U.S. patent application number 12/852180 was filed with the patent office on 2011-02-03 for polynucleotide analysis using combinatorial pcr.
This patent application is currently assigned to Point-2-Point Genomics Limited. Invention is credited to Brendan James Hamill.
Application Number | 20110028340 12/852180 |
Document ID | / |
Family ID | 9912044 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110028340 |
Kind Code |
A1 |
Hamill; Brendan James |
February 3, 2011 |
POLYNUCLEOTIDE ANALYSIS USING COMBINATORIAL PCR
Abstract
The invention comprises a two-step process for analysis of
polynucleotides by chain extension of multiple polynucleotide
primers attached to solid supports by first performing PCR of the
samples in the presence of multiple oligonucleotides in solution,
the oligonucleotides of both sets being similar or identical. This
produces immobilized single-strand polynucleotides containing
genetic sequence data derived from sample molecules. In a second
step, support-bound polynucleotides are interrogated by
hybridization with a single labeled oligonucleotide probe or by
second-strand synthesis with a primer-dependent polymerase using an
oligonucleotide primer and nucleotide monomers, in which either or
both of the primer and nucleotide monomers are labeled.
Incorporation of label demonstrates the presence of two separate
defined-sequence primers within the sample polynucleotide. The
presence or absence within the sample of the multiple combinations
of primers is demonstrable in a single experiment by use of
suitable apparatus, such as an oligonucleotide array.
Inventors: |
Hamill; Brendan James;
(Fife, GB) |
Correspondence
Address: |
ROPES & GRAY LLP;IPRM - Floor 43
PRUDENTIAL TOWER, 800 BOYLSTON STREET
BOSTON
MA
02199-3600
US
|
Assignee: |
Point-2-Point Genomics
Limited
Edinburgh
GB
|
Family ID: |
9912044 |
Appl. No.: |
12/852180 |
Filed: |
August 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10473796 |
Sep 30, 2003 |
7771975 |
|
|
PCT/GB02/01489 |
Mar 28, 2002 |
|
|
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12852180 |
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Current U.S.
Class: |
506/9 |
Current CPC
Class: |
B01J 2219/00317
20130101; B01J 2219/00527 20130101; B01J 2219/00689 20130101; B01J
2219/00319 20130101; B01J 2219/00612 20130101; C12Q 1/6837
20130101; B01J 2219/00702 20130101; C40B 40/06 20130101; B01J
2219/00695 20130101; B01J 2219/0059 20130101; B01J 2219/0054
20130101; C12P 19/34 20130101; B01J 2219/00596 20130101; B01J
2219/00722 20130101; B01J 2219/00605 20130101; C40B 60/14 20130101;
C12Q 1/6837 20130101; B01J 2219/00626 20130101; B01J 2219/00585
20130101; B01L 7/52 20130101; C40B 70/00 20130101; C12Q 2531/113
20130101; C12Q 2565/537 20130101; B01J 2219/00659 20130101; B01L
3/50851 20130101 |
Class at
Publication: |
506/9 |
International
Class: |
C40B 30/04 20060101
C40B030/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 2000 |
GB |
0027959.6 |
Nov 23, 2000 |
GB |
0028541.1 |
Apr 2, 2001 |
GB |
0108182.7 |
Claims
1. A process for the analysis of a nucleic acid sample so as to
determine the simultaneous presence in the sample of two or more
target sequences each bounded by first and second oligonucleotide
primer sequences, said process comprising the steps of: (i)
providing a first multiplicity of different oligonucleotide
primers, each covalently anchored at its 5'-terminus to a discrete,
individually identifiable, solid support surface, wherein the first
multiplicity comprises the first and second oligonucleotide primer
sequences, (ii) providing a second multiplicity of oligonucleotide
primers, in solution, wherein the nucleotide sequences of
respective members of said first and second oligonucleotide primer
multiplicities are substantially identical to each other, (iii)
performing a polymerase chain reaction amplification on said sample
in the presence of both said first and second oligonucleotide
primer multiplicities, so as to convert said anchored
oligonucleotide primers to anchored polynucleotide chain extension
products, (iv) separating said solid support surface anchored
polynucleotide chain extension products from the resulting
solution, and (v) determining, for each said separated solid
support-linked chain extension product, which of said second
multiplicity of oligonucleotide primers is a complementary sequence
to a portion of a said chain extension product, whereby the
presence of the two or more target sequences is detected in the
sample.
2. The process of claim 1 wherein the determination of which of
said second multiplicity of oligonucleotide primers is a
complementary sequence to a portion of said anchored chain
extension product is performed by probing serially with different
labelled oligonucleotides, each having a sequence corresponding to
that of a respective one of said second multiplicity of
oligonucleotide primers, with removal of any bound labeled
oligonucleotide after each probing step.
3. The process of claim 1 wherein the determination of which of
said second multiplicity of oligonucleotide primers is a
complementary sequence to a portion of said anchored chain
extension product is effected by a serial multiple probing process
using primer-directed complementary strand synthesis with different
oligonucleotide primers, each having a sequence corresponding to
that of one of said second multiplicity of oligonucleotide primers
together with labeled nucleotide monomers, with removal of any
bound complementary strand after each probing step.
