U.S. patent application number 11/030391 was filed with the patent office on 2005-08-25 for arrays and their reading.
Invention is credited to Schembri, Carol T., Tam, Kimberly L..
Application Number | 20050186598 11/030391 |
Document ID | / |
Family ID | 34218166 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186598 |
Kind Code |
A1 |
Schembri, Carol T. ; et
al. |
August 25, 2005 |
Arrays and their reading
Abstract
A method of reading an array of moieties such as polynucleotides
(for example, DNA) on at least a portion of a surface of a
transparent slide which is opposite a first portion on the opposing
surface, which array has been previously exposed to a sample. The
method may include mounting the slide on a slide holder and
retaining the slide thereon in a mounted position in which the
holder does not contact the previously exposed array. The holder is
then inserted into an array reader and the array read. A holder and
slides which can be used in the method are also provided.
Inventors: |
Schembri, Carol T.; (San
Mateo, CA) ; Tam, Kimberly L.; (Daly City,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
Intellectual Property Administration
Legal Department, DL429
P. O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
34218166 |
Appl. No.: |
11/030391 |
Filed: |
January 5, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11030391 |
Jan 5, 2005 |
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09671966 |
Sep 27, 2000 |
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6864097 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 702/19 |
Current CPC
Class: |
B01L 2300/0822 20130101;
Y10T 436/112499 20150115; B01L 9/52 20130101; B01L 2300/0819
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 702/019 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C12M 001/34 |
Claims
1-42. (canceled)
43. A holder having a body comprising: (a) opposing side portions;
(b) a channel between the side portions comprising an open end for
receiving a transparent slide having opposing front and rear
surfaces and carrying one or more arrays of moieties exposed to a
sample on at least a portion of the rear surface of the slide, the
channel further having a bottom surface forming a backer member;
(c) clamp members which can be resiliently urged against edge
portions of the slide for retaining the slide in the channel and
the moieties out of contact with the backer member when the rear
surface of the slide faces the backer member.
44. The holder of claim 43, wherein the clamp members comprise a
fixed clamp member set and a movable clamp member set, wherein the
fixed clamp member set is fixed to the body side portions while the
movable set is movable to an open position away from the fixed set
for receiving edge portions of the slide between the fixed and
movable clamp members and can be urged against the edge portions
for mounting the slide within the holder.
45. The holder of claim 43 wherein the moieties on the rear side of
the slide are spaced from the backer member by between 0.1 mm to 10
mm, when the slide is mounted within the holder.
46. The holder of claim 43 wherein the backer is opaque.
47. The holder of claim 43, additionally comprising two spaced
apart guides extending from the body adjacent the opposing side
portions so as to guide the sliding of the slide into a mounted
position.
48. The holder of claim 43, wherein opposed sides of the channel
comprises ledges.
49. The holder of claim 44, additionally comprising a control
member set positioned on the holder outside the channel for moving
the movable clamp set to the open position.
50. The holder of claim 44, wherein the control member set
comprises a button.
51. The holder of claim 48, wherein the ledges act as clamp
members.
52. The holder of claim 51, wherein the ledges are movable by a
control member set to an open position.
53. The holder of claim 47, wherein the guides comprise ledges for
sliding the slide along.
54. The holder of claim 43, wherein the holder is configured for
insertion into an array reader.
55. A method of reading one or more arrays of moieties on at least
a portion of a rear surface of a transparent slide having opposing
front and rear surfaces, which array has been previously exposed to
a sample, the method comprising: (a) mounting the slide in a slide
holder according to claim 1 and retaining the slide thereon in a
mounted position in which the previously exposed array faces, and
is spaced apart from, the backer member of the holder without the
array contacting the holder, (b) inserting the holder into an array
reader and reading the array.
56. The method of claim 55, wherein the clamp members of the sets
are resiliently urged towards one another.
57. The method of claim 55, wherein the clamp members comprise a
fixed clamp member set and a movable clamp member set, wherein the
fixed clamp member set is fixed to the body side portions while the
movable member set is movable to an open position away from the
fixed member set for receiving edge portions of the slide between
the fixed and movable clamp members.
58. The method of claim 57, wherein the holder additionally
comprises a control member set positioned on the holder outside the
channel and wherein the control member set is moved to move the
movable clamp set to the open position.
59. The method of claim 55, wherein the holder additionally
comprises two spaced apart guides extending from the body adjacent
the opposing side portions and wherein the slide is slid into a
mounted position along the guides.
60. The method of claim 59, wherein the channel further comprises a
closed end and the slide is slid along the guides up to the closed
end.
61. The method of claim 59, additionally comprising removing the
slide from the holder after the reading.
62. The method of claim 55, wherein the slide comprises an
identification code on the front surface and the method further
comprises reading the identification code.
