U.S. patent application number 09/886779 was filed with the patent office on 2002-06-20 for nucleic acid arrays and methods of synthesis.
This patent application is currently assigned to Trustees of Boston University. Invention is credited to Cantor, Charles, Hatch, Anson, Misasi, John, Sabanayagam, Chandran R., Sano, Takeshi.
Application Number | 20020076716 09/886779 |
Document ID | / |
Family ID | 26765389 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076716 |
Kind Code |
A1 |
Sabanayagam, Chandran R. ;
et al. |
June 20, 2002 |
Nucleic acid arrays and methods of synthesis
Abstract
The present invention generally relates to high density nucleic
acid arrays and methods of synthesizing nucleic acid sequences on a
solid surface. Specifically, the present invention contemplates the
use of stabilized nucleic acid primer sequences immobilized on
solid surfaces, and circular nucleic acid sequence templates
combined with the use of isothermal rolling circle amplification to
thereby increase nucleic acid sequence concentrations in a sample
or on an array of nucleic acid sequences.
Inventors: |
Sabanayagam, Chandran R.;
(Allston, MA) ; Sano, Takeshi; (Needham, MA)
; Misasi, John; (Syracuse, NY) ; Hatch, Anson;
(Seattle, WA) ; Cantor, Charles; (Del Mar,
CA) |
Correspondence
Address: |
NIXON PEABODY LLP
101 FEDERAL ST
BOSTON
MA
02110
US
|
Assignee: |
Trustees of Boston
University
|
Family ID: |
26765389 |
Appl. No.: |
09/886779 |
Filed: |
June 21, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09886779 |
Jun 21, 2001 |
|
|
|
09287781 |
Apr 8, 1999 |
|
|
|
6284497 |
|
|
|
|
60081254 |
Apr 9, 1998 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/91.2; 438/1 |
Current CPC
Class: |
B01J 2219/00722
20130101; B01J 2219/00529 20130101; C12P 19/34 20130101; B01J
2219/00659 20130101; C07H 21/00 20130101; B01J 2219/00608 20130101;
B01J 2219/0063 20130101; B01J 2219/00637 20130101; B01J 2219/00612
20130101; C12Q 2531/125 20130101; C12Q 2531/101 20130101; C12Q
2565/537 20130101; C12Q 2531/101 20130101; B01J 2219/00527
20130101; C12Q 2565/537 20130101; C07B 2200/11 20130101; Y10T
436/143333 20150115; C12Q 2531/125 20130101; C12Q 1/6853 20130101;
B01J 2219/0061 20130101; C40B 40/06 20130101; C12Q 1/6853 20130101;
C12Q 1/6846 20130101; C12Q 1/6846 20130101; B01J 2219/00596
20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 438/1; 435/91.2 |
International
Class: |
C12Q 001/68; C12M
001/34; C12P 019/34 |
Goverment Interests
[0002] The development of the present invention was supported under
Contract Number DE-FG02-93ER61609 awarded by the Department of
Energy. The United States Government may have certain rights in
this invention.
Claims
We claim:
1. A method of generating an array, comprising: a) providing: i) a
solid support comprising a plurality of positions for
oligonucleotides, said positions defined by x and y coordinates;
ii) a plurality of identical oligonucleotides, each oligonucleotide
comprising a sequence; and iii) a plurality of unique circular DNA
templates, each circular DNA template comprising a sequence of
interest and a region complementary to at least a portion of said
sequence of said oligonucleotides, said sequence of interest being
different for each circular template; b) immobilizing one
oligonucleotide from said plurality of identical oligonucleotides
in each of said positions on said solid support to create an
ordered array comprising a plurality of identical immobilized
oligonucleotides; c) adding to each immobilized oligonucleotide of
said ordered array a circular DNA template from said plurality of
said unique circular DNA templates under conditions such that said
immobilized oligonucleotide hybridizes to said circular DNA
template to create a plurality of primed circular templates, each
primed circular template comprising a different sequence of
interest; and d) extending each of said primed circular templates
to create an extended immobilized oligonucleotide comprising at
least two copies of said sequence of interest, thereby generating
an ordered redundant array.
2. The method of claim 1, wherein said oligonucleotides are
immobilized on a solid surface by a chemical linkage.
3. The method of claim 1, wherein said oligonucleotides are
immobilized on said solid surface by the 5' end of said
oligonucleotides.
4. The method of claim 1, wherein said oligonucleotides are
approximately 17 bases in length.
5. The method of claim 1 wherein said solid surface is selected
from a group of materials comprising silicon, metal, and glass.
6. The method of claim 1 wherein said immobilized oligonucleotides
are attached to a complimentary nucleic acid stabilizer
sequence.
7. The method of claim 1, wherein said circular nucleic acid
template is bacteriophage DNA.
8. The method of claim 1, wherein said circular nucleic acid
template is non-bacteriophage DNA.
9. The method of claim 1, wherein said extending in step (d) is
achieved with a polymerase.
10. The method of claim 9, wherein said polymerase is selected from
a group comprising E. coli. DNA polymerase I, a fragment of E.
coli. DNA polymerase I, or .PHI.29 DNA polymerase.
11. An ordered redundant array of immobilized oligonucleotides
produced according to the method of claim 1.
12. A method of hybridizing target nucleic acid fragments,
comprising: a) providing i) the ordered redundant array of extended
immobilized oligonucleotides of claim 1; and ii) a plurality of
fragments of a target nucleic acid; and b) bringing said fragments
of said target nucleic acid into contact with said array under
conditions such that at least one of said fragments hybridizes to
one of said extended immobilized oligonucleotides on said
array.
13. A method of generating an array capable of hybridizing to
fragments of a target nucleic acid, comprising: a) providing: i) a
solid support comprising positions for oligonucleotides, said
positions defined by x and y coordinates; ii) a plurality of
oligonucleotides, each oligonucleotide comprising a sequence
complementary to a different portion of the sequence of said target
nucleic acid; and iii) a plurality of corresponding circular DNA
templates, each circular DNA template comprising a different
portion of the sequence of said target; b) immobilizing each of
said oligonucleotides in one of said positions on said solid
support to create an ordered array comprising a plurality of
immobilized oligonucleotides; c) adding to each immobilized
oligonucleotide of said ordered array a corresponding circular DNA
template under conditions such that said immobilized
oligonucleotide hybridizes to said corresponding circular DNA
template to create a plurality of primed circular templates; and d)
extending said primed circular templates to create an ordered
redundant array of extended immobilized oligonucleotides, each
extended immobilized oligonucleotide comprising at least two copies
of said portion of said sequence of said target nucleic acid.
14. The method of claim 13, wherein said oligonucleotides are
immobilized on a solid surface by a chemical linkage.
15. The method of claim 13, wherein said oligonucleotides are
immobilized on said solid surface by the 5' end of said
oligonucleotides.
16. The method of claim 13, wherein said oligonucleotides are
approximately 17 bases in length.
17. The method of claim 13 wherein said solid surface is selected
from a group of materials comprising silicon, metal, and glass.
18. The method of claim 13 wherein said immobilized
oligonucleotides are attached to a complimentary nucleic acid
stabilizer sequence.
19. The method of claim 13, wherein said circular nucleic acid
template is bacteriophage DNA.
20. The method of claim 13, wherein said circular nucleic acid
template is non-bacteriophage DNA.
21. The method of claim 13, wherein said extending in step (d) is
achieved with a polymerase.
22. The method of claim 21, wherein said polymerase is selected
from a group comprising E. coli. DNA polymerase I, a fragment of E.
coli. DNA polymerase I, or .PHI.29 DNA polymerase.
23. An ordered redundant array of immobilized oligonucleotides
produced according to the method of claim 13.
24. A method of hybridizing target nucleic acid fragments,
comprising: a) providing i) the ordered redundant array of extended
immobilized oligonucleotides of claim 13; and ii) a plurality of
fragments of a target nucleic acid; and b) bringing said fragments
of said target nucleic acid into contact with said array under
conditions such that at least one of said fragments hybridizes to
one of said extended immobilized oligonucleotides on said array.