4. The process of claim 1, wherein the solid support surface
comprises a multiplicity of solid support zones, each solid support
zone comprising the first multiplicity of anchored
oligonucleotides, which solid support zones are processable
separately from each other, such that all possible combinations of
primers may be utilized in a single analysis, and wherein the
determination of which of said second multiplicity of
oligonucleotide primers is a complementary sequence to a portion of
said solid support-linked chain extension product is effected by a
parallel multiple probing at each one of said multiplicity of solid
support zones with different labeled oligonucleotides for each of
said multiplicity of solid support zones, each having a sequence
corresponding to that of a respective one of said second
multiplicity of oligonucleotide primers.
5. The process of claim 1, wherein the solid support surface
comprises a multiplicity of solid support zones, each solid support
zone comprising the first multiplicity of anchored
oligonucleotides, which solid support zones are processable
separately from each other, such that all possible combinations of
primers may be utilized in a single analysis, and wherein the
determination of which of said second multiplicity of
oligonucleotide primers is a complementary sequence to a portion of
said solid support-linked chain extension product is effected by a
parallel multiple probing process at each one of said multiplicity
of solid support zones using primer-directed complementary strand
synthesis with a third multiplicity of oligonucleotide primers
being used for each of said multiplicity of solid support zones,
each having a sequence corresponding to that of one of said second
multiplicity of oligonucleotide primers, together with labeled
nucleotide monomers.
6. The process of claim 1 wherein the solid support-anchored
oligonucleotide primers are anchored to discrete solid support
surfaces provided in a microarray device.
7. The process of claim 1 wherein the solid support-anchored
oligonucleotide primers are anchored to discrete solid support
surfaces provided on respective beads bearing identification
indicia.
8. The process of claim 1 wherein the oligonucleotide primers
contain internucleotide linkages selected from phosphodiesters,
phosphorothioates and methyl phosphonates.
9. The process of claim 1 wherein the nucleic acid analyte is
selected from single-stranded DNA, double-stranded DNA, and
messenger RNA.
10. A process for the analysis of a nucleic acid sample so as to
determine the simultaneous presence in the sample of two or more
target sequences each bounded by first and second oligonucleotide
primer sequences, said process comprising the steps of: (i)
providing a first multiplicity of different oligonucleotide
primers, each adapted for covalently anchoring at its 5'-terminus
to a solid support surface, wherein the first multiplicity
comprises the first and second oligonucleotide primer sequences,
(ii) providing a second multiplicity of oligonucleotide primers, in
solution, wherein the nucleotide sequences of respective members of
said first and second oligonucleotide primer multiplicities are
substantially identical to each other, (iii) providing a solid
support surface comprising a multiplicity of individually
identifiable support zones, every one of which is subdivided into
discrete, individually identifiable loci, (iv) covalently anchoring
each member of the first multiplicity of oligonucleotide primers to
an individually identifiable locus of each individually
identifiable support zone, (v) for every individually identifiable
support zone comprising said first multiplicity of anchored
oligonucleotide primers, performing a polymerase chain reaction
amplification on said sample in the presence of said second
multiplicity of oligonucleotide primers, so as to convert said
anchored oligonucleotide primers to anchored polynucleotide chain
extension products, (vi) separating said solid support surface
covalently anchored polynucleotide chain extension products from
the resulting solutions, and (vii) determining, for each said
covalently anchored chain extension product, which of said second
multiplicity of oligonucleotide primers is a complementary sequence
to a portion of said chain extension product, whereby amplification
products from all possible combinations of primers are generated in
a single analysis, and the presence of the two or more target
sequences is detected in the sample.
11. The process of claim 10 wherein the determination of which of
said second multiplicity of oligonucleotide primers is a
complementary sequence to a portion of said covalently anchored
chain extension products is performed by probing with a different
labeled oligonucleotide for each of said individually identifiable
support zones, each labeled oligonucleotide having a sequence
corresponding to that of a respective one of said second
multiplicity of oligonucleotide primers.
12. The process of claim 10, wherein the determination of which of
said second multiplicity of oligonucleotide primers is a
complementary sequence to a portion of said covalently anchored
chain extension products is effected by a parallel multiple probing
process at each one of said individually identifiable zones using
primer-directed complementary strand synthesis with a third
multiplicity of oligonucleotide primers, a different primer from
said third multiplicity of oligonucleotide primers being used for
each of said multiplicity of individually identifiable support
zones, each having a sequence corresponding to that of one of said
second multiplicity of oligonucleotide primers, together with
labeled nucleotide monomers.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/473,796, filed Sep. 30, 2003, which is a
national stage filing under 35 U.S.C. 371 of International
Application No. PCT/GB02/01489, filed Mar. 28, 2002, which claims
priority from United Kingdom Patent Application No. 0108182.7,
filed Apr. 2, 2001. The entire contents of all applications are
hereby incorporated by reference. International Application No.
PCT/GB02/01489 was published under PCT Article 21(2) in
English.
[0002] This invention relates to methods and apparatus for analysis
of biological materials. In particular the invention relates to
methods and apparatus for the analysis of DNA and RNA using the
polymerase chain reaction (PCR).
[0003] The polymerase chain reaction is a technique which is well
known to those skilled in the art of biochemical research, is
widely used for isolation and analysis of biological samples and is
described in many publications, for example "PCR" by C. R. Newton
and A. Graham, published by Bios scientific publishers, Oxford
1997.