Description
[0001] This application claims priority to copending application
Ser. No. 09/671,966, filed Sep. 27, 2000, under 35 U.S.C. 120, the
entirety of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to slides holding multiple moieties
to be read, and in particular to arrays such as polynucleotide
arrays (for example, DNA arrays), which are useful in diagnostic,
screening, gene expression analysis, and other applications.
BACKGROUND OF THE INVENTION
[0003] Polynucleotide arrays (such as DNA or RNA arrays), are known
and are used, for example, as diagnostic or screening tools. Such
arrays include regions of usually different sequence
polynucleotides arranged in a predetermined configuration on a
substrate. These regions (sometimes referenced as "features") are
positioned at respective locations ("addresses") on the substrate.
In use, the arrays, when exposed to a sample, will exhibit an
observed binding or hybridization pattern. This binding pattern can
be detected upon interrogating the array. For example, all
polynucleotide targets (for example, DNA) in the sample can be
labeled with a suitable label (such as a fluorescent dye), and the
fluorescence pattern on the array accurately observed following
exposure to the sample. Assuming that the different sequence
polynucleotides were correctly deposited in accordance with the
predetermined configuration, then the observed binding pattern will
be indicative of the presence and/or concentration of one or more
polynucleotide components of the sample.
[0004] Biopolymer arrays can be fabricated by depositing previously
obtained biopolymers (such as from synthesis or natural sources)
onto a substrate, or by in situ synthesis methods. Methods of
depositing obtained biopolymers include dispensing droplets to a
substrate from dispensers such as pin or capillaries (such as
described in U.S. Pat. No. 5,807,522) or such as pulse jets (such
as a piezoelectric inkjet head, as described in PCT publications WO
95/25116 and WO 98/41531, and elsewhere). For in situ fabrication
methods, multiple different reagent droplets are deposited from
drop dispensers at a given target location in order to form the
final feature (hence a probe of the feature is synthesized on the
array substrate). The in situ fabrication methods include those
described in U.S. Pat. No. 5,449,754 for synthesizing peptide
arrays, and described in WO 98/41531 and the references cited
therein for polynucleotides. The in situ method for fabricating a
polynucleotide array typically follows, at each of the multiple
different addresses at which features are to be formed, the same
conventional iterative sequence used in forming polynucleotides
from nucleoside reagents on a support by means of known chemistry.
This iterative sequence is as follows: (a) coupling a selected
nucleoside through a phosphite linkage to a functionalized support
in the first iteration, or a nucleoside bound to the substrate
(i.e. the nucleoside-modified substrate) in subsequent iterations;
(b) optionally, but preferably, blocking unreacted hydroxyl groups
on the substrate bound nucleoside; (c) oxidizing the phosphite
linkage of step (a) to form a phosphate linkage; and (d) removing
the protecting group ("deprotection") from the now substrate bound
nucleoside coupled in step (a), to generate a reactive site for the
next cycle of these steps. The functionalized support (in the first
cycle) or deprotected coupled nucleoside (in subsequent cycles)
provides a substrate bound moiety with a linking group for forming
the phosphite linkage with a next nucleoside to be coupled in step
(a). Final deprotection of nucleoside bases can be accomplished
using alkaline conditions such as ammonium hydroxide, in a known
manner.
[0005] The foregoing chemistry of the synthesis of polynucleotides
is described in detail, for example, in Caruthers, Science 230:
281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356;
Hunkapillar et al., Nature 310: 105-110, 1984; and in "Synthesis of
Oligonucleotide Derivatives in Design and Targeted Reaction of
Oligonucleotide Derivatives", CRC Press, Boca Raton, Fla., pages
100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S.
Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and
elsewhere
[0006] Polynucleotide arrays have previously been provided in two
formats. In one format, the array is provided as part of a package
in which the array itself is disposed on a first side of a glass or
other transparent substrate. This substrate is fixed (such as by
adhesive) to a housing with the array facing the interior of a
chamber formed between the substrate and housing. An inlet and
outlet may be provided to introduce and remove sample and wash
liquids to and from the chamber during use of the array. The entire
package may then be inserted into a laser scanner, and the sample
exposed array may be read through a second side of the
substrate.
