titrated for temperature, buffer composition, time, and other
important parameters in an antibody interaction.
[0921] C. Cell Classification Scheme
[0922] The present invention can be used for cell classification
using fingerprinting type technology as described above in the
polypeptide fingerprint. Classes of cells are typically defined by
the presence of common functions which are usually reflected by
structural features. Thus, a plant cell is classified differently
from an animal cell by a number of structural features. Given an
unknown cell, the present invention provides improved means for
distinguishing the different cell types. Once a cell classification
scheme is developed and the structural features which define it are
identified using the present invention, homogeneous cell population
expressing these features may be separated from others. Standard
cell sorters may be coupled with recognition reagents and labels
which can distinguish various cell types.
[0923] a. T-Cell Classes
[0924] T-cell classes are defined on the basis of expression of
particular antigens characteristic of each class. For example,
mouse T-cell differentiation markers include the LY antigens. With
the plurality of different antigens which may be tested using
antibody or other recognition reagents, new populations and classes
of cells may be defined. For example, different neural cell types
may be defined on the basis of cell surface antigens. Different
tissue types will be defined on the basis of tissue specific
antigens. Developmental cell classes will be similarly defined. All
of these screenings can make use of the VLSIPS substrates with
specific recognition molecules attached thereto. The substrates are
exposed to the cell types directly, assaying for attachment of
cells to specific regions, or are exposed to products of a
population of cells, e.g., a supernatant, or a cell lysate.
[0925] Once a cell classification scheme has been correlated with
specific. structural markers therein, reagents which recognize
those features may be developed and used in a fluorescence
activated cell sorter as described, e.g., in Dangl, J. and
Herzenberg (1982) J. Immunological Methods, 52: 1-14; and Becton
Dickinson, Fluorescence Activated Cell Sorters Division, San Jose,
Calif. This will provide a homogeneous population of cells whose
function has been defined by structure.
[0926] b. B-Cell Classes
[0927] The present cell classification scheme may also be used to
determine specific B-cell classes. For example, B-cells specific
for producing IgM, IgG, IgD, IgE, and IgA may be defined by the
internal expression of specific mRNA sequences encoding each type
of immunoglobulin. The classification scheme may depend on either
extracellularly expressed markers which are correlated as being
diagnostic of specific stages in development, or intracellular mRNA
sequences which indicate particular functions.
[0928] D. Temporal Development Scheme
[0929] 1. Developmental Antigens
[0930] The present fingerprinting invention also allows cell
classification by expression of developmental antigens. For
example, a lymphocyte stem cell expresses a particular combination
of antigens. As the lymphocyte develops through a program
developmental scheme, at various stages it expresses particular
antigens which are diagnostic of particular stages in development.
Again, the fingerprinting methodology allows for the definition of
specific structural features which are diagnostic of developmental
or functional features which will allow classification of cells
into temporal developmental classes. Cells, products of those
cells, or lysates of those cells will be assayed to determine the
developmental stage of the source cells. In this manner, once a
developmental stage is defined, specific synchronized populations
of cells will be selected out of another population. These
synchronized populations may be very important in determining the
biological mechanisms of development.
[0931] 2. Developmental nRNA Expression
[0932] Besides expressed antigens, the present invention also
allows for fingerprinting of the mRNA population of a cell. In this
fashion, the mRNA population, which should be a good determinant of
developmental stage, will be correlated with other structural
features of the cell. In this manner, cells at specific
developmental stages will be characterized by the intracellular
environment, as well as the extracellular environment.
[0933] The present invention also allows the combination of
definitions based, in part, upon antigens and, in part, upon mRNA
expression.
[0934] In one embodiment, the two may be combined in a single
incubation step. A particular incubation condition may be found
which is compatible with both hybridization recognition and
non-hybridization recognition molecules. Thus, e.g., an incubation
condition may be selected which allows both specificity of antibody
binding and specificity of nucleic acid hybridization. This allows
simultaneous performance of both types of interactions on a single
matrix. Again, where developmental mRNA patterns are correlated
with structural features, or with probes which are able to
hybridize to intracellular mRNA populations, a cell sorter may be
used to sort specifically those cells having desired mRNA
population patterns.
[0935] E. Diagnostic Tests
[0936] The present invention also provides the ability to perform
diagnostic tests. Diagnostic tests typically are based upon a
fingerprint type assay, which tests for the presence of specific
diagnostic structural features. Thus, the present invention
provides means for viral strain identification, bacterial strain
identification, and other diagnostic tests using positionally
defined specific reagents. The present invention also allows for
determining a spectrum of allergies, diagnosing a biological sample
for any or all of the above, and testing for many other
conditions.