[0004] In general, the polymerase chain reaction makes use of two
or more oligonucleotides which serve as primers for a
primer-dependent DNA polymerase enzyme. This enzyme is used to
produce multiple copies of double-stranded DNA molecules present in
an analyte. By selection of suitable primers matching specific
regions of the target DNA, selective amplification of those regions
may be effected by repetitive cycling of the reaction mixture
through a temperature profile which includes the steps of
denaturing of double-stranded DNA, annealing of oligonucleotide
primers to the denaturated template DNA and primer extension using
a thermostable DNA polymerase.
[0005] The use of oligonucleotides immobilised on solid supports as
primers for PCR has been described in a number of documents. For
example, U.S. Pat. No. 5,656,462 describes a method of synthesising
a polynucleotide immobilised support on PCR microplates, and World
Patent No. WO 9932654 describes a method for performing RT-PCR on
oligonucleotide-immobilised PCR microplates. In both these patent
documents, the same oligonucleotide is immobilised on the surface
of all the wells of the microplate in order to allow parallel
processing of multiple samples of analytes.
[0006] U.S. Pat. No. 5,770,358 describes the use of PCR to amplify
oligonucleotide "tags" bound to a solid-support bead in order to
discriminate between different populations of such beads.
[0007] U.S. Pat. No. 5,916,776 describes a method for amplification
of a target nucleic acid in which copies of a first strand are
captured on a solid support at a first location and moved to a
second location at which copies of the first strand are
regenerated. Procedures for practising the method by use of
magnetic particles and microfluidic devices are also disclosed. A
further microfluidic device is described by the same applicants in
U.S. Pat. No. 5,939,291.
SUMMARY OF THE INVENTION
[0008] The present invention provides processes and apparatus for
analysis of DNA molecules by a combinatorial method which makes use
of multiple oligonucleotide primers immobilised on solid
supports.
[0009] Advantageously, the present invention provides a method and
apparatus for analysis of DNA samples which requires no prior
knowledge of the sequence information contained in the sample DNA,
provides a large volume of data from a single experiment, and uses
standard components and materials and is capable of
mass-production.
[0010] According to the present invention, there is provided a
process for transcription or amplification of sequence information
from genetic material by chain extension of oligonucleotide primers
characterised in that during the performance of the process:
[0011] (i) at least two oligonucleotide primers are in solution,
and
[0012] (ii) at least two other oligonucleotide primers are attached
to solid supports, and
[0013] (iii) the nucleotide-sequence of at least one
solid-support-bound oligonucleotide is partially or completely
identical with or complementary to the nucleotide-sequence of at
least one oligonucleotide in solution.
[0014] The present invention further provides a process for
transcription or amplification of sequence information from a
polynucleotide by chain extension of oligonucleotide primers
characterised in that during the performance of the process:
[0015] (i) at least two oligonucleotide primers are in solution,
and
[0016] (ii) at least one other oligonucleotide primer is attached
to a solid support, and
[0017] (iii) the nucleotide-sequence of at least one
solid-support-bound oligonucleotide is partially or completely
identical with or complementary to part of the nucleotide-sequence
of the polynucleotide.
[0018] The present invention further provides an apparatus for the
analysis of analytes which comprises a multiplicity of solid
support surfaces, a multiplicity of oligonucleotides or
polynucleotides attached to said solid support surfaces, such that
each surface bears a single oligonucleotide or polynucleotide,
identification means allowing each of said solid-support surfaces
to be distinguished from other solid support surfaces, containment
means allowing said solid-support surfaces to be contacted with
solutions of analytes, enzymes or reagents, and closure means to
allow agitation, heating or cooling without leakage.
[0019] The invention further provides an apparatus for the analysis
of analytes which comprises a single solid support surface
segregated into zones, a multiplicity of oligonucleotides or
polynucleotides attached to said zones, such that each zone bears a
single oligonucleotide or polynucleotide, identification means
allowing each of said zones to be distinguished from other zones,
containment means allowing said solid-support surface to be
contacted with solutions of analytes, enzymes or reagents, and
closure means to allow agitation, heating or cooling without
leakage.
[0020] The invention further provides an apparatus as hereinbefore
defined further comprising containment means allowing any
individual solid-support surface to be contacted with a specific
analyte, enzyme or reagent without so contacting other
solid-support surfaces, such that a different analyte, enzyme or
reagent may be contacted with every individual solid-support
surface.
[0021] The invention further provides apparatus as hereinbefore
defined in which each solid support-surface or zone of said surface
bears multiple oligonucleotides or polynucleotides in combination,
such that each said surface or zone bears a different combination
of oligonucleotides or polynucleotides, said combination comprising
a sub-set of the totality of oligonucleotides or polynucleotides
attached to all said surfaces or zones of the apparatus.
[0022] The invention further provides apparatus comprising a single
solid support surface segregated into zones as hereinbefore defined
further comprising division means allowing each of said zones to be
physically separated from other zones, thereby enabling the entire
support surface to be physically divided into individual zones.