[0007] In another format, the array is present on an unmounted
glass or other transparent slide substrate. This array is then
exposed to a sample optionally using a temporary housing to form a
chamber with the array substrate. The slide may then be placed in a
laser scanner to read the exposed array. Most slide scanners
require that the user manually insert the slide into a holder
within the scanner. Some scanners allow the slide to rest on a
surface while others clamp it to a known location using various
types of guides. The present invention realizes that this technique
creates a number of potential problems. First, since the array
itself is unprotected it is subject to damage. Any damage is
extremely undesirable for a number of reasons. For example, slight
damage, such as fingerprints or scratches may occur to the sample
exposed array which is not noticed. Such damage could lead to
incorrect readings with serious consequences in interpretation of
results. Also, it is not uncommon for the slides to be broken
during insertion or removal from these scanners. Slide glass is
easily chipped or broken. Losing a slide at this stage of the
experiment can be extremely costly. Typically, the arrayed slides
cost several hundred dollars and may involve long lead times. The
samples under test may be from tumors or other hard-to-obtain
sources. The fluorescent dyes typically employed are currently
quite expensive. Therefore, a broken slide represents the loss of
many hundreds of dollars and many hours of work. Thus, the present
invention realizes that it is preferred to have a safer method and
means of handling these slides. Furthermore, given that the
individual features within the arrays on the surface of such slides
are on the order of 10 to 120 microns in size and the importance of
gathering all possible fluorescent signal, it is desirable to
reference and hold these slides precisely. However, the present
invention further realizes that precision placement usually
involves firm surfaces and forcibly clamping the slides, which
actions can result in slide breakage or array damage. If the slide
is simply placed into a chamber to avoid clamping, large positional
tolerances are needed which reduce the detection quality of the
signals from the surface. Gathering all possible fluorescent signal
from each feature on the array also requires that other sources of
noise are minimized.
[0008] It would be desirable then to provide a means which could
protect moieties, such as an exposed array, carried on a slide and
protect the slide itself from breakage, which is relatively easy to
use without requiring extensive manipulations of the slide, and
which can aid in precisely positioning the slide (and hence the
moieties) in a reader for reading of the exposed array.
SUMMARY OF THE INVENTION
[0009] The present invention then, provides in one aspect, a method
of reading an array of moieties on at least a portion of a surface
of a transparent slide which is opposite a first portion on an
opposite surface, which array has been previously exposed to a
sample. The method includes mounting the slide on a slide holder
and retaining the slide thereon in a mounted position without the
array contacting the holder. The holder is then inserted into an
array reader and the array read. In one embodiment of the method,
the moieties may be on at least a portion of a rear surface of a
transparent slide which is opposite a first portion on the front
surface, which array has been previously exposed to a sample. In
this embodiment the slide when in the mounted position has the
exposed array facing a backer member of the holder without the
array contacting the holder. The backer member is preferably has a
very low in intrinsic fluoresence or is located far enough from the
array to render any such fluroesence insignificant. In either
situation, the backer member contributes less than 20% or 10%, and
preferably less than 5% or less than 1% or 0.5% (or even less than
0.1%) to the strongest signal which can be obtained from a region
(such as a feature) on the slide.
[0010] Optionally, the array may be read through the front side of
the slide. The reading, for example, may include directing a light
beam through the slide from the front side and onto the array on
the rear side, and detecting a resulting signal from the array
which has passed from the rear side through the slide and out the
slide front side. The holder may further include front and rear
clamp sets which can be moved apart to receive the slide between
the sets. In this case, the slide is retained in the mounted
position by the clamp sets being urged (such as resiliently, for
example by one or more springs) against portions of the front and
rear surfaces, respectively. The clamp sets may, for example, be
urged against the slide front and rear surfaces of a mounted slide
at positions adjacent a periphery of that slide. Alternatively, the
array may be read on the front side when the slide is positioned in
the holder with the array facing forward (that is, away from the
holder).
[0011] The holder may in one embodiment have a body with side
portions and a channel intermediate the side portions, which
channel extends in a direction between ends of the body. In this
case, the backer member may be a bottom surface of the channel. In
such a configuration, the front and rear clamp member sets may have
their members disposed about the channel, and one of those sets may
have its members fixed to the body side portions while the other
set is movable to an open position away from the fixed set. For
example, the front clamp member set may be fixed to the body side
portions and the rear clamp member set may be movable. In this
case, the slide is retained in the mounted position by being urged
against the fixed clamp member set.
[0012] The method may use a holder with a control member set
positioned on an accessible location on the holder, for example at
a position outside the channel, which control member set is moved
to move the movable clamp set to the open position. In the case
where the movable clamp set is the rear clamp member set, the
control member may simply be moved rearward to move the rear clamp
member set to the open position.
[0013] In the holder configuration where the body has the channel
as described above, the slide may be mounted on the holder by
sliding the slide in an endways direction of the channel and into
the mounted position in which a leading end of the slide abuts the
closed end of the channel. Clamp member sets positioned about the
channel, may be held in the open position during such a mounting
procedure (for example, by the control member set rearward).