[0937] 1. Viral Identification
[0938] The present invention provides reagents and methodology for
identifying viral strains. The specific reagents may be either
antibodies or recognition proteins which bind to specific viral
epitopes preferably surface exposed, but may make use of internal
epitopes, e.g., in a denatured viral sample. In an alternative
embodiment, the viral genome may be probed for specific sequences
which are characteristic of particular viral strains. As above, a
combination of the two may be performed simultaneously in a single
interaction step, or in separate tests, e.g., for both genetic
characteristics and epitope characteristics.
[0939] 2. Bacterial Identification
[0940] Similar techniques will be applicable to identifying a
bacterial source. This may be useful in diagnosing bacterial
infections, or in classifying sources of particular bacterial
species. For example, the bacterial assay may be useful in
determining the natural range of survivability of particular
strains of bacteria across regions of the country or in different
ecological niches.
[0941] 3. Other Microbiological Identifications
[0942] The present invention provides means for diagnosis of other
microbiological and other species, e.g., protozoal species and
parasitic species in a biological sample, but also provides the
means for assaying a combination of different infections. For
example, a biological specimen may be assayed for the presence of
any or all of these microbiological species. In human diagnostic
uses, typical samples will be blood, sputum, stool, urine, or other
samples.
[0943] 4. Allergy Tests
[0944] An immobilized set of antigens may be attached to a solid
substrate and, instead of the standard skin reaction tests, a blood
sample may be assayed on such a substrate to determine the presence
of antibodies, e.g., IgE or other type antibodies, which may be
diagnostic of an allergic or immunological susceptibility. A
standard radioallergosorbent test (RAST) may be used to check a
much larger population of antigens.
[0945] In addition, an allergy like test may be used to diagnose
the immunological history of a particular individual. For example,
by testing the circulating antibodies in a blood sample, which
reflects the immunological history and memory of an individual, it
may be determined what infections may not have been historically
presented to the immune system. In this manner, it may be possible
to specifically supplement an immune system for a short period of
time with IgG fractions made up of specific types of gamma
globulins. Thus, hepatitis gamma globulin injections may be better
designed for a particular environment to which a person is expected
to be exposed. This also provides the ability to identify
genetically equivalent individuals who have immunologically
different experiences. Thus, a blood sample from an individual who
has a particular combination of circulating antibodies will likely
be different from the combination of circulating antibodies found
in a genetically similar or identical individual. This could allow
for the distinction between clones of particular animals, e.g.,
mice, rats, or other animals. globulin injections may be better
designed for a particular environment which a person is expected to
be exposed. This also provides the ability to identify genetically
equivalent individuals who have immunologically different
experiences. Thus, a blood sample from an individual who has a
particular combination of circulating antibodies will likely be
different from the combination of circulating antibodies found in a
genetically similar or identical individual. This could allow for
the distinction between clones of particular animals, e.g., mice,
rats, or other animals.
[0946] F. Individual Identification
[0947] The present invention provides the ability to fingerprint
and identify a genetic individual. This individual may be a
bacterial or lower microorganism, as described above in diagnostic
tests, or of a plant or animal. An individual may be identified
genetically or immunologically, as described.
[0948] 1. Genetic
[0949] Genetic fingerprinting has been utilized in comparing
different related species in Southern hybridization blots. Genetic
fingerprinting has also been used in forensic studies, see, e.g.,
Morris et al. (1989) J. Forensic Science, 34: 1311-1317, and
references cited therein. As described above, an individual may be
identified genetically by a sufficiently large number of probes.
The likelihood that another individual would have an identical
pattern over a sufficiently large number of probes may be
statistically negligible. However, it is often quite important that
a large number of probes be used where the statistical probability
of matching is desired to be particularly low. In fact, the probes
will optimally be selected for having high heterogeneity among the
population. In addition, the fingerprint method may make use of the
pattern of homologies indicated by a series of more and more
stringent washes. Then, each position has both a sequence
specificity and a homology measurement, the combination of which
greatly increases the number of dimensions and the statistical
likelihood of a perfect pattern match with another genetic
individual.
[0950] 2. Immunological
[0951] As indicated above in the diagnostic tests, it is possible
to identify a particular immune system within a genetically
homogeneous class of organisms by virtue of their immunological
history. For example, a large colony of cloned mice may be
distinguishable by virtue of each immunological history. For
example, one mouse may have had an immunological response to
exposure to antigen A to which her genetically identical sibling
may have not been exposed. By virtue of this differential history,
the first of the pair will likely have a high antibody titer
against the antigen A whereas her genetically identical sibling
will have not had a response to that antigen by virtue of never
having been exposed to it. For this reason, immune systems may be
identified by their immunological memories. Thus, immunological
experience may also be a means for identifying a particular
individual at a particular moment in her lifetime.