Description
PRIOR APPLICATIONS
[0001] The present application claims priority from United States
Provisional Patent Application Serial No. 60/081,254, filed Apr. 9,
1998. This prior application is incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention generally relates to high density
nucleic acid arrays and methods of synthesizing oligonucleotides on
a solid surface. Specifically, the present invention contemplates
the use of stabilized nucleic acid primer sequences immobilized on
solid surfaces, and circular nucleic acid sequence templates
combined with the use of isothermal rolling circle amplification to
thereby increase oligonucleotide concentrations in a sample or on
an array of oligonucleotides.
BACKGROUND OF THE INVENTION
[0004] It is estimated that the human genome encodes from 60,000 to
100,000 different genes, and that certain mutations in the genome
lead to dysfunctional proteins, giving rise to a multitude of
diseases. Assays capable of detecting the presence of particular
mutations in a DNA sample are of substantial importance in
forensics, medicine, epidemiology, public health, and in the
prediction and diagnosis of disease. Such assays can be used, for
example, to identify the causal agent of an infectious disease, to
predict the likelihood that an individual will suffer from a
genetic disease, to determine the purity of drinking water or milk,
or to identify tissue samples.
[0005] Technologies are presently available that automate the
processing and interpretation of such assays. For example, U.S.
Pat. No. 5,874,219 to Rava, et al., teaches processing multiple
chip assays by providing biological chips comprising molecular
probe arrays. The biological chip is subjected to manipulation by
fluid handling devices that automatically perform steps to carry
out reactions between target molecules in the samples and probes.
The chip is further subjecting to a reader that examines the probe
arrays to detect any reactions between target molecules and probes.
While this sophisticated technology is useful, the sensitivity of
detection assays generally is often limited by the concentration at
which a particular target nucleic acid molecule is present in a
sample. Thus, methods that are capable of amplifying the
concentration of nucleic acid molecules must be developed as
important adjuncts to detection assays.
[0006] Methods of synthesizing desired single stranded DNA
sequences are well known to those of skill in the art. In
particular, methods of synthesizing oligonucleotides are found in,
for example, Oligonucleotide Synthesis: A Practical Approach, Gait,
ed., IRL Press, Oxford (1984). Methods of forming large arrays of
oligonucleotides, peptides and other polymer sequences have been
devised. Of particular note, Pirrung et al., U.S. Pat. No.
5,143,854, incorporated herein by reference, disclose methods of
forming arrays of peptides, oligonucleotides and other polymer
sequences using, for example, light-directed synthesis techniques.
However, the above techniques produces only a relatively low
concentrations of DNA; that is, the number of DNA on the array is
limited to surface area.
[0007] One approach for overcoming the limitation of DNA
concentration is to selectively amplify the nucleic acid molecule
whose detection is desired prior to performing the assay.
Recombinant DNA methodologies capable of amplifying purified
nucleic acid fragments in vivo have long been recognized.
Typically, such methodologies involve the introduction of the
nucleic acid fragment into a DNA or RNA vector, the clonal
amplification of the vector, and the recovery of the amplified
nucleic acid fragment. An example of such methodologies are
provided by, for example, Molecular Cloning, A Laboratory, Manual,
2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press, 1989), incorporated herein by reference. However,
these methods are limited because the concentration of a target
molecule in a sample under evaluation is so low that it cannot be
readily cloned.
[0008] In an effort to solve such limitations, other methods of in
vitro nucleic acid amplification have been developed that employ
template directed extension. In such methods, the nucleic acid
molecule is used as a template for extension of a nucleic acid
primer in a reaction catalyzed by polymerase. One such template
extension method is the "polymerase chain reaction" ("PCR"); see
Mullis, K. et al., Cold Spring Harbor, Symp. Quant. Biol.,
51:263-273 (1986), incorporated herein by reference. PCR technology
has several deficiencies. First, it requires the preparation of two
different primers which hybridize to two oligonucleotide sequences
of the target sequence flanking the region that is to be amplified.
The concentration of the two primers can be rate limiting for the
reaction. A disparity between the concentrations of the two primers
can greatly reduce the overall yield of the reaction. The reaction
conditions chosen must be such that both primers "prime" with
similar efficiency. Since the two primers necessarily have
different sequences, this requirement can constrain the choice of
primers and require considerable experimentation. Finally, PCR
requires the thermocycling of the molecules being amplified. The
thermocycling requirement attenuates the overall rate of
amplification because further extension of a primer ceases when the
sample is heated to denature double-stranded nucleic acid
molecules. Thus, to the extent that the extension of any primer
molecule has not been completed prior to the next heating step of
the cycle, the rate of amplification is impaired.
[0009] Other known nucleic acid amplification procedures include
transcription-based amplification systems; for example, see Kwoh D.
et al.,Proc. Natl. Acad. Sci. (U.S.A.), 86:1173 (1989). These
methods are limited in that the amplification procedures depend on
the time spent for all molecules to have finished a step in a
cycling method. Particular molecules used to perform the method
have different enzymatic rates. Molecules with slower enzymatic
rates would slow down molecules with faster enzymatic rates in the
cycle. This slowing down of the faster acting enzymes leads to a
lower exponent of amplification, and hence, a lower concentration
of DNA. Examples of others systems developed to amplify nucleotide
sequences are described in U.S. Pat. No. 5,854,033 to Lizardi,
incorporated herein by reference. Lizardi, however, does not
describe solid surface immobilization of the primers used for
extension, as the Lizardi method is performed in solution. This
reference is therefore limited because it does not allow for the
immobilization of the oligonucleotides, does not form an array, and
hence suffers from the same deficiencies as the other methods
described above.
[0010] Clearly, there is a great need for DNA arrays that allow for
higher concentrations of DNA. Furthermore, approaches are needed to
synthesize the arrays and particular target nucleic acid molecules
at increased concentrations.
DEFINITIONS
[0011] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, for purposes of the present invention, the following
terms are defined below.
[0012] The term "rolling circle amplification" ("RCA") as used
herein describes a method of DNA replication and amplification that
results in a strand of nucleic acid comprising one or more copies
of a sequence that is a complimentary to a sequence of the original
circular DNA. This process for amplifying (generating complimentary
copies) comprises hybridizing an oligonucleotide primer to the
circular target DNA, followed by isothermal cycling (e.g., in the
presence of a ligase and a DNA polymerase). A single round of
amplification using RCA results in a large amplification of the
sequences in the circular target to obtain a high concentration the
desired oligonucleotide on a single strand of nucleic acid. Because
the desired nucleic acid sequence becomes the predominant sequence
(in terms of concentration) in the mixture, it is said to be "RCA
amplified". With RCA, it is possible to amplify a single copy of a
particular nucleic acid sequence to a level detectable by several
different methodologies (e.g., hybridization with a labeled probe;
incorporation of biotinylated primers followed by avidin-enzyme
conjugate detection; incorporation of .sup.32P-labeled
deoxynucleotide triphosphates, such as dCTP or dATP, into the
amplified segment). In particular, the resulting nucleic acid
comprising amplified nucleic acid sequences created by the RCA
process are, themselves, efficient templates for subsequent RCA
amplifications. Solid Surface Rolling Circle Amplification
("ssRCA") refers to RCA that occurs when the oligonucleotide which
hybridizes to the circular DNA is attached to a solid surface. The
term "RCA product" as used herein refers to the resultant nucleic
acid comprising at least three (and preferably many more) copies of
the desired sequence contained within the circular DNA.
[0013] The term "high density" as used herein refers to the high
number of nucleic acid repeated sequences that may be obtained by
the methods of the present invention. The term "nucleic acid
repeated sequences" as used herein refers to the sequential
repeating of a given nucleic acid sequence that is achieved by the
amplification of the rolling circle amplification arrays or methods
of the present invention. For example, the concentration of a
target species in a sample under evaluation is increased do to the
amplification of the template directed repeating extension. High
density in the present application is not dependant on the surface
density of the oligonucleotides; rather, density is volume
dependant ("volume density"). The definition of density in the
present invention therefore defines the volume density of
oligonucleotides in terms of the "Z" plane, or three-dimensional
space, as opposed to the prior art attempts to define density in
the "X/Y", or two-dimensional plane. Because density is not limited
by the physical constraints of the two-dimensional surface, the
potential number of oligonucleotides on the array is much greater.