[0014] The holder used in the method may additionally have two
spaced apart guides extending from the body adjacent respective
sides of the channel. With this configuration the slide may be slid
into the mounted position along the guides, the guides being
dimensioned such that when the slide is in the mounted position a
trailing end of the slide is positioned between the guides. During
any mounting of the slide portions of the slide, portions of the
slide front and rear surfaces may be gripped (such as with a user's
fingers) and the gripped portions used to then slide the slide into
the mounted position. The guides, in such case may be dimensioned
such that the gripped portions are positioned between the guides
when the slide is in the mounted position. The method may
optionally additionally include removing the slide from the mounted
position, which removing includes gripping portions of the slide
front and rear surfaces which are between the guides and using the
gripped portions to slide the slide in an endways direction
opposite to that in which the slide was slid during the slide
mounting.
[0015] The present invention also provides a holder for a slide,
which holder has any of the features already described above. In
one embodiment, the holder may include a backer member and the
clamp sets as described above. The clamp members may be positioned
such that the holder can receive and retain a slide having an area
of no more than 200 cm.sup.2 (or no more than 100 cm.sup.2 or even
no more than 50 or 40 cm.sup.2). The holder may also be dimensioned
such that the moieties on the rear side of the mounted slide are
spaced from the backer member by between 0.1 mm to 10 mm (or more
preferably between 0.5 and 5 or 3 mm). The holder itself may have
various shapes, for example, rectangular. In one aspect, the holder
will have a maximum area of a side which is no more than 300
cm.sup.2 (or preferably no more than 200 cm.sup.2 or 100 cm.sup.2).
When a channel is present in the holder it may, for example, be no
wider than 20 cm.sup.2 (or no wider than 15 cm.sup.2, 10 cm.sup.2,
or 5 cm.sup.2).
[0016] The present invention further provides a transparent slide
having opposed front and rear surfaces, the slide carrying moieties
such as an array of biopolymers on a rear surface, and an
identification code on a front surface. The identification code
may, for example, be a bar code, which is printed on an opaque
label attached to the front side of the slide. A method of reading
an array on such a slide is also provided, where the array is read
through the front surface and the identification code is read from
a front side.
[0017] Alternatively, as mentioned above, the holder may be used
with an array of the mounted slide located on the a forward facing
surface (that is, away from the holder). While the surface
protection benefit is lost in this configuration, the benefits of
ease of handling and physical protection of the slide are retained.
One may wish to read the array on a forward facing side of the
slide to allow for various opaque slides, mirrored slides or to
avoid the issues of thickness variations in transparent slides
(that is, arrays on a backward facing surface of a slide which are
read through the slide from the front side, may be on different
focal planes of the scanner in the case where the thicknesses of
the transparent slide vary).
[0018] Different embodiments of methods and devices of the present
invention can provide any or more of a number of useful features.
For example, moieties on the slide (such as the exposed array) can
be protected from damage and the slide itself protected from
breakage. Background signals during array reading may be reduced by
the use of the backer member. Further, it may be relatively easy to
use devices of the present invention and extensive manipulations of
the slide may be avoided, while relatively precise positioning of
the slide (and hence the moieties) in a reader may be obtained for
assisting in the reading of the exposed array.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Embodiments of the invention will now be described with
reference to the drawings, in which:
[0020] FIG. 1 illustrates a slide carrying an array, of the present
invention, and such as may be used in a holder and methods of the
present invention;
[0021] FIG. 2 is an enlarged view of a portion of FIG. 1 showing
ideal spots or features;
[0022] FIG. 3 is an enlarged illustration of a portion of the
substrate in FIG. 2;
[0023] FIG. 4 is a front view of a holder of the present
invention;
[0024] FIG. 5 is a leading end elevation of the holder of FIG.
1;
[0025] FIG. 6 is a view the same as that of FIG. 4 but showing a
slide of FIG. 1 being slid into the mounted position on the
holder;
[0026] FIG. 7 is the same as FIG. 6 but showing the slide in the
mounted position on the holder;
[0027] FIG. 8 is a leading end elevation of the holder with mounted
slide;
[0028] FIG. 9 is an exploded view of the holder of FIG. 1;
[0029] FIGS. 10 and 11 are more detailed view of some of the
components shown in FIG. 8; and
[0030] FIG. 12 illustrates scanning of a slide mounted in the
holder of FIG. 1.