[0952] This same immunological screening may be used for other
sorts of identifiable biological products. For example, an
individual may be identified by her combination of expressed
proteins. These proteins may reflect a physiological state of the
individual, and would thus be useful in certain circumstances where
diagnostic tests may be performed. For example, an individual may
be identified, in part, by the presence of particular metabolic
products.
[0953] In fact, a plant origin may be determined by virtue of
having within its genome an unnatural sequence introduced to it by
genetic breeders. Thus, a marker nucleic acid sequence may be
introduced as a means to determine whether a genetic strain of a
plant or animal originated from another particular source.
[0954] G. Genetic Screening
[0955] 1. Test Alleles with Markers
[0956] The present invention provides for the ability to screen for
genetic variations of individuals. For example, a number of genetic
diseases are linked with specific alleles. See, e.g., Scriber, C.
et al. (eds.) (1989) The Metabolic Bases of Inherited Disease,
McGraw-Hill, New York. In one embodiment, cystic fibrosis has been
correlated with a specific gene, see, Gregory et al. (1990) Nature,
347: 382-386. A number of alleles are correlated with specific
genetic deficiencies. See, e.g., McKusick, V. (1990) Genetic
Inheritance in Man: Catalogs of Autosomal Dominant, Autosomal
Recessive, and X-linked Phenotypes, Johns Hopkins University Press,
Baltimore; Ott, J. (1985) Analysis of Human Genetic Linkage, Johns
Hopkins University Press, Baltimore; Track, R. et al. (1989)
Banbury Report 32: DNA Technology and Forensic Science, Cold Spring
Harbor Press, New York; each of which is hereby incorporated herein
by reference.
[0957] 2. Amniocentesis
[0958] Typically, amniocentesis is used to determine whether
chromosome translocations have occurred. The mapping procedure may
provide the means for determining whether these translocations have
occurred, and for detecting particular alleles of various
markers.
[0959] III. Mapping
[0960] A. Positionally Located Clones
[0961] The present invention allows for the positional location of
specific clones useful for mapping. For example, caged biotin may
be used for specifically positioning a probe to a location on a
matrix pattern.
[0962] In addition, the specific probes may be positionally
directed to specific locations on a substrate by targeting. For
example, polypeptide specific recognition reagents may be attached
to oligonucleotide sequences which can be complementarily targeted
to specific locations on a VLSIPS.TM. Technology substrate.
Hybridization conditions, as applied for oligonucleotide probes,
will be used to target the reagents to locations on a substrate
having complementary oligonucleotides synthesized thereon. In
another embodiment, oligonucleotide probes may be attached to
specific polypeptide targeting reagents such as an antigen or
antibody. These reagents can be directed towards a complementary
antigen or antibody already attached to a VLSIPS.TM. Technology
substrate.
[0963] In another embodiment, an unnatural nucleotide which does
not interfere with natural nucleotide complementary hybridization
may be used to target oligonucleotides to particular positions on a
substrate. Unnatural optical isomers of natural nucleotides should
be ideal candidates.
[0964] In this way, short probes may be used to determine the
mapping of long targets or long targets may be used to map the
position of shorter probes. See, e.g., Craig et al. 1990 Nuc. Acids
Res., 18: 2653-2660.
[0965] B. Positionally Defined Clones
[0966] Positionally defined clones may be transferred to a new
substrate by either physical transfer or by synthetic means.
Synthetic means may involve either a production of the probe on the
substrate using the VLSIPSF Technology synthetic methods, or may
involve the attachment of a targeting sequence made by VLSIPS.TM.
Technology synthetic methods which will target that positionally
defined clone to a position on a new substrate. Both methods will
provide a substrate having a number of positionally defined probes
useful in mapping.
[0967] IX. Conclusion
[0968] The present inventions provide greatly improved methods and
apparatus for synthesis of polymers on substrates. It is to be
understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reviewing the above description.
By way of example, the invention has been described primarily with
reference to the use of photoremovable protective groups, but it
will be readily recognized by those of skill in the art that
sources of radiation other than light could also be used. For
example, in some embodiments it may be desirable to use protective
groups which are sensitive to electron beam irradiation, x-ray
irradiation, in combination with electron beam lithograph, or x-ray
lithography techniques. Alternatively, the group could be removed
by exposure to an electric current. The scope of the invention
should, therefore, be determined not with reference to the above
description, but should instead be determined with reference to the
appended claims, along with the full scope of equivalents to which
such claims are entitled.
[0969] All publications and patent applications referred to herein
are incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually incorporated by reference. The present invention now
being fully described, it will be apparent to one of ordinary skill
in the art that many changes and modifications can be made thereto
without departing from the spirit or scope of the appended
claims.