The terms "ordered redundant array" and "ordered array" as used
herein, refer to the orientation of nucleic acid sequences on the Z
plane, or three-dimensional space, or in the in the X/Y, or
two-dimensional plane, respectively.
[0014] An ordered redundant array is "redundant" in the sense that
sequences of interest (e.g., used for hybridization) are repeated
in the array. Rather than achieving this redundacy by adding
repeated sequences to the X/Y plane of the solid support, the
present invention contemplates achieving redundancy by introducing
repeating sequences in the growing strand (in the Z dimension) as
the primer is extended using the circular template.
[0015] The term "nucleic acid repeat sequence" as used herein, can
be used interchangeably, and has the same meaning, as the term
"concatamer". A DNA concatamer consists of two or more DNA
fragments which have been joined to produce a single DNA chain.
This product can be single-stranded or double-stranded DNA.
Usually, concatamers consist of a specific nucleotide sequence
which is repeated. Concatamers usually consist of several to
hundreds of repeats. A "dimer" is defined as two repeats. A
"trimer" is defined as three repeats. A "tetramer" is defined as
four repeats. Concatamers are usually more than several repeats.
For example, if the monomeric nucleotide sequence is: (N1-N2-N3- .
. . -Nn), where N1 through Nn define a specific nucleotide
sequence, then (N1-N2-N3- . . . Nn)m is a concatamer if that
sequence contains m repeats. The total length of the concatamer is
thus n.times. m. The volume density of the present invention can be
calculated by multiplying the number of concatamers in a given
oligonucleotide by the number of oligonucleotides attached to the
solid surface. The only limitations on the volume density are the
respective half life of the polymerases, or the amount of precursor
deoxyribonucleotides or ribonucleotides that are added during the
polymerase reaction.
[0016] The term "hybridization" as used herein involve the
annealing of a complementary sequence to the target nucleic acid
(the sequence to be detected). The ability of two polymers of
nucleic acid containing complementary sequences to find each other
and anneal through base pairing interaction is a well-recognized
phenomenon. The initial observations of the "hybridization" process
by Marmur and Lane, Proc. Natl. Acad. Sci. USA, 46:453 (1960) and
Doty et al., Proc. Natl. Acad. Sci. USA, 46:461 (1960) have been
followed by the refinement of this process into an essential tool
of modern biology. The term "secondary hybridization" as used
herein refers to the annealing of probe or tagging molecules to the
extended "nucleic acid repeated sequences" or "concatamers" of the
present invention.
[0017] The term "complementary" or "substantially complementary" as
used herein refers to the hybridization or base pairing between
nucleotides or nucleic acids, such as, for instance, between the
two strands of a double stranded DNA molecule or between an
oligonucleotide primer and a primer binding site on a single
stranded nucleic acid to be sequenced or amplified. Complementary
nucleotides are, generally, A and T (or A and U), or C and G. Two
single stranded RNA or DNA molecules are said to be substantially
complementary when the nucleotides of one strand, optimally aligned
and compared and with appropriate nucleotide insertions or
deletions, pair with at least about 80% of the nucleotides of the
other strand, usually at least about 90% to 95%, and more
preferably from about 98 to 100%. Alternatively, substantial
complementarity exists when an RNA or DNA strand will hybridize
under selective hybridization conditions to its complement.
Typically, selective hybridization will occur when there is at
least about 65% complementarity over a stretch of at least 14 to 25
nucleotides, preferably at least about 75%, more preferably at
least about 90% complementarity. See M. Kanehisa, Nucleic Acids
Res., 12:203 (1984), incorporated herein by reference. The term "at
least a portion of" as used herein, refers to the complimentarity
between a circular DNA template and an oligonucleotide primer of at
least one base pair.
[0018] Partially complementary sequences will hybridize under low
stringency conditions. This is not to say that conditions of low
stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific (i.e., selective) interaction. The
absence of non-specific binding may be tested by the use of a
second target which lacks even a partial degree of complementarity
(e.g., less than about 30% identity); in the absence of
non-specific binding the probe will not hybridize to the second
non-complementary target.
[0019] Low stringency conditions when used in reference to nucleic
acid hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of 5.times.
SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.multidot.H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times. Denhardt.times.s reagent [50.times. Denhardt.times.s
contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA
(Fraction V; Sigma)] and 100 .mu.g/ml denatured salmon sperm DNA
followed by washing in a solution comprising 5.times. SSPE, 0.1%
SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0020] High stringency conditions when used in reference to nucleic
acid hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of 5.times.
SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.multidot.H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times. SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0021] When used in reference to nucleic acid hybridization the art
knows well that numerous equivalent conditions may be employed to
comprise either low or high stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of
either low or high stringency hybridization different from, but
equivalent to, the above listed conditions.
[0022] "Stringency" when used in reference to nucleic acid
hybridization typically occurs in a range from about
T.sub.m-5.degree. C. (5.degree. C. below the T.sub.m of the probe)
to about 20.degree. C. to 25.degree. C. below T.sub.m. As will be
understood by those of skill in the art, a stringent hybridization
can be used to identify or detect identical polynucleotide
sequences or to identify or detect similar or related
polynucleotide sequences. Under "stringent conditions" a nucleic
acid sequence of interest will hybridize to its exact complement
and closely related sequences.
[0023] The term "nucleic acid sequence" as used herein is defined
as a molecule comprised of two or more deoxyribonucleotides or
ribonucleotides. The exact length of the sequence will depend on
many factors, which in turn depends on the ultimate function or use
of the sequence. The sequence may be generated in any manner,
including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof. Due to the amplifying
nature of the present invention, the number of deoxyribonucleotides
or ribonucleotides bases within a nucleic acid sequence may be
virtually unlimited. The term "oligonucleotide," as used herein, is
interchangeably synonymous with the term "nucleic acid
sequence".
[0024] The term "primer" as used herein refers to a sequence of
nucleic acid attached to a solid surface, and used for rolling
circle amplification. The primer may be complimentary or
substantially complimentary to a portion of the circular
template.
[0025] The term "Solid Surface" as used herein refers to a material
having a rigid or semi-rigid surface. Such materials will
preferably take the form of chips, plates, slides, small beads,
pellets, disks or other convenient forms, although other forms may
be used. In some embodiments, at least one surface of the solid
surface will be substantially flat. In other embodiments, a roughly
spherical shape is preferred.
[0026] The term "nucleic acid sequence of interest" refers to any
nucleic acid sequence the manipulation of which may be deemed
desirable for any reason by one of ordinary skill in the art (e.g.,
for nucleic acid sequence amplification or detection purposes). The
term "sequence of interest being different" refers to a comparison
of the base sequence of at least two nucleic acid molecules. The
differences may be single base differences or may involve many
bases.
SUMMARY OF THE INVENTION
[0027] Arrays of oligonucleotides have previously been proposed for
sequencing target nucleic acid by hybridization. In the prior
methods, groups of contiguous bases are determined simultaneously
by hybridization, rather than by sequencing one base at a time
using conventional Sanger sequencing. The approach utilizes
oligonucleotides (of length n) immobilized on a solid support in
large numbers in an ordered array. The array is designed such that
the sequences of all of the oligonucleotides (collectively)
represents the complete sequence of the target. Thus the target can
be fractionated into smaller pieces which can hybridize to the
oligonucleotides on the array.
[0028] One important drawback to current approaches for such arrays
is the inconvenience of chemically synthesizing each distinct
oligonucleotide necessary to represent (collectively) the entire
target sequence, particularly if such oligos are long (e.g.,
greater than twenty nucleotides). Moreover, since space on the
solid support is limited--and yet large numbers of such
oligonucleotides are needed--there is little room for redundancy,
i.e., an array containing two identical nucleotide sequences.