[0031] To facilitate understanding, identical reference numerals
have been used, where practical, to designate identical elements
that are common to the figures.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0032] In the present application, unless a contrary intention
appears, the following terms refer to the indicated
characteristics. A "biopolymer" is a polymer of one or more types
of repeating units. Biopolymers are typically found in biological
systems (although they may be made synthetically) and particularly
include peptides or polynucleotides, as well as such compounds
composed of or containing amino acid analogs or non-amino acid
groups, or nucleotide analogs or non-nucleotide groups. This
includes polynucleotides in which the conventional backbone has
been replaced with a non-naturally occurring or synthetic backbone,
and nucleic acids (or synthetic or naturally occurring analogs) in
which one or more of the conventional bases has been replaced with
a group (natural or synthetic) capable of participating in
Watson-Crick type hydrogen bonding interactions. Polynucleotides
include single or multiple stranded configurations, where one or
more of the strands may or may not be completely aligned with
another. A "nucleotide" refers to a sub-unit of a nucleic acid and
has a phosphate group, a 5 carbon sugar and a nitrogen containing
base, as well as functional analogs (whether synthetic or naturally
occurring) of such sub-units which in the polymer form (as a
polynucleotide) can hybridize with naturally occurring
polynucleotides in a sequence specific manner analogous to that of
two naturally occurring polynucleotides. For example, a
"biopolymer" includes DNA (including cDNA), RNA, oligonucleotides,
and PNA and other polynucleotides as described in U.S. Pat. No.
5,948,902 and references cited therein (all of which are
incorporated herein by reference), regardless of the source. An
"oligonucleotide" generally refers to a nucleotide multimer of
about 10 to 100 nucleotides in length, while a "polynucleotide"
includes a nucleotide multimer having any number of nucleotides. A
"biomonomer" references a single unit, which can be linked with the
same or other biomonomers to form a biopolymer (for example, a
single amino acid or nucleotide with two linking groups one or both
of which may have removable protecting groups). A "peptide" is used
to refer to an amino acid multimer of any length (for example, more
than 10, 10 to 100, or more amino acid units). A biomonomer fluid
or biopolymer fluid reference a liquid containing either a
biomonomer or biopolymer, respectively (typically in solution).
[0033] A "set" or "sub-set" of any item (for example, a set of
features) may contain one or more than one of the item (for
example, a set of clamp members may contain one or more such
members). An "array", unless a contrary intention appears, includes
any one, two or three dimensional arrangement of addressable
regions bearing a particular chemical moiety or moieties (for
example, biopolymers such as polynucleotide sequences) associated
with that region. An array is "addressable" in that it has multiple
regions of different moieties (for example, different
polynucleotide sequences) such that a region (a "feature" or "spot"
of the array) at a particular predetermined location (an "address")
on the array will detect a particular target or class of targets
(although a feature may incidentally detect non-targets of that
feature). Array features are typically, but need not be, separated
by intervening spaces. In the case of an array, the "target" will
be referenced as a moiety in a mobile phase (typically fluid), to
be detected by probes ("target probes") which are bound to the
substrate at the various regions. However, either of the "target"
or "target probes" may be the one which is to be evaluated by the
other (thus, either one could be an unknown mixture of
polynucleotides to be evaluated by binding with the other). An
"array layout" refers collectively to one or more characteristics
of the features, such as feature positioning, one or more feature
dimensions, and some indication of a moiety at a given location.
"Hybridizing" and "binding", with respect to polynucleotides, are
used interchangeably. When one item is indicated as being "remote"
from another, this is referenced that the two items are at least in
different buildings, and may be at least one mile, ten miles, or at
least one hundred miles apart.
[0034] It will also be appreciated that throughout the present
application, that words such as "front", "rear", "back", "leading",
"trailing", "top", "upper", and "lower", are all used in a relative
sense only. "Fluid" is used herein to reference a liquid. Reference
to a singular item, includes the possibility that there are plural
of the same items present. Furthermore, when one thing is "slid" or
"moved" or the like, with respect to another, this implies relative
motion only such that either thing or both might actually be moved
in relation to the other. All patents and other cited references
are incorporated into this application by reference.
[0035] Referring first to FIGS. 1-3, typically methods and
apparatus of the present invention generate or use a contiguous
planar transparent slide 110 carrying an array 112 disposed on a
rear surface 111b of substrate 110. It will be appreciated though,
that more than one array (any of which are the same or different)
may be present on rear surface 111b, with or without spacing
between such arrays. Note that one or more arrays 112 together will
only cover a portion of the rear surface 111b, with regions of the
rear surface 111b adjacent the opposed sides 113c, 113d and leading
end 113a and trailing end 113b of slide 110, not being covered by
any array 112. A front surface 111a of the slide 110 does not carry
any arrays 112. Each array 112 can be designed for testing against
any type of sample, whether a trial sample, reference sample, a
combination of them, or a known mixture of polynucleotides (in
which latter case the arrays may be composed of features carrying
unknown sequences to be evaluated). Slide 110 may be of any shape,
and any holder used with it adapted accordingly, although slide 110
will typically be rectangular in practice. Array 112 contains
multiple spots or features 116 of biopolymers in the form of
polynucleotides. A typical array may contain from more than ten,
more than one hundred, more than one thousand or ten thousand
features, or even more than from one hundred thousand features. All
of the features 116 may be different, or some or all could be the
same. In the case where array 112 is formed by the conventional in
situ or deposition of previously obtained moieties, as described
above, by depositing for each feature at least one droplet of
reagent such as by using a pulse jet such as an inkjet type head,
interfeature areas 117 will typically be present which do not carry
any polynucleotide. It will be appreciated though, that the
interfeature areas 117 could be of various sizes and
configurations. Each feature carries a predetermined polynucleotide
(which includes the possibility of mixtures of polynucleotides). As
per usual, A, C, G, T represent the usual nucleotides. It will be
understood that there may be a linker molecule (not shown) of any
known types between the rear surface 111b and the first nucleotide.