[0970] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
34 1 5 PRT Artificial Sequence Synthesized Peptide Sequence 1 Tyr
Gly Gly Phe Leu 1 5 2 4 PRT Artificial Sequence Synthesized Peptide
Sequence 2 Gly Gly Phe Leu 1 3 5 PRT Artificial Sequence
Synthesized Peptide Sequence 3 Pro Gly Gly Phe Leu 1 5 4 6 PRT
Artificial Sequence Synthesized Peptide Sequence 4 Tyr Pro Gly Gly
Phe Leu 1 5 5 5 PRT Artificial Sequence Synthesized Peptide
Sequence 5 Tyr Ala Gly Phe Leu 1 5 6 5 PRT Artificial Sequence
Synthesized Peptide Sequence 6 Tyr Ser Gly Phe Leu 1 5 7 5 PRT
Artificial Sequence Synthesized Peptide Sequence 7 Leu Gly Gly Phe
Leu 1 5 8 5 PRT Artificial Sequence Synthesized Peptide Sequence 8
Phe Gly Gly Phe Leu 1 5 9 5 PRT Artificial Sequence Synthesized
Peptide Sequence 9 Leu Ala Gly Phe Leu 1 5 10 5 PRT Artificial
Sequence Synthesized Peptide Sequence 10 Phe Ala Gly Phe Leu 1 5 11
5 PRT Artificial Sequence Synthesized Peptide Sequence 11 Trp Gly
Gly Phe Leu 1 5 12 5 PRT Artificial Sequence Synthesized Peptide
Sequence 12 Tyr Pro Gly Phe Leu 1 5 13 5 PRT Artificial Sequence
Synthesized Peptide Sequence 13 Leu Pro Gly Phe Leu 1 5 14 5 PRT
Artificial Sequence Synthesized Peptide Sequence 14 Trp Pro Gly Phe
Leu 1 5 15 5 PRT Artificial Sequence Synthesized Peptide Sequence
15 Trp Ala Gly Phe Leu 1 5 16 5 PRT Artificial Sequence Synthesized
Peptide Sequence 16 Leu Ser Gly Phe Leu 1 5 17 5 PRT Artificial
Sequence Synthesized Peptide Sequence 17 Phe Ser Gly Phe Leu 1 5 18
5 PRT Artificial Sequence Synthesized Peptide Sequence 18 Trp Ser
Gly Phe Leu 1 5 19 5 PRT Artificial Sequence Synthesized Peptide
Sequence 19 Phe Pro Gly Phe Leu 1 5 20 5 PRT Artificial Sequence
Synthesized Peptide Sequence 20 Tyr Xaa Gly Phe Leu 1 5 21 5 PRT
Artificial Sequence Synthesized Peptide Sequence 21 Tyr Xaa Gly Phe
Leu 1 5 22 5 PRT Artificial Sequence Synthesized Peptide Sequence
22 Tyr Xaa Gly Phe Leu 1 5 23 5 PRT Artificial Sequence Synthesized
Peptide Sequence 23 Xaa Gly Gly Phe Leu 1 5 24 5 PRT Artificial
Sequence Synthesized Peptide Sequence 24 Xaa Gly Gly Phe Leu 1 5 25
5 PRT Artificial Sequence Synthesized Peptide Sequence 25 Xaa Xaa
Gly Phe Leu 1 5 26 5 PRT Artificial Sequence Synthesized Peptide
Sequence 26 Xaa Gly Gly Phe Leu 1 5 27 5 PRT Artificial Sequence
Synthesized Peptide Sequence 27 Xaa Xaa Gly Phe Leu 1 5 28 5 PRT
Artificial Sequence Synthesized Peptide Sequence 28 Xaa Xaa Gly Phe
Leu 1 5 29 5 PRT Artificial Sequence Synthesized Peptide Sequence
29 Xaa Xaa Gly Phe Leu 1 5 30 5 PRT Artificial Sequence Synthesized
Peptide Sequence 30 Xaa Xaa Gly Phe Leu 1 5 31 5 PRT Artificial
Sequence Synthesized Peptide Sequence 31 Xaa Xaa Gly Phe Leu 1 5 32
5 PRT Artificial Sequence Synthesized Peptide Sequence 32 Xaa Xaa
Gly Phe Leu 1 5 33 5 PRT Artificial Sequence Synthesized Peptide
Sequence 33 Xaa Xaa Gly Phe Leu 1 5 34 5 PRT Artificial Sequence
Synthesized Peptide Sequence 34 Xaa Xaa Gly Phe Leu 1 5
* * * * *