[0029] The present invention contemplates solving both problems by
utilizing circular nucleic acid in the production of the array. The
method contemplates a solid support with positions for
oligonucleotides defined by x and y coordinates. At each position
(e.g., x1, y1; x1, y2; etc.), a oligonucleotide is immobilized. In
one embodiment (see FIG. 1A), the same oligonucleotide (i.e., an
oligonucleotide with the same generic nucleotide sequence) is
immobilized in every position (or nearly every position, with some
positions left empty or for controls) on the solid support. In this
embodiment, a circular DNA template comprising i) a region having a
sequence complementary to at least a portion of said generic
oligonucleotide (shown in FIG. 1A as AAAACC), and ii) a region
comprising a sequence of interest (shown in FIG. 1A as QQQQetc.) is
employed. The region having a sequence complementary to at least a
portion of said generic oligonucleotide permits hybridization of
the circular template to the immobilized oligonucleotide (FIG. 1A
is merely illustrative and is not meant to limit the sequence or
length of the sequence of this hybridizing region; indeed, regions
larger than six nucleotides are preferred). The sequence of
interest may comprise a portion of the sequence of a target of
interest (e.g., cancer gene, histocompatibility gene, etc.). To
create an array with diverse sequences, a circular DNA template is
added at each position (e.g., by a robot), wherein each circular
DNA template added has a unique sequence of interest (e.g., a
different sequence corresponding to a unique portion of a target).
Each circular DNA template is added under conditions such that the
circular DNA template hybridizes with the generic immobilized
oligonucleotide, said immobilized oligonucleotide thereafter being
extended by a polymerase to create a unique extended nucleic acid
strand at each position on the solid support, such extended strands
comprising two or more (and more typically three or more, and morc
preferably, ten or more, and still more preferably more than fifty)
copies of the sequence of interest. Thereby, an array is created
with redundancy in the z dimension (i.e., out of the x and y plane
of the solid support). Variations on this first embodiment, include
(but are not limited to) circular templates with more than one copy
of the sequence of interest (see the larger circular template in
FIG. 1A, which is merely illustrative and not intended to limit
such templates to comprising only two copies, i.e., greater than
two copies is also contemplated). Of course, such larger templates
still need a region that will hybridize to the generic immobilized
oligonucleotide. Such larger templates may (or may not) contain
other regions such as regions that separate each copy of the
sequence of interest (such a separating region is depicted in FIG.
1A as WWWW, the number of nucleotides "W" being variable between 0
and 100). The invention contemplates that such regions that
separate each copy of the sequence of interest can be additional
regions that can hybridize to the generic immobilized
oligonucleotide (e.g. the WWWW of FIG. 1A could be replaced with
yet another region defined by AAAACC).
[0030] FIG. 1B shows an alternative embodiment, wherein a generic
immobilized oligonucleotide is not employed. In this case, each
immobilized oligonucleotide comprises a region comprising a
different sequence (FIG. 1B is merely illustrative, showing one
such oligonucleotide with one such unique sequence), each different
sequence being complementary to a sequence of interest on a
circular template. The circular DNA template comprises i) a first
region comprising a sequence of interest (shown in FIG. 1B as
ACGATAAAACC) and ii) a second region (shown in FIG. 1B as QQQQetc.)
is employed. Because each immobilized oligonucleotide is unique,
the region having a sequence complementary to at least a portion of
the circular template permits hybridization only to the
"corresponding" circular template; thus, the region permitting
hybridization on the circular template is also the sequence of
interest (FIG. 1B is merely illustrative and is not meant to limit
the sequence or length of the sequence of this hybridizing region;
indeed, regions larger than thirteen nucleotides are preferred).
Each circular DNA template is added under conditions such that the
circular DNA template hybridizes and thereafter the oligonucleotide
is extended by a polymerase to create a unique extended nucleic
acid strand at each position on the solid support, such extended
strands comprising two or more (and more typically three or more,
and more preferably, ten or more, and still more preferably more
than fifty) copies of the sequence of interest. Thereby, an array
is created with redundancy in the z dimension (i.e., out of the x
and y plane of the solid support). Variations on this first
embodiment, include (but are not limited to) circular templates
with more than one copy of the sequence of interest (see the larger
circular template in FIG. 1B, which is merely illustrative and not
meant to limit the invention on only a certain number of copies).
Such larger templates may (or may not) contain other regions such
as regions that separate each copy of the sequence of interest
(such a separating region is depicted in FIG. 1B as WWWW, the
number of nucleotides "W" being variable between 0 and 100). The
number of such other regions not being limited to the number of
copies (although it may be convenient to insert one such region
between each copy of the sequence of interest).
[0031] The present invention makes available novel nucleic acid
arrays, and novel methods to synthesize nucleic acid sequences on
solid surfaces using nucleic acid primer sequences, circular
nucleic acid template sequences, and isothermal rolling-circle
amplification, as well as methods of using the arrays and methods
for the detection of nucleic acid sequences.
[0032] The present invention contemplates an array of nucleic acid
sequences, comprising a solid support having at least one surface;
and a plurality of nucleic acid sequences attached to said surface
of said solid support, wherein each said nucleic acid sequence is
attached to said surface on different physical areas of said
surface, and each nucleic acid sequence may contain sequentially
identical or different deoxyribonucleotide or ribonucleotide bases.
It is not intended that the present invention be limited to
identical nucleic acid sequence within the arrays. A variety of
arrays are contemplated. For example, the nucleic acid sequences of
the present invention may be sequentially identical or different
within the array. The present invention also contemplates varying
sizes of the circular template.
[0033] Another embodiment of the present invention is to provide
nucleic acid arrays that are produced by a process comprising the
steps of providing circular single-stranded nucleic acid templates
having a sequence, and immobilized linear partially single-stranded
nucleic acid oligonucleotide primers having a sequence
complementary to at least a portion of said sequence of said
circular single-stranded nucleic acid templates, and mixing said
circular single-stranded nucleic acid templates with said partially
single-stranded nucleic acid oligonucleotide primers to create a
mixture under conditions such that at least a portion of said
circular single-stranded nucleic acid templates hybridize to said
partially single-stranded oligonucleotide primers, and treating
said mixture under conditions such that said immobilized linear
partially single-stranded nucleic acid primers are extended. It is
not intended that the present invention be limited to the exact
conditions in the above process to produce the nucleic acid arrays.
A variety of conditions are contemplated. For example, the arrays
of the present invention may be produced with simple condition
modifications known to those skilled in the art (e.g., varying
nucleic acid sequences, polymerase types, ligase types, or surface
types). Furthermore, It is not intended that the present invention
be limited to the exact sequence of method steps described in the
above process to produce the nucleic acid arrays. Other steps are
contemplated. For example, upon primer sequence attachment to a
solid surface, a second primer sequence may be hybridized to the
first primer, followed by the addition of circular or semi-circular
template sequences to be hybridized to the second primer sequence.
RCA can then be carried out as in the first description above, and
the resulting concatamer visualized via a florescent tagging or
other detection method. The resulting array is therefore
contemplated as a product of this series of steps.
[0034] Another embodiment of the present invention is to provide a
method of determining the amount of specific template nucleic acid
sequences present in a sample where the signal level measured is
proportional to the amount of a template sequence in a sample and
where the ratio of signal levels measured for different template
sequences substantially matches the ratio of the amount of the
different template sequences present in the sample.
[0035] Another embodiment of the present invention is to provide a
method of detecting and determining the amount of multiple specific
template nucleic acid sequences in a single sample where the ratio
of signal levels measured for different template nucleic acid
sequences substantially matches the ratio of the amount of the
different template nucleic acid sequences present in the
sample.
[0036] Another embodiment of the present invention is to provide a
method of detecting the presence of template nucleic acid sequences
representing individual alleles of a template genetic element.