[0023] The invention further provides a process for the preparation
of multiple polynucleotides attached to a plurality of support
surfaces which comprises the steps of:
[0024] (i) providing a plurality of support surfaces
[0025] (ii) covalently linking one or more oligonucleotides to each
of said surfaces by means of a stable linker moiety covalently
linked to the 5'-terminus of each oligonucleotide and covalently
linked to the support surface, such that each of said surfaces
bears a different oligonucleotide or combination of
oligonucleotides,
[0026] (iii) contacting each of said support surfaces with one or
more additional oligonucleotides and a nucleic acid or mixture of
nucleic acids in the presence of an enzyme or enzymes together with
enzyme substrates and co-factors so as to transfer genetic sequence
data from the nucleic acid or mixture of nucleic acids to the
oligonucleotides of the support surface,
[0027] (iv) optionally heating or altering the pH of said
solid-supported oligonucleotide or oligonucleotides, mixture of
oligonucleotides and nucleic acids, enzyme or enzymes, substrates
and co-factors so as to effect dissociation of double-stranded
molecules,
[0028] (v) optionally cooling or altering the pH of said
solid-supported oligonucleotide or oligonucleotides, mixture of
oligonucleotides and nucleic acids, enzyme or enzymes, substrates
and co-factors so as to effect further transfer of genetic sequence
data from the nucleic acid or mixture of nucleic acids to the
oligonucleotides of the support surface, and
[0029] (vi) optionally repeating steps (iv) to (v) one or more
times in a cyclic manner, so as to effect conversion of individual
oligonucleotides of the support surface to polynucleotides
containing genetic sequence data derived from the nucleic acid or
mixture of nucleic acids.
[0030] The invention further provides a process for the preparation
of multiple polynucleotides attached to a support surface which
comprises the steps of:
[0031] (i) providing a support surface
[0032] (ii) segregating the surface into discrete zones
[0033] (iii) covalently linking a plurality of oligonucleotides to
said zones by means of a stable linker moiety covalently linked to
the 5'-terminus of each oligonucleotide and covalently linked to
the support surface, such that each individual zone bears a
different oligonucleotide or combination of oligonucleotides,
[0034] (iv) contacting said support surface with one or more
additional oligonucleotides and a nucleic acid or mixture of
nucleic acids in the presence of an enzyme or enzymes together with
enzyme substrates and co-factors so as to transfer genetic sequence
data from the nucleic acid or mixture of nucleic acids to the
oligonucleotides of the support surface,
[0035] (v) optionally heating or altering the pH of said
solid-support-bound oligonucleotide or oligonucleotides, mixture of
oligonucleotides and nucleic acids, enzyme or enzymes, substrates
and co-factors so as to effect dissociation of double-stranded
molecules,
[0036] (vi) optionally cooling or altering the pH of said
solid-support-bound oligonucleotide or oligonucleotides, mixture of
oligonucleotides and nucleic acids, enzyme or enzymes, substrates
and co-factors so as to effect further transfer of genetic sequence
data from the nucleic acid or mixture of nucleic acids to the
oligonucleotides of the support surface, and
[0037] (vii) optionally repeating steps (v) to (vi) one or more
times in a cyclic manner, so as to effect conversion of individual
oligonucleotides of the support surface to polynucleotides
containing genetic sequence data derived from the nucleic acid or
mixture of nucleic acids.
[0038] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0039] (i) applying the process as hereinbefore defined in order to
produce a support surface or plurality of support surfaces bearing
multiple polynucleotides containing genetic sequence data derived
from the nucleic acid or mixture of nucleic acids,
[0040] (ii) contacting at least one of said support surfaces with
one or more oligonucleotide or polynucleotide probes capable of
hybridising to polynucleotides of the support surface or
surfaces,
[0041] (iii) washing the support surface or surfaces to remove
unhybridised oligonucleotide or polynucleotide probes,
[0042] (iv) measuring the quantity of oligonucleotide or
polynucleotide probe hybridised to each polynucleotide of the
support surface or surfaces, and
[0043] (v) optionally washing the support surface or surfaces under
conditions sufficient to remove hybridised oligonucleotide or
polynucleotide probes and repeating steps (ii) to (iv) one or more
times using a different oligonucleotide or polynucleotide probe or
probes in each iteration.
[0044] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0045] (i) applying the process as hereinbefore defined in order to
produce a support surface or plurality of support surfaces bearing
multiple polynucleotides containing genetic sequence data derived
from the nucleic acid or mixture of nucleic acids,
[0046] (ii) contacting at least one of said support surfaces with
one or more oligonucleotide primers in the presence of an enzyme or
enzymes together with enzyme substrates and co-factors so as to
produce one or more polynucleotides complementary to the
polynucleotides of the support surface or surfaces,
[0047] (iii) optionally heating or altering the pH of said support
surface or surfaces, oligonucleotide primer or primers, enzyme or
enzymes, substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0048] (iv) optionally cooling or altering the pH of said support
surface or surfaces, oligonucleotide primer or primers, enzyme or
enzymes, substrates and co-factors so as to produce further
polynucleotides complementary to the polynucleotides of the support
surface or surfaces,
[0049] (v) optionally repeating steps (iii) to (iv) one or more
times in a cyclic manner, so as to effect conversion of the primer
oligonucleotide or oligonucleotides to polynucleotides
complementary to the polynucleotides of the support surface or
surfaces,
[0050] (vi) hybridising said complementary polynucleotides to the
polynucleotides of the support surface or surfaces,
[0051] (vii) washing the support surface or surfaces to remove
unhybridised polynucleotides,
[0052] (viii) measuring the quantity of complementary
polynucleotide hybridised to each polynucleotide of the support
surface or surfaces, and
[0053] (ix) optionally washing the support surface or surfaces
under conditions sufficient to remove hybridised complementary
polynucleotides and repeating steps (ii) to (viii) one or more
times using a different oligonucleotide primer or primers in each
iteration.