However, as mentioned above, the array 112 may optionally be on the
front surface 111a.
[0036] Slide 110 also carries on front surface 110a, an
identification code in the form of bar code 115 printed on an
opaque substrate in the form of a paper label attached by adhesive
to front side 110a. By "opaque" in this context is referenced that
the means used to read bar code 115 (typically a laser beam) cannot
read code 115 through the label without reading errors. Typically
this means that less than 60% or even less than 50%, 30%, 20% or
10% of the signal from the code passes through the substrate. Bar
code 115 contains an identification of array 112 and either
contains or is associated with, array layout or layout error
information in a manner such as described in U.S. patent
applications [GMS to insert patent applications Ser. Nos.],
incorporated herein by reference.
[0037] For the purposes of the discussions below, it will be
assumed (unless the contrary is indicated) that the array 112 is a
polynucleotide array formed by the deposition of previously
obtained polynucleotides using pulse jet deposition units. However,
it will be appreciated that an array of other polymers or chemical
moieties generally, whether formed by multiple cycle in situ
methods adding one or more monomers per cycle, or deposition of
previously obtained moieties, or by other methods, may be present
instead.
[0038] Turning now to FIG. 4-7, a holder 10 of the present
invention will now be described in more detail. Holder 10 has a
body which is generally rectangular in shape and includes two
opposed side portions 14 with a channel 18 positioned therebetween,
and extending in a direction between ends 12a, 12b of the body.
Channel 18 has a bottom surface 32 which acts as a backer member,
and has a closed leading end 26 and an open trailing end 26b.
Opposed sides 20 of channel 18 have ledges 22 running the length of
the sides 20. Portions of ledges 22 act as a movable set of rear
clamp members, as will shortly be described. Four tabs 30
positioned about channel 18, have outside portions 34 attached to
side portions 14 and inside portions 36 which extend over ledges 22
and are slightly spaced therefrom in a normal position of ledges
22. Inside portions 36 act as a front set of fixed clamp members
which are fixed to side portions 14. Positioned outside channel 18
on a front side of holder 10, is a control member set consisting of
two control members in the form of buttons 40 each of which is
positioned and movable within an opening 15 in a front surface 16
of a corresponding side portion 14. Each control member is
connected to channel 18 (including ledges 22) such that moving the
control members rearward (into the page, as viewed in FIG. 4)
causes the channel 18 to also move rearward, thereby moving ledges
18 (portions of which, beneath inside portions 36 of tabs 30, act
as the rear clamp member set) away from portions 36 of tabs 30
(which act as the fixed front clamp member set) to an open
position. That is, pressing down on buttons 40 (as viewed in FIG.
4) moves the clamp member sets to an open position. Four springs 72
(seen in FIG. 9) resiliently urge the channel 18 and hence ledges
22 forward toward one another (thereby urging the rear clamp
member, composed of portions of ledges 22, to the normal
position).
[0039] Two spaced apart guides 50 extend from a trailing end of the
holder body adjacent respective sides of channel 18. Each guide
includes a trailing end 50 and a ledge 54 approximately aligned
with a corresponding ledge 22 when the set of ledges 22 (rear clamp
member set) is in the open position.
[0040] The holder as described, is used to mount slide 110 in a
manner as will now be described. First, the array 112 will have
typically been previously exposed to a fluid sample which is to be
tested for moieties (such as polynucleotides) which may bind (for
example, hybridize) to the moieties (such as polynucleotides) at
one or more features. The moieties to be tested may be labeled with
fluorescent dyes in a known manner. The array 112 may then be
washed and dried in preparation for reading. At this point a user
will typically grip opposing portions of the front and rear
surfaces of slide 110 toward the trailing end 113b using their
thumb and forefinger. Buttons 40 can then be pressed rearward (into
the page as viewed in FIG. 4) to move channel 18 and attached
ledges 22 rearward thereby moving the clamp member sets to the open
position. Note that when in the open position, the distance between
the ledges 22 (movable rear clamp member set) and portions 36
(fixed front clamp member set) is greater than the thickness of
slide 110. Leading edge 113a of slide 110 can then be positioned
between guides 50 with opposite edges of slide 110 resting on
ledges 54 of guides 50, with rear surface 111b (and hence array
112) facing rearward) and bar code 115 facing forward. Slide 110
can then be slid using the gripped portions in an endways direction
120 (see FIG. 6) along ledges 54 of guides 50 and then along ledges
22 of channel 18, between the open clamp member sets, until leading
edge 113a of slide 110 abuts leading edge 26 of channel 18 at which
point slide 110 is in the mounted position (as shown in FIGS. 7 and
8).