[0037] Another embodiment of the present invention contemplates the
use of molecular stabilizer nucleic acid sequences (e.g.,
stabilized with a second primer thereby forming a partially
double-stranded and partially single-stranded primer) to reduce
stearic hindrance of a nucleic acid primer sequence and thereby
increase fidelity of the isothermal rolling circle amplification of
DNA sequences. The present invention is not intended to be limited
by any specific stabilizer length or configuration. A variety of
lengths and configurations are contemplated. For example, a
stabilizer nucleic acid sequence may be long or short, single or
double stranded, or be comprised of any type of nucleic acid,
including polypeptide nucleic acid (PNA).
[0038] Another embodiment of the present invention contemplates an
allele-specific nucleic acid template sequence circularization,
mediated by DNA ligase. A DNA template is considered circularized
or "closed" when perfect hybridization between the template
sequence and the nucleic acid primer sequence allows ligase to
covalently circularize the template. Mismatches around the ligation
site prevent template circularization, resulting in a
non-circularized or "open" template. DNA polymerase is then used to
preferentially amplify the closed templates, via ssRCA. The present
invention is not intended to be limited by any specific ligation
site hybridization. A variety of ligation site hybridizations are
contemplated. For example, hybridization can be achieved by any
base pairs that will bind to their complimentary base, regardless
of the sequence order.
[0039] Another embodiment of the present invention contemplates the
detection of single-nucleotide polymorphisms (SNP). Successful SNP
detection can be performed by three coupled steps: hybridization of
the primer nucleic acid sequence to nucleic acid template sequence,
proofreading by DNA ligase, and replication of the template
sequence (see FIG. 2). The invention contemplates a nucleic acid
template sequence, P1, that was designed to circularize via
hybridization on two immobilized nucleic acid primer sequences, T1
and T2. T1 and T2 differ by only a single nucleotide such that the
P1/T1 complex forms 30 contiguous base pairs, while the P1/T2
complex contains a C:T mismatch at the 5'-terminus of P1. This
sequence recognition step is similar to hybridization on arrays,
and it is difficult to distinguish between the P1/T1 and P1/T2
complexes because, in general, 3'- and 5'-end mismatches do not
greatly affect duplex stability. The proofreading step is mediated
by DNA ligase, because this enzyme is sensitive to single-base
mismatches. When hybridized around the ligation point, the
3'-hydroxyl and 5'-phosphate of P1 can be successfully joined to
form a covalently-closed circular template. Thus, DNA ligase can be
used as a proofreading enzyme to change the topological structure
of the template in a (+) or (-) type reaction. The (+) being the
"closed" nucleic acid template sequence, and the (-) constituting
the "open" nucleic acid template sequence. The ssRCA is achieved by
DNA polymerase in an extension reaction primed by the 3'-hydroxyl
of the primer sequences. In the case of circularized (closed)
templates, extension occurs via ssRCA. In contrast, only partial
extension is possible with non-circularized (open) templates. Using
isotopically labeled nucleotides during the extension reaction, a
10.sup.2-10.sup.3 fold more nucleotide incorporation with P1/T1
than with 5'- or 3'-mismatched template/primer complexes was
observed. However, the present invention is not limited to the
specific sequence of steps described above. Different ordering of
the steps in the method are also contemplated. For example, upon
primer sequence attachment to a solid surface, a second primer
sequence may be hybridized to the first primer, followed by the
addition of circular or semi-circular template sequences to be
hybridized to the second primer sequence. RCA can then be carried
out as in the first description above, and the resulting concatamer
visualized via a florescent tagging or other detection method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A and 1B are schematic representations of
oligonucleotide primers and oligonucleotide templates. For much
greater description of these Figures, see the summary of the
invention section, supra.
[0041] FIG. 2 is a schematic representation of Solid Surface
Rolling Circle Amplification ("ssRCA"). The nucleic acid primer
sequence is attached first to a surface and when a circular nucleic
acid template is added, the primer hybridizes to the template. The
template is then replicated by DNA polymerase (i.e., the primer is
extended).
[0042] FIG. 3A is a diagram of Oligonucleotide sequences used in
RCA. The 74-base template nucleic acid sequences, P1 and P2 were
purchased with a 5'-phosphate, PAGE purified from Bio-Synthesis,
Inc. The 53-base primer nucleic acid sequences, T1 and T2 were
purchased with a 5'-biotin modification, HPLC purified from Operon
Technologies. The 5'-15 bases, and 3'-15 bases of P1 are
complementary to positions 23-38, and 39-44 of T1, respectively.
The P1/T2 complex contained a C:T mismatch at the 5'-terminus of
P1; the P2/T2 complex is similar in sequence to P1/T2, except that
the C:T mismatch occurs at the 3'-terminus of P2. The single
stranded nucleic acid sequence, A12 (Operon Technologies), is
complementary to the 5'-end of T1 and T2, and is used for
structural support in chip-based experiments.
[0043] FIGS. 3B and 3C depict the effect of polymerization using a
circular (ligated) template sequence as compared to polymerization
on an uncircularized (non-ligated) template sequence. FIG. 3B
illustrates that as RCA continues around the circular template
sequence, the RCA product is displaced and rolls providing
essentially unlimited concatamer length. FIG. 3C shows the results
of DNA polymerase on an uncircularized (non-ligated) template
sequence. As can bee seen, only a partial primer extension is
achieved in this instance. The difference in ligated versus
non-ligated sequences is monitored using .sup.32P-labeled
nucleotides which are incorporated into the polymerizing DNA. Thus
a ligated template is observed as having more radioactivity after
DNA polymerization compared to an unligated template.
[0044] FIG. 4A represents the kinetics of RCA produced by .PHI.29
or E. coli DNA polymerases. RCA amplifications were resolved on a
0.8% agarose gel, and stained with SYBRII, which is a DNA
intercalating dye which can stain both double-stranded and
single-stranded DNAs, but shows enhanced fluorescence when bound to
duplexes. The incubation times were 0, 1/4, 1/2, 1, 3, 6 and 24 hr,
respectively. One kb and 100 bp makers were loaded in the first and
last lanes, respectively. RCA was performed using ligated (+) or
non-ligated (-) P1 nucleic acid template sequences as indicated at
the bottom of the image, and T1 nucleic acid primer sequences.
[0045] FIG. 4B represents the fluorescence intensity measurement of
RCA concatamers that was determined by scanning the concatamers
with a Molecular Dynamics STORM imager. The x-axis represents RCA
incubation times, and the y-axis is florescence. The readings were
taken by the hybridization of 5'-fluorescein-labeled (F)
oligonucleotide (5'-FAACTAATACACCAA) to RCA concatamers immobilized
on nitrocellulose membranes by UV crosslinking. Hybridization was
carried out overnight at 25.degree. C. in 6.times. SSC buffer,
followed by a 15 min wash in 6.times. SSC, and a 15 min wash in
2.times. SSC.
[0046] FIG. 5 depicts autoradiograms that show a solid surface
format using a patterned streptavidin microwell chip. Each chip
comprised of six microwells. Two target oligonucleotide sequences
differing by a single base substitution were spotted in triplicate
in the microwells. The following assays were performed in parallel
on the immobilized target sequences. Assay volumes were 25 ml. A
hybridization chamber was placed over the chip to contain the
solutions. First, the chip was incubated with templates overnight,
followed by a wash. Then, ligation was performed for 30 min,
followed by a wash. Lastly, DNA polymerase and nucleotides,
including [a-.sup.32P]-dTTP were added and incubated for 6 hours.
FIG. 5 is an autoradiogram of the chip reaction using (A) .PHI.29
DNA polymerase or (B) E. coli DNA polymerase. The left columns in
the autoradiograms are wells containing primer sequences which
hybridize perfectly to the template sequences; the right column
contain primers which create a 5'-end mismatch with the templates.
Although both polymerases yielded approximately 3-fold differences
between the matched and mismatched targets, .PHI.29 DNA polymerase
generated more, or longer concatamers than E. coli polymerase.