[0054] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0055] (i) providing a multiplicity of support surfaces each
equipped with unique identification means,
[0056] (ii) covalently linking each of a set of n oligonucleotides,
where n is an integral number greater than unity, to one of said
support surfaces by means of a stable linker moiety covalently
linked to the 5'-terminus of each oligonucleotide and covalently
linked to the support surface, such that each individual
oligonucleotide is attached to an individual support surface,
[0057] (iii) contacting all of said support-surfaces with a
solution containing a mixture of all n oligonucleotides and the
nucleic acid or mixture of nucleic acids to be analysed in the
presence of an enzyme or enzymes together with enzyme substrates
and co-factors so as to transfer genetic sequence data from the
nucleic acid or mixture of nucleic acids to the oligonucleotides
bound to the support surfaces,
[0058] (iv) optionally heating or altering the pH of said support
surfaces, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0059] (v) optionally cooling or altering the pH of said support
surfaces, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect further transfer
of genetic sequence data from the nucleic acid or mixture of
nucleic acids to the oligonucleotides of the support surface,
[0060] (vi) optionally repeating steps (iv) and (v) one or more
times in a cyclic manner, so as to produce n sets of support-bound
polynucleotides by effecting conversion of individual support-bound
oligonucleotides to polynucleotides containing genetic sequence
data derived from the mixtures of nucleic acids,
[0061] (vii) washing said support surfaces under denaturing
conditions to denature double-stranded molecules and remove
oligonucleotides or polynucleotides hybridised to said
support-bound polynucleotides,
[0062] (viii) contacting one or more of said n support surfaces
with a solution containing one of said n oligonucleotide primers in
the presence of an enzyme or enzymes together with enzyme
substrates and co-factors so as to produce polynucleotides
complementary to the polynucleotides bound to said support
surfaces,
[0063] (ix) optionally heating or altering the pH of said support
surfaces, oligonucleotide primers, enzyme or enzymes, substrates
and co-factors so as to effect dissociation of double-stranded
molecules,
[0064] (x) optionally cooling or altering the pH of said support
surfaces, oligonucleotide primers, enzyme or enzymes, substrates
and co-factors so as to produce further polynucleotides
complementary to the polynucleotides bound to said support
surfaces,
[0065] (xi) optionally repeating steps (ix) to (x) one or more
times in a cyclic manner, so as to effect conversion of the primer
oligonucleotide to polynucleotides complementary to the
polynucleotides bound to said support surfaces,
[0066] (xii) hybridising said complementary polynucleotides to the
polynucleotides bound to said support surfaces,
[0067] (xiii) washing said support surfaces under non-denaturing
conditions to remove unhybridised polynucleotides,
[0068] (xiv) separating each of said support surfaces from other
support surfaces,
[0069] (xv) washing each of said support surfaces under denaturing
conditions to denature double-stranded molecules and release
hybridised polynucleotides into solution,
[0070] (xvi) analysing the hybridised polynucleotides released into
solution from each said support surface, and
[0071] (xvii) optionally repeating steps (viii) to (xvi) one or
more times using a different member of the said set of n
oligonucleotide primers in each iteration.
[0072] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0073] (i) providing a multiplicity of support surfaces each
equipped with unique identification means,
[0074] (ii) covalently linking each of a set of n oligonucleotides,
where n is an integral number greater than unity, to one of said
support surfaces by means of a stable linker moiety covalently
linked to the 5'-terminus of each oligonucleotide and covalently
linked to the support surface, such that each individual
oligonucleotide is attached to an individual support surface,
[0075] (iii) contacting all of said support-surfaces with a
solution containing a mixture of all n oligonucleotides and the
nucleic acid or mixture of nucleic acids to be analysed in the
presence of an enzyme or enzymes together with enzyme substrates
and co-factors so as to transfer genetic sequence data from the
nucleic acid or mixture of nucleic acids to the oligonucleotides
bound to the support surfaces,
[0076] (iv) optionally heating or altering the pH of said support
surfaces, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0077] (v) optionally cooling or altering the pH of said support
surfaces, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect further transfer
of genetic sequence data from the nucleic acid or mixture of
nucleic acids to the oligonucleotides of the support surface,
[0078] (vi) optionally repeating steps (iv) and (v) one or more
times in a cyclic manner, so as to produce n sets of support-bound
polynucleotides by effecting conversion of individual support-bound
oligonucleotides to polynucleotides containing genetic sequence
data derived from the mixtures of nucleic acids,
[0079] (vii) washing said support surfaces under denaturing
conditions to denature double-stranded molecules and remove
oligonucleotides or polynucleotides hybridised to said
support-bound polynucleotides,
[0080] (viii) individually contacting each of said n support
surfaces with a solution containing one of said n oligonucleotide
primers in the presence of an enzyme or enzymes together with
enzyme substrates and co-factors so as to produce polynucleotides
complementary to the polynucleotides bound to said support surface,
such that every one of said n support surfaces is contacted with a
unique member of the set of n oligonucleotide primers,
[0081] (ix) optionally heating or altering the pH of every said
support surface, oligonucleotide primer, enzyme or enzymes,
substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0082] (x) optionally cooling or altering the pH of every said
support surface, oligonucleotide primer, enzyme or enzymes,
substrates and co-factors so as to produce further polynucleotides
complementary to the polynucleotides bound to said support
surface,
[0083] (xi) optionally repeating steps (ix) to (x) one or more
times in a cyclic manner, so as to effect conversion of the primer
oligonucleotide to polynucleotides complementary to the
polynucleotides bound to said support surface,
[0084] (xii) hybridising said complementary polynucleotides to the
polynucleotides bound to said support surface,
[0085] (xiii) washing said support surface under non-denaturing
conditions to remove unhybridised polynucleotides,
[0086] (xiv) washing said support surface under denaturing
conditions to denature double-stranded molecules and release
hybridised polynucleotides into solution,
[0087] (xv) analysing the hybridised polynucleotides released into
solution from each said support surface, and
[0088] (xvi) optionally repeating steps (viii) to (xv) one or more
times using for each of said n support surfaces a different member
of the said set of n oligonucleotide primers in each iteration.