[0041] Slide 110 is retained in the mounted position by releasing
buttons 40. Springs 72 then urge ledges 22 (rear clamp member sets)
against portions 36 (front clamp member sets), the urging of the
clamp member sets against side edge portions of slide 110 causing
the slide 110 to be retained in the mounted position. Since the
rear movable clamp member set urges slide 110 against the fixed
front clamp member set, this helps ensure that array 112 is in a
known fixed position relative to the holder for reading of the
array. Note that when in the mounted position, rear surface 111b
(and hence array 112) is spaced apart from bottom surface 32 (which
acts as the backer member). Note also that when slide 110 is in the
mounted position, the clamp members, and any other portion of the
holder, do not contact array 112 or a portion of front surface 111a
which is opposite array 112. Also, when the slide 110 is in the
mounted position, trailing end 113b is positioned between guides
50. This helps protect trailing end 113b from breakage.
Furthermore, the gripped position will be between guides 50. The
fact that guides 50 extend away from the remainder of the holder
such that there are no surfaces or members between guides 50,
allows a user to continue to maintain a hold on the gripped
portions of the slide 110 until it is in the mounted position at
which point the gripped portions will also be between guides 50.
The array 112 of the mounted slide is spaced apart from surface 32
(backer member). This allows backer member 32 to protect array 112
of the mounted slide, while the spacing between backer member 32
and array 112 maintains backer member out of the plane of focus of
a reader (which will focus on the plane in which array 112 lies on
the rear surface 111b). This reduces the detection of any
fluorescence which might occur from the backer member in response
to an interrogating light.
[0042] The holder 10 with mounted slide may then be inserted into a
reader, such as a laser scanner, which has a suitable mounting
means for receiving and releasably retaining the holder in a known
position. The scanner should be able to read the location and
intensity of fluorescence at each feature of an array following
exposure to a fluorescently labeled sample (such as a
polynucleotide containing sample). For example, such a scanner may
be similar to the GENEARRAY scanner available from Agilent
Technologies, Inc., Palo Alto, Calif. The array 12 may then be read
through front side 110a of slide 110 in a manner illustrated in
FIG. 11. In particular, a scanning interrogating laser beam 150 is
directed through a beam splitter 155 and then through front side
110a and scanned across array 12. Resulting fluorescent signals
from the array which have passed back through slide 110 and out
through front side 110a may then be detected at detector 160.
Results from the interrogation can be processed such as by
rejecting a reading for a feature which is below a predetermined
threshold and/or forming conclusions based on the pattern read from
the array (such as whether or not a particular target sequence may
have been present in the sample). The results of the interrogation
or processing can be forwarded (such as by communication) to a
remote location if desired, for further use. The bar code 115 is
read from the front side of slide 110 by bar code reader 170.
Information from the read bar code 115 can be used to retrieve
array layout information which can be used in the reading and/or
processing of the interrogation results, in a manner as described
in U.S. patent application Ser. No. [GMS to insert Ser. Nos. of two
patent applications].
[0043] After the reading of array 112 is complete, the holder may
be removed from the scanner. A user may now remove slide 110 for
storage or disposal. To remove the slide from the mounted position,
the user depresses the two buttons 40 on the slide holder to open
the clamp member set, and grips opposite portions of the front and
back surfaces of slide 110 at positions between guides 50. The
gripped portions may then be used to slide the slide out of holder
10 in an endways direction 140 opposite that of direction 120.
[0044] As previously mentioned though, the slide can be mounted
with the array 112 facing forward. For example, the slide is
mounted as shown in the FIGS. 6 through 8 but array 112 is on the
front side 111a of slide 110. Such a forward facing slide can be
read directly from the front side without the signal from the array
having to pass through the slide (as it does in the arrangment
described in connection with FIGS. 6-8 and 12). In such a case, the
bar code may be on the front or rear side of slide 110 and bar code
reader 170 can be positioned to read bar code 115 accordingly.