[0047] FIG. 6 illustrates a phosphorimager scan of three wells of
enzymatically-enhanced DNA arrays after primer extension with
.PHI.29 DNA polymerase in the presence of [.alpha.-32.sub.p] dTTP.
Primer T1 and T2 were immobilized in the left and middle wells,
respectively, and the right well (Bkg) was left blank for a
background reference. P2 was used as the template for hybridization
and circularization. The plot above the image shows the counts for
each pixel in the scan. Total radioactive counts for T1, T2 and the
background were 3,180,000, 255,000 and 43,000, respectively. This
indicates that the discrimination of single-based polymorphisms was
increased at a 12 fold rate from T1 over T2, which in turn
increases the volume density of the nucleic acid sequences.
DESCRIPTION OF THE INVENTION
[0048] Some of the reagents used in the practice of the present
invention can be made using conventional techniques of molecular
biology. Such techniques are described in the literature. For
example, see Molecular Cloning, A Laboratory, Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). In the
experimental disclosure which follows, the following abbreviations
apply: eq (equivalents); M (Molar); .mu.M (micromolar); N (Normal);
mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol
(nanomoles); gm (grams); mg (milligrams); .mu.g (micrograms); ng
(nanograms); L (liters); ml (milliliters); .mu.l (microliters); cm
(centimeters); mm (millimeters); .mu.m (micrometers); nm
(nanometers); .degree. C. (degrees Centigrade); hr (hour); sec
(second); min (minutes).
[0049] The present invention contemplates a method of generating an
array, comprising providing a solid support comprising a plurality
of positions for oligonucleotides, the positions defined by x and y
coordinates; a plurality of identical oligonucleotides, each
oligonucleotide comprising a sequence; and a plurality of unique
circular DNA templates, each circular DNA template comprising a
sequence of interest and a region complementary to at least a
portion of the sequence of the oligonucleotides, the sequence of
interest being different for each circular template; immobilizing
one oligonucleotide from the plurality of identical
oligonucleotides in each of the positions on the solid support to
create an ordered array comprising a plurality of identical
immobilized oligonucleotides; adding to each immobilized
oligonucleotide of the ordered array a circular DNA template from
the plurality of the unique circular DNA templates under conditions
such that the immobilized oligonucleotide hybridizes to the
circular DNA template to create a plurality of primed circular
templates, each primed circular template comprising a different
sequence of interest; and extending each of the primed circular
templates to create an extended immobilized oligonucleotide
comprising at least two copies of the sequence of interest, thereby
generating an ordered redundant array.
[0050] In one embodiment of the present invention, oligonucleotides
that are immobilized by the 5' end on a solid surface by a chemical
linkage are contemplated. the oligonucleotides may approximately 17
bases in length, although other lengths are also contemplated.
[0051] In one embodiment of the present invention, the solid
surface is selected from a group of materials comprising silicon,
metal, and glass.
[0052] In another embodiment of the present invention, the
immobilized oligonucleotides are attached to a complimentary
nucleic acid stabilizer sequence.
[0053] In another embodiment of the present invention, the circular
nucleic acid template is bacteriophage DNA, or non-bacteriophage
DNA.
[0054] In another embodiment of the present invention, the
extending step is achieved with a polymerase, wherein the
polymerase is selected from a group comprising E. coli. DNA
polymerase I, a fragment of E. coli. DNA polymerase I, or .PHI.29
DNA polymerase.
[0055] In another embodiment of the present invention, an ordered
redundant array of immobilized oligonucleotides produced according
to the above method is contemplated.
[0056] In another embodiment of the present invention, a method of
hybridizing target nucleic acid fragments is contemplated providing
the ordered redundant array of extended immobilized
oligonucleotides of the above methods, a plurality of fragments of
a target nucleic acid; and bringing the fragments of the target
nucleic acid into contact with the array under conditions such that
at least one of the fragments hybridizes to one of the extended
immobilized oligonucleotides on the array.
[0057] In another embodiment of the present invention, a method of
generating an array capable of hybridizing to fragments of a target
nucleic acid is contemplated, comprising providing a solid support
comprising positions for oligonucleotides, the positions defined by
x and y coordinates; a plurality of oligonucleotides, each
oligonucleotide comprising a sequence complementary to a different
portion of the sequence of the target nucleic acid; and a plurality
of corresponding circular DNA templates, each circular DNA template
comprising a different portion of the sequence of the target;
immobilizing each of the oligonucleotides in one of the positions
on the solid support to create an ordered array comprising a
plurality of immobilized oligonucleotides; adding to each
immobilized oligonucleotide of the ordered array a corresponding
circular DNA template under conditions such that the immobilized
oligonucleotide hybridizes to the corresponding circular DNA
template to create a plurality of primed circular templates; and
extending the primed circular templates to create an ordered
redundant array of extended immobilized oligonucleotides, each
extended immobilized oligonucleotide comprising at least two copies
of the portion of the sequence of the target nucleic acid.
[0058] In one embodiment of the present invention, oligonucleotides
that are immobilized by the 5' end on a solid surface by a chemical
linkage are contemplated. the oligonucleotides may approximately 17
bases in length, although other lengths are also contemplated.
[0059] In one embodiment of the present invention, the solid
surface is selected from a group of materials comprising silicon,
metal, and glass.
[0060] In another embodiment of the present invention, the
immobilized oligonucleotides are attached to a complimentary
nucleic acid stabilizer sequence.
[0061] In another embodiment of the present invention, the circular
nucleic acid template is bacteriophage DNA, or non-bacteriophage
DNA.
[0062] In another embodiment of the present invention, the
extending step is achieved with a polymerase, wherein the
polymerase is selected from a group comprising E. coli. DNA
polymerase I, a fragment of E. coli. DNA polymerase I, or .PHI.29
DNA polymerase.
[0063] In another embodiment of the present invention, an ordered
redundant array of immobilized oligonucleotides produced according
to the above method is contemplated.
[0064] In another embodiment of the present invention, a method of
hybridizing target nucleic acid fragments, is contemplated
comprising the ordered redundant array of extended immobilized
oligonucleotides of the above methods; a plurality of fragments of
a target nucleic acid; and bringing the fragments of the target
nucleic acid into contact with the array under conditions such that
at least one of the fragments hybridizes to one of the extended
immobilized oligonucleotides on the array.
[0065] However, it is not intended that the present invention be
limited to specific examples of oligonucleotide primers or
templates, specific reagents, or specific solid surfaces. A variety
of oligonucleotide primers or templates, specific reagents, and
specific solid surfaces are contemplated.
[0066] Importantly, it is not necessary to the successful use of
the compositions, products, and methods of the present invention
that one understand the precise mechanism by which the invention is
achieved.
EXPERIMENTAL
[0067] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
Example 1
Isolation of m13mp18 Circular DNA Templates
[0068] In this example, Escherichia coli bacteria were inoculated
with m13mp18 bacteriophage (M13). The infected cells were grown
overnight allowing the phage to multiply. Then the single stranded
nucleic acid template sequences were harvested by centrifugation.
As follows:
[0069] (a) Prepare LB Media.
[0070] (b) Grow E. coli TG1 on 1% LB-Agar plates for 1 day at
37.degree. C.
[0071] (c) Prepare 2.times. TY Media.
[0072] (d) Grow an isolated colony of TG1 in 2.times. TY at
37.degree. C. overnight.
[0073] (e) Transfer 500 ml of TG1 to 25 ml 2.times. TY and
inoculate with 1 ml of M13mp18 stock. Grow for 5 hours at
37.degree. C.
[0074] (f) Centrifuge at 15000 g for 15 min. Save supernatant.
[0075] (g) Add 1 g PEG 800 and 0.75 g NaCl. Stir for 40 mins.
[0076] (h) Centrifuge at 10000 g at 4.degree. C. for 20 min.
[0077] (i) Add 1:8 ml Tris-HCl to dissolve and then collect in
tubes.