[0089] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0090] (i) providing a support surface,
[0091] (ii) segregating the surface into n discrete zones, where n
is an integral number greater than unity,
[0092] (iii) providing each of said zones with unique
identification means,
[0093] (iv) covalently linking each of a set of n oligonucleotides
to one of said zones by means of a stable linker moiety covalently
linked to the 5'-terminus of each oligonucleotide and covalently
linked to the support surface, such that each individual
oligonucleotide is attached to an individual zone of the support
surface,
[0094] (v) contacting said support-surface with a solution
containing a mixture of all n oligonucleotides and the nucleic acid
or mixture of nucleic acids to be analysed in the presence of an
enzyme or enzymes together with enzyme substrates and co-factors so
as to transfer genetic sequence data from the nucleic acid or
mixture of nucleic acids to the oligonucleotides attached to the
support surface,
[0095] (vi) optionally heating or altering the pH of said support
surface, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0096] (vii) optionally cooling or altering the pH of said support
surface, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect further transfer
of genetic sequence data from the nucleic acid or mixture of
nucleic acids to the oligonucleotides of the support surface,
[0097] (viii) optionally repeating steps (vi) and (vii) one or more
times in a cyclic manner, so as to produce n sets of support-bound
polynucleotides by effecting conversion of individual support-bound
oligonucleotides to polynucleotides containing genetic sequence
data derived from the mixtures of nucleic acids,
[0098] (ix) washing said support zones under denaturing conditions
to denature double-stranded molecules and remove oligonucleotides
or polynucleotides hybridised to said support-bound
polynucleotides,
[0099] (x) contacting one or more of said n support zones with a
solution containing one of said n oligonucleotide primers in the
presence of an enzyme or enzymes together with enzyme substrates
and co-factors so as to produce polynucleotides complementary to
the polynucleotides bound to said support zones,
[0100] (xi) optionally heating or altering the pH of said support
zones, oligonucleotide primer, enzyme or enzymes, substrates and
co-factors so as to effect dissociation of double-stranded
molecules,
[0101] (xii) optionally cooling or altering the pH of said support
zones, oligonucleotide primer, enzyme or enzymes, substrates and
co-factors so as to produce further polynucleotides complementary
to the polynucleotides bound to said support zones,
[0102] (xiii) optionally repeating steps (xi) to (xii) one or more
times in a cyclic manner, so as to effect conversion of the primer
oligonucleotide to polynucleotides complementary to the
polynucleotides bound to said support zones,
[0103] (xiv) hybridising said complementary polynucleotides to the
polynucleotides bound to said support zones,
[0104] (xv) washing said support zones under non-denaturing
conditions to remove unhybridised polynucleotides,
[0105] (xvi) separating each of said support zones from other
support zones,
[0106] (xvii) washing each said support zone under denaturing
conditions to denature double-stranded molecules and release
hybridised polynucleotides into solution,
[0107] (xviii) analysing the hybridised polynucleotides released
into solution from each said support zone, and
[0108] (xix) optionally repeating steps (x) to (xviii) one or more
times using in each iteration a solution of a different
oligonucleotide primer selected from the set of n oligonucleotide
primers.
[0109] The invention further provides a process for the analysis of
a nucleic acid or mixture of nucleic acids which comprises the
steps of:
[0110] (i) providing a support surface,
[0111] (ii) segregating the surface into n discrete zones, where n
is an integral number greater than unity,
[0112] (iii) providing each of said zones with unique
identification means,
[0113] (iv) covalently linking each of a set of n oligonucleotides
to one of said zones by means of a stable linker moiety covalently
linked to the 5'-terminus of each oligonucleotide and covalently
linked to the support surface, such that each individual
oligonucleotide is attached to an individual zone of the support
surface,
[0114] (v) contacting said support-surface with a solution
containing a mixture of all n oligonucleotides and the nucleic acid
or mixture of nucleic acids to be analysed in the presence of an
enzyme or enzymes together with enzyme substrates and co-factors so
as to transfer genetic sequence data from the nucleic acid or
mixture of nucleic acids to the oligonucleotides attached to the
support surface,
[0115] (vi) optionally heating or altering the pH of said support
surface, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect dissociation of
double-stranded molecules,
[0116] (vii) optionally cooling or altering the pH of said support
surface, mixture of oligonucleotides and nucleic acids, enzyme or
enzymes, substrates and co-factors so as to effect further transfer
of genetic sequence data from the nucleic acid or mixture of
nucleic acids to the oligonucleotides of the support surface,
[0117] (viii) optionally repeating steps (vi) and (vii) one or more
times in a cyclic manner, so as to produce n sets of support-bound
polynucleotides by effecting conversion of individual support-bound
oligonucleotides to polynucleotides containing genetic sequence
data derived from the mixtures of nucleic acids,
[0118] (ix) washing said support surface under denaturing
conditions to denature double-stranded molecules and remove
oligonucleotides or polynucleotides hybridised to said
support-bound polynucleotides,
[0119] (x) physically dividing the support surface to form n
individually-identifiable support zones each bearing a set of
support-bound polynucleotides derived by extension of one of the n
support-bound oligonucleotide primers,
[0120] (xi) individually contacting every one of said n support