[0045] The holder 10 is preferably made in three molded sections
from an opaque plastic, such as black ABS plastic (although other
materials could be used), as illustrated in FIG. 9. In this manner
a channel section 70 is interposed between a front section 60 and
rear section 80. Rear and front views of channel section 70 are
illustrated in more detail in FIGS. 9 and 10, respectively. Channel
section is mounted to be free floating between sections 60, 80,
with buttons 40 retained and movable forwardly and rearwardly
within openings 15. The four springs 72 are retained in openings 74
in a rear side of channel section 70, as best seen in FIG. 10. For
ease of manufacturing, sections 60 and 80 of the holder 10 are
preferably ultrasonically welded together. Alternatives include
adhesive bonding, solvent welding, molded-in snap fit joints and
the use of fasteners such as screws. Springs 72 resiliently urge
channel section 70 forward, and hence urge buttons 40 and channel
18 forward into the normal position. There is enough spring force
behind to ensure that the slide will not move when loads of up to
30 times the force of gravity are applied to the channel in the
rearward direction. The color of holder 10 is preferably black to
minimize any fluorescent noise or signal contribution from holder
10. Also, holder 10 being opaque prevents any interrogating light
from being scattered around inside the scanner. In this context, by
the holder being "opaque" is referenced that it typically transmits
less than 40%, and preferably less than 10% or 5%, and more
preferably less than 2%, of an interrogating light.
[0046] It will be appreciated that both flexible and rigid slides
may be used, provided such slide is not flexible as would prevent
the clamp member sets from positioning the array in a known fixed
position with reference to the holder. Preferred slide materials
provide physical support for the deposited material and endure the
conditions of the deposition process and of any subsequent
treatment or handling or processing that may be encountered in the
use of the particular array. The array substrate may take any of a
variety of configurations ranging from simple to complex. In many
embodiments, the slide will be shaped generally as a rectangular
solid, having a length in the range about 5 mm to 100 cm, usually
about 10 mm to 25 cm, more usually about 10 mm to 15 cm; a width in
the range about 4 mm to 25 cm, usually about 4 mm to 10 cm and more
usually about 5 mm to 5 cm; and a thickness in the range about 0.01
mm to 5.0 mm, usually from about 0.1 mm to 2 mm and more usually
from about 0.2 to 1 mm.
[0047] In the present invention, any of a variety of geometries of
arrays 112 on a slide 110 may be used, other than rectilinear rows
and columns, when multiple arrays 112 are present. For example,
such arrays can be arranged in a sequence of curvilinear rows
across the substrate surface (for example, a sequence of concentric
circles or semi-circles of spots), and the like. Similarly, the
pattern of features 116 may be varied from the rectilinear rows and
columns of spots in FIG. 2 to include, for example, a sequence of
curvilinear rows across the substrate surface (for example, a
sequence of concentric circles or semi-circles of spots), and the
like. The configuration of the arrays and their features may be
selected according to manufacturing, handling, and use
considerations.
[0048] The slide may be fabricated from any of a variety of
materials but is typically transparent. In this context, by
"transparent" is referenced permitting the signal from features to
pass therethrough without substantial attenuation and also
permitting any interrogating radiation to pass therethrough without
substantial attenuation. By "without substantial attenuation" may
include, for example, without a loss of more than 40% or more
preferably without a loss of more than 30%, 20% or 10%. The
interrogating radiation and signal may for example be visible,
ultraviolet or infrared light. In certain embodiments, such as for
example where production of binding pair arrays for use in research
and related applications is desired, the materials from which the
substrate may be fabricated should ideally exhibit a low level of
non-specific binding during hybridization events. Suitable rigid
substrates may include: glass (which term is used to include
silica) and suitable plastics. Should a front array location be
used, additional rigid, non-transparent substrates may be
considered, such as silicon, mirrored surfaces, opaque plastics,
membranes and laminates.
[0049] The substrate surface onto which the polynucleotide
compositions or other moieties is deposited may be smooth or
substantially planar, or have irregularities, such as depressions
or elevations. The surface may be modified with one or more
different layers of compounds that serve to modify the properties
of the surface in a desirable manner. Such modification layers,
when present, will generally range in thickness from a
monomolecular thickness to about 1 mm, usually from a monomolecular
thickness to about 0.1 mm and more usually from a monomolecular
thickness to about 0.001 mm. Modification layers of interest
include: inorganic and organic layers such as metals, metal oxides,
polymers, small organic molecules and the like. Polymeric layers of
interest include layers of: peptides, proteins, polynucleic acids
or mimetics thereof (for example, peptide nucleic acids and the
like); polysaccharides, phospholipids, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneamines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and
the like, where the polymers may be hetero- or homopolymeric, and
may or may not have separate functional moieties attached thereto
(for example, conjugated).
[0050] Various further modifications to the particular embodiments
described above are, of course, possible. Accordingly, the present
invention is not limited to the particular embodiments described in
detail above.
* * * * *