[0078] (j) Add 150 ml phenol (pH 8). Vortex 30 sec. Let stand 1
min. Repeat. Centrifuge at 12000 g at 4.degree. C. for 2 min.
Remove top layer to new tube.
[0079] (k) Add 150 ml chloroform. Vortex briefly. Let stand.
Centrifuge at 12000 g at 4.degree. C.
[0080] (l) Transfer upper phase to new tubes that contain 900 ml
25:1 EtOH:3 M Sodium.
[0081] (m) Recover precipitate by centrifuging at 12000 g at
4.degree. C. for 10 min.
[0082] (n) Gently aspirate and recentrifuge (.about.15 sec) to
remove residual supernate.
[0083] (o) Add 200 ml 70% EtOH. Vortex briefly. Centrifuge and
immediately remove supernatant as above.
[0084] (p) Use speedvac to dry pellet of residual EtOH.
[0085] (q) Dissolve in 20 ml TE (pH 8.0).
[0086] (r) Repeat from step (j).
[0087] (s) Store at -20.degree. C.
[0088] The single stranded template sequences that are harvested
can be used as nucleic acid template sequences according to the
method of the present invention.
Example 2
Creation of Long Primers for ssRCA
[0089] This example involves two small PCR primers that were
selected and used to make a longer primer suitable for ssRCA. The
primers are selected so that the 5' end of the long primer could be
biotinylated and that 3' end will have .about.20 bases that are
complementary to M13. This example involved the creation of the 226
base RCA primer known as C9. C9 is a hybrid primer that was
constructed by performing PCR on a M13 derivative called MGP1-2.
The 5' region of C9 is mostly native T7 polymerase DNA and the 3
prime region is M13 DNA.
[0090] (a) Two primers were selected using computer program PRIME
from GCG computer package.
[0091] (i) P1: 5'-CAA TTT CAC ACA GGC CCA AG
[0092] (ii) P2: 5'-XCG TAA GAC TCA TGC TCA AGC X=Biotin
[0093] (b) PCR Reaction Mixture:
1 dNTP Primer 1 Primer 2 MGP MgCl.sub.2 10X PCR AmpliTaq Total [2.5
mM] [10 mM] [10 mM] 1-2 1:70 [25 mM] Buffer 1:8 Volume Final 375 mM
1 mM 1 mM 0.6 2.5 mM 1.25 X 5 U 20 ml Concl. ng/ml
[0094] (c) Thermal Controller Program:
2 Step Time Temp 1 1 min. 90.degree. C. 2 1 min. 56.degree. C. 3 1
min. 65.degree. C. 4 30 sec 90.degree. C. 5 25 times to step 2 6 7
min 72.degree. C. 7 99 hr. 4.degree. C. 8 END
[0095] The long primer sequences can be used as nucleic acid primer
sequences according to the method of the present invention.
Example 3
Creation of Short Primers for ssRCA
[0096] In similar reaction conditions as in Example 2, a shorter 17
base DNA primer was also made. It was biotinylated on the 5' end
and was entirely complimentary to m13mp18 DNA. A 17 base long
primer was used in a solid surface RCA reaction run in a 1% agarose
gel (results not shown). In these experiments, one lane was loaded
with single stranded circular M13 DNA which was used as a standard
of 7.25 kb, while other lanes were represented ssRCA reactions, and
still other lanes contained positive and negative controls (i.e.,
there were multiple positive control and multiple negative control
lanes used in this experiment). For the lanes containing the ssRCA
reactions, smearing and collection of DNA in the wells was
observed, as was the case of in the positive control lanes. The
negative control lanes generally showed no collection of DNA in the
wells. However, in two lanes containing negative controls,
collection of DNA was observed. The smearing is indicative of an
RCA reaction, where product sizes vary. Also, the collection of DNA
in the wells indicates a high molecular weight product (>20 kb).
The DNA collection in the wells of two lanes loaded with negative
control material was much smaller than that seen in the other RCA
and ssRCA lanes. In addition, the Reaction yield was higher with
the shorter primer. The short primer sequences of this example can
be used as nucleic acid primer sequences according to the method of
the present invention.
Example 4
Attachment of Single Stranded Primers to M-280 Stv Coated Magnetic
Beads
[0097] In this example, a protocol is demonstrated to purify a
biotinylated nucleic acid primer sequence from the complementary
nonbiotinylated nucleic acid sequence, and attach the biotinylated
nucleic acid sequence primer to the M-280 Stv coated magnetic beads
(Dynal Inc.). As follows:
[0098] (a) Wash 1.5 mg M-280 Stv coated magnetic beads (Dynal Inc.)
three times in TE(pH 8.0) on magnetic separator. Resuspend beads in
30 ml TE (pH 8.0). Store at 4.degree. C.
[0099] (b) Bring 100 pmoles of primer to final concentration of
0.01 M NaOH.
[0100] (c) Boil primer mix for 10 minutes in water bath.
[0101] (d) Immediately cool on ice for 2 min.
[0102] (e) To bead mixture of step (a), add primer mix.
[0103] (f) Let stand for 20 seconds on magnetic separator. Then
remove supernatant.
[0104] (g) Wash twice with TE (pH 8.0). Resuspend in 30 ml TE. (pH
8.0).
[0105] (h) Store at 4.degree. C.
[0106] This example represents the immobilization of a nucleic acid
primer sequence to a solid surface.
Example 5
ssRCA Reaction
[0107] This is an example of the protocol for the ssRCA reaction.
In this reaction, the single stranded DNA primer that was attached
to the bead in Example 4 is extended via ssRCA into a long linear
DNA molecule that is a concatamer of M13 DNA. This product remains
attached to the bead.
[0108] (a) ssRCA Reaction Mixture:
3 dNTP 10X PCR DNA Pol I @ [2.5 mM] Primer M13 1:4 Buffer Step 2
Total Volume Final .225 mM 1 mM 200 1 X 10U 100 ml Concl. ng/ml
[0109] (b) Thermal Controller Program:
4 Step Time/Temp. 1 3 min./60.degree. C. 2 3 min./60.degree. C. 3 7
hr./37.degree. C. 4 99 hr./4.degree. C. 5 END
Example 6
Template Circularization aqnd RCA in Solution
[0110] The sequences and base paring of a P1/T1 complex are shown
in FIG. 3A. P1 circularization was performed in solution by
combining equimolar ratios of P1 and T1 to a final concentration of
5 mM in TE (10 mM Tris-Cl, 1 mM EDTA) supplemented with 0.1 M NaCl,
heating the mixture to 95.degree. C., followed by slowly cooling to
room temperature. Ten pmol of the P1/T1 complex and 400 U T4 DNA
ligase (New England Biolabs) were suspended in 50 ml of T4 DNA
ligase buffer supplied by the manufacturer. The cocktail was
incubated at 16.degree. C. for 30 min in a PCR thermocycler, and
the reaction was terminated by heating the samples to 75.degree. C.
for 15 min. Non-ligated controls were made by omitting ligase from
the above reaction.
[0111] Both circular and linear P1 templates were used as templates
for RCA. P1 template sequences were circularized on T1 primer
sequences using T4 DNA ligase. Ligation products were analyzed on
12% denaturing polyacrylamide gels; circularized P1 sequence
product was observed as a supershifted band from the linear form.
Approximately 60% of the P1 templates were circularized when
equimolar ratios of P1 and T1 were ligated for 30 min at 16.
Aliquots of either linear or circular P1 were used as templates for
RCA reactions. The kinetics of RCA was explored by different
incubation times of the P1/T1 complexes with E. coli DNA polymerase
I or .PHI.29 DNA polymerase. FIG. 4A shows the resulting
amplification resolved on a 0.8% agarose gel. The gel was stained
with SYBRII (FMC), a single-strand specific nucleic acid dye. The
RCA amplification generated from DNA polymerase I and .PHI.29 DNA
polymerase showed remarkable differences. Note that circularized P1
sequence RCA products increase over time, for incubation periods up
to 24 hr. DNA polymerase I also generated amplifications with
linear P1/T1 complexes after long incubation periods (between 6 and
24 hr), but not nearly at the same rate as the .PHI.29 DNA
polymerase; see FIG. 4B.