zones with a solution containing one of said n oligonucleotide
primers in the presence of an enzyme or enzymes together with
enzyme substrates and co-factors so as to produce polynucleotides
complementary to the polynucleotides bound to said support zone,
such that every one of said n support zones is contacted with a
unique member of the set of n oligonucleotide primers,
[0121] (xii) optionally heating or altering the pH of every said
support zone, oligonucleotide primer, enzyme or enzymes, substrates
and co-factors so as to effect dissociation of double-stranded
molecules,
[0122] (xiii) optionally cooling or altering the pH of every said
support zone, oligonucleotide primer, enzyme or enzymes, substrates
and co-factors so as to produce further polynucleotides
complementary to the polynucleotides bound to said support
zone,
[0123] (xiv) optionally repeating steps (xii) to (xiii) one or more
times in a cyclic manner, so as to effect conversion of the primer
oligonucleotide to polynucleotides complementary to the
polynucleotides bound to said support zone,
[0124] (xv) hybridising said complementary polynucleotides to the
polynucleotides bound to said support zone,
[0125] (xvi) washing said support zone under non-denaturing
conditions to remove unhybridised polynucleotides,
[0126] (xvii) washing said support zone under denaturing conditions
to denature double-stranded molecules and release hybridised
polynucleotides into solution,
[0127] (xviii) analysing the hybridised polynucleotides released
into solution from each said support zone, and
[0128] (xix) optionally repeating steps (xi) to (xviii) one or more
times using for each of said n support zones a different member of
the said set of n oligonucleotide primers in each iteration.
[0129] The invention further provides a process for the analysis of
two or more distinct mixtures of nucleic acids which comprises the
steps of:
[0130] (i) applying the processes as hereinbefore defined in order
to produce a set of support surfaces or zones bearing a first set
of immobilised polynucleotides containing genetic sequence data
derived from a first mixture of nucleic acids, together with a
first set of polynucleotides complementary to said first set of
immobilised polynucleotides,
[0131] (ii) applying the processes as hereinbefore defined in order
to produce a set of support surfaces or zones bearing a second set
of immobilised polynucleotides containing genetic sequence data
derived from a second mixture of nucleic acids, together with a
second set of polynucleotides complementary to said second set of
immobilised polynucleotides,
[0132] (iii) hybridising said first set of complementary
polynucleotides to said first set of immobilised
polynucleotides,
[0133] (iv) individually releasing and analysing the complementary
polynucleotides hybridised to each of said first set of immobilised
polynucleotides,
[0134] (v) hybridising said second set of complementary
polynucleotides to said second set of immobilised
polynucleotides
[0135] (vi) individually releasing and analysing the complementary
polynucleotides hybridised to each of said second set of
immobilised polynucleotides,
[0136] (vii) optionally hybridising one or more members of said
first set of complementary polynucleotides to one or more members
of said second set of immobilised polynucleotides, individually
releasing and analysing the complementary polynucleotides
hybridised to each of said second set of immobilised
polynucleotides,
[0137] (viii) optionally hybridising one or more members of said
second set of complementary polynucleotides to one or more members
of said first set of immobilised polynucleotides, individually
releasing and analysing the complementary polynucleotides
hybridised to each of said first set of immobilised
polynucleotides, and
[0138] (ix) optionally repeating steps (ii) and (v) to (viii) for
further mixtures of nucleic acids.
[0139] The invention further provides a process as hereinbefore
defined in which a single support surface segregated into zones is
physically divided into discrete zones after completing at least
one step of the process and each such zone is processed separately
during subsequent steps of the process.
[0140] The invention further provides a process as hereinbefore
defined in which the stable linker moiety is covalently linked to
the 5'-terminus of each oligonucleotide by means of covalent
bonding to one or more atoms of the carbohydrate moiety of the
5'-terminal nucleotide.
[0141] The invention further provides a process as hereinbefore
defined in which the stable linker moiety is covalently linked to
the 5'-terminus of each oligonucleotide by means of covalent
bonding to one or more atoms of the heterocyclic moiety of the
5'-terminal nucleotide.
[0142] The invention further provides a process as hereinbefore
defined in which the stable linker moiety comprises a
covalently-bound functionality with a chemical equivalence of
between 100 and 5000 picomoles per square millimetre of support
surface.
[0143] The invention further provides a process as hereinbefore
defined in which the stable linker moiety is thermally stable in
aqueous solution at 95 deg. C. The invention further provides a
process as hereinbefore defined in which the stable linker moiety
is hydrolytically stable in aqueous solution between pH 4 and pH
11.
[0144] The invention further provides a process as hereinbefore
defined in which the oligonucleotides contain internucleotide
linkages selected from the group comprising phosphodiesters,
phosphorothioates and methyl phosphonates.
[0145] The invention further provides a process as hereinbefore
defined in which one or more oligonucleotides contain labelled
molecules selected from the group comprising fluorescent molecules,
luminescent molecules, biotin-labelled molecules, radiolabelled
molecules and enzyme conjugates.
[0146] The invention further provides a process as hereinbefore
defined in which the nucleic acid or mixture of nucleic acids to be
analysed is selected from the group comprising single-stranded DNA
molecules, double-stranded DNA molecules and messenger RNA
molecules.