Example 7
Fabrication of Streptavidin-Coated Microwells
[0112] Microwells were etched in silicon. A 3" diameter silicon
wafer contained approximately 250 microwells, with dimensions 2
mm.times.2 mm.times.200 mm deep, and spaced 2 mm apart. Biotin was
discretely patterned inside the microwells using photolithography
that is well known in the art. Briefly, the micromachined wafers
were silanized and an ethanolic solution of photoactivatable biotin
(Pierce) was deposited on the wafers and allowed to evaporate.
Photoactivatable biotin forms covalent bonds with nearby organic
moieties upon exposure to UV light. A photomask was placed over the
wafer such that only the microwells were exposed to UV light. The
irradiated wafer was washed and then incubated with streptavidin,
which only binds inside the biotinylated microwells. Because of
streptavidin's tetrameric structure, two biotin-binding sites are
used to immobilize the protein, leaving the remaining sites
available to bind biotinylated oligonucleotides. Each well has a
500 nl volume.
Example 8
Target Immobilization and RCA Performed on Silicon Chips
[0113] This example involves the 18-base oligonucleotide, A12,
which is complementary to the first 5'-18 bases of T1 and T2, and
serves as a structural support (i.e., stabilizer) to lift the
nucleic acid strands from the surface. Equimolar ratios of T1/A12
and T2/A12 were suspended at a final concentration of 5 mM in TE
supplemented with 0.1 M NaCl, heated to 95.degree. C., followed by
slowly cooling to room temperature. Five hundred nl (1250 fmol) of
both DNA complexes were spotted inside streptavidin-coated
microwells. The chip was saturated with 50 mM biotin in SPE buffer
(0.1 M Na-phosphate, pH 6.6, 1M NaCl), then washed 4.times.15 min
in SPE buffer at 37.degree. C., rinsed briefly with deionized
H.sub.2O, and air dried. A hybridization slide chamber (CoverWell
PC50, Grace Bio-labs) was placed over the chip and secured with
small paper binding clips. Two small holes were punctured in the
hybridization chamber and served as fluid inlet and outlet ports.
Hybridization was performed by injecting 85 ml of the indicated
template solution suspended in TE supplemented with 0.1 M NaCl. The
injection ports were sealed, and the chip was incubated for 12 hr
at 37.degree. C. The chip was washed 4.times.15 min in SPE buffer
at 37.degree. C., followed by a rinse in deionized H.sub.2O. A new
hybridization chamber was placed on the chip and 85 ml of ligation
cocktail was injected onto the chip and incubated for 30 min. The
chip was washed 4.times.15 min in SPE buffer at 37.degree. C.,
rinsed in deionized H.sub.2O, and DNA polymerase cocktail was
added. The chip was washed 4.times.15 min in SPE buffer before
exposing to phosphorimaging plates. A Molecular Dynamics STORM
imager was used to scan the plates, and analysis was performed
using software provided by the manufacturer. Thus, this example
represents the immobilization of a nucleic acid primer sequence to
a solid surface, and further amplification of the nucleic acid
sequences on that surface.
Example 9
Single Nucleotide Polymorphism Detection on Silicon Chips
[0114] In this example, it is shown that the ability of .PHI.29 to
generate significant amounts of RCA product using circularized
templates is equally applicable to amplify surface-bound primer
nucleic acid sequences. Two biotinylated primer nucleic acid
sequences, T1 and T2, differing only at position 38, were
immobilized inside streptavidin-coated microwells. The 5'-terminus
of the P1 template aligns with target positions 38, forming a C:G
basepair and a C:T mismatch with T1 and T2, respectively. The P2/T2
complex contained a C:T mismatch at the 3'-terminus of P2 and
position 38 of T2. Chip-based SNP detection was performed in three
steps. The chips were incubated overnight with the templates in a
solution of TE supplemented with 0.1 M NaCl, and washed 4.times.15
min with SPE buffer to remove non-hybridized templates. Ligation
was performed with T4 DNA ligase for 30 min at 37.degree. C.
followed by a wash. Chips were then incubated with DNA polymerase
in the presence of dNTPs and [a-32P] dTTP for 12 hr at 31.degree.
C. The chip was washed to remove unincorporated nucleotides, before
radioactive imaging. Line profiles were determined using ImageQuant
software (Molecular Dynamics). In general, even the mismatched
template/target complexes gave significant signals, and optimal
conditions for SNP discrimination were determined by using
different polymerases, varying the template concentrations, and
adjusting the ligation conditions. FIGS. 5A and 5B show
autoradiograms of the chips with line profiles above the images.
The specific reaction conditions are given in the Figures.
[0115] As shown in the Figure, .PHI.29 DNA polymerase was more
stringent and efficient in amplifying closed switches (i.e.,
circularized templates) than E. coli DNA polymerase I.
[0116] The ligation conditions which produced the best
discrimination between matched and mismatched templates consisted
of 4 U/ml T4 DNA ligase, 1 M NaCl, and incubation at 37.degree. C.
for 30 min. Under our most stringent conditions, the two primer
sequences could be distinguished with a signal-to-noise ratio of
.about.103.
Example 10
Increased Volume Density of Nucleic Acid Sequences
[0117] As demonstrated by FIG. 6, a phosphorimager scan of three
wells of enzymatically-enhanced DNA arrays after primer extension
with .PHI.29 DNA polymerase in the presence of [.alpha.-32P] dTTP.
Primers T1 and T2 were immobilized in the left and middle wells,
respectively, and the right well (Bkg) was left blank for a
background reference. P2 was used as the template for hybridization
and circularization. .PHI.29 DNA polymerase was used to amplify the
primer T1 well, and DNA polymerase I was used to amplify the primer
T2 well. The plot above the image shows the counts for each pixel
in the scan. Total radioactive counts for T1, T2 and the background
were 3,180,000, 255,000 and 43,000, respectively. This indicates
that the discrimination of single-based polymorphisms was increased
at a 12 fold rate from T1 over T2, which in turn indicates an
increased volume density of the nucleic acid sequences. Notice that
the reaction yield (hence, volume density) was higher with the
.PHI.29 DNA polymerase that was used.
[0118] Based on the above disclosure, embodiments, and experiments,
those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
Sequence CWU 1
1
11 1 15 DNA Artificial Sequence Description of Artificial
Sequencerecombinant 1 attatgctat tttgg 15 2 6 DNA Artificial
Sequence Description of Artificial Sequencerecombinant 2 aaaacc 6 3
11 DNA Artificial Sequence Description of Artificial
Sequencerecombinant 3 acgataaaac c 11 4 74 DNA Artificial Sequence
Description of Artificial Sequencerecombinant 4 ctgtcatcat
ttgtgaacta atacaccaat aactaataca ccaataacta atacaccaac 60
gcttggctat ccat 74 5 53 DNA Artificial Sequence Description of
Artificial Sequencerecombinant 5 cctaaactca cggcgatgaa cgccacaaat
gatgacagat ggatagccaa gcg 53 6 53 DNA Artificial Sequence
Description of Artificial Sequencerecombinant 6 cctaaactca
cggcgatgaa cgccacaaat gatgacatat ggatagccaa gcg 53 7 74 DNA
Artificial Sequence Description of Artificial Sequencerecombinant 7
tgtcatcatt tgtgaactaa tacaccaata actaatacac caataactaa tacaccaacg
60 cttggctatc catc 74 8 18 DNA Artificial Sequence Description of
Artificial Sequencerecombinant 8 catcgccgtg agtttagg 18 9 14 DNA
Artificial Sequence Description of Artificial Sequencerecombinant 9
aactaataca ccaa 14 10 20 DNA Artificial Sequence Description of
Artificial Sequencerecombinant 10 caatttcaca caggcccaag 20 11 20
DNA Artificial Sequence Description of Artificial
Sequencerecombinant 11 cgtaagactc atgctcaagc 20
* * * * *