[0147] The invention further provides a process as hereinbefore
defined in which the enzyme or enzymes are selected from the group
comprising DNA polymerases, thermostable DNA polymerases, reverse
transcriptases, thermostable reverse transcriptases, DNA ligases
and thermostable DNA ligases.
[0148] The invention further provides a process as hereinbefore
defined in which the enzyme substrates and co-factors are selected
from the group comprising nucleoside triphosphates, fluorescent
nucleoside triphosphates, luminescent nucleoside triphosphates,
biotin-labelled nucleoside triphosphates, amino-allyl nucleoside
triphosphates, radiolabelled nucleoside triphosphates, metal salts,
buffering agents, chelating agents, chaotropic agents and nuclease
inhibitors.
[0149] The invention further provides an apparatus as hereinbefore
defined in which the support surface, containment means and closure
means comprise separate components adapted to be assembled to form
the apparatus.
[0150] The invention further provides an apparatus as hereinbefore
defined in which each set of immobilised oligonucleotides or
polynucleotides further comprises control substances or indicia
adapted for identification, alignment or calibration purposes.
[0151] The invention further provides apparatus and processes as
hereinbefore defined further comprising automated handling
equipment allowing high-throughput analysis of DNA samples under
computer control.
[0152] The invention further provides a computer program comprising
software code portions for controlling apparatus adapted to perform
the process of the invention as hereinbefore defined.
[0153] The invention further provides a computer program comprising
software code portions for predicting the outcome of the process of
the invention as hereinbefore defined for any given combination of
oligonucleotides in relation to the presence or absence in an
analyte of one or more polynucleotides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0154] Specific embodiments of the invention will now be described,
by way of example, with reference to the accompanying drawings, in
which: --
[0155] FIG. 1 shows a radio-frequency transponder sealed in a glass
envelope whose surface may be used as a solid support surface in
the practice of the invention.
[0156] FIG. 2 shows a glass plate divided into zones which may be
used as a solid support surface in the practice of the
invention.
[0157] FIG. 3 shows a silicon wafer divided into zones which may be
used as a solid support surface in the practice of the
invention.
[0158] FIG. 4 shows schematically a process for performing PCR on
multiple solid supports.
[0159] FIG. 5 shows schematically a process for generating labelled
polynucleotides which are complementary to immobilised
polynucleotides produced by PCR.
[0160] FIG. 6 shows schematically procedures for analysis of
complementary polynucleotides.
[0161] FIG. 7 shows schematically a process for performing PCR on a
single solid support subdivided into zones.
[0162] FIG. 8 shows schematically a process for generating labelled
complementary sequences after separating a single solid support
into individual zones.
[0163] FIG. 9 shows, in plan view, a 96-well plate in which each
well contains an oligonucleotide array.
[0164] FIG. 10 shows, in plan view, an individual oligonucleotide
array.
[0165] FIG. 11 shows, in exploded view, the components of a modular
96-well plate.
[0166] FIGS. 12 to 15 show, in sectional view, the assembly of the
components of a modular 96-well plate mounted between heating
blocks.
[0167] FIG. 16 shows, in sectional view, the detailed construction
of an individual well of a 96-well plate.
[0168] FIG. 17 shows a table of oligonucleotide sequences which may
be used in the practice of the invention.
[0169] FIG. 18 shows schematically a process for distinguishing
between two DNA samples by practising the process of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0170] In a preferred embodiment of the apparatus of the invention,
the support surface is the external glass surface of a
radio-frequency tag (RF tag) as illustrated in FIG. 1. Referring to
the drawing, this consists of a microelectronic radio-frequency
transponder circuit 1 equipped with an antenna coil 2 wound on a
ferrite core 3. The entire assembly is hermetically sealed in a
glass envelope 4 whose outer surface 5 serves as the support
surface for immobilised oligonucleotides in the practice of the
invention.
[0171] In a second preferred embodiment of the apparatus of the
invention, the support surface is the surface of a glass plate
divided into zones as illustrated in FIG. 2. Referring to the
drawing, the surface of the glass plate is divided into zones by
means of a grid of lines 6 which are scribed into the surface, for
example by use of a glass knife or a diamond pencil, or etched into
the surface by chemical treatment, for example with hydrofluoric
acid, or by laser ablation. Zones are also equipped with unique
identifying marks 7.
[0172] In a third preferred embodiment of the apparatus of the
invention, the support surface is the surface of a silicon wafer
divided into zones as illustrated in FIG. 3. Referring to the
drawing, the surface of the silicon wafer is divided into zones by
means of a grid of lines 8 which are formed in the surface, for
example by laser ablation. Zones are also equipped with unique
identifying marks 9 in the form of a two-dimensional bar code
formed by laser ablation. Prior to use of the wafer as a
solid-support for oligonucleotide immobilisation, a layer of
silicon oxide is formed on the surface of the wafer using standard
microelectronic fabrication methods.
[0173] A preferred method of practising the invention is
illustrated schematically in FIG. 4. According to this method, a
set 10 of oligonucleotide primers in solution is used in
conjunction with a set 11 of the same oligonucleotide primers each
of which is attached to one of a set of individual support surfaces
12 to 19 inclusive. Each of the solid support surfaces may be a
discrete entity, for example an RF tag as hereinbefore described
and illustrated in FIG. 1, or may be a zone of a larger surface,
for example the glass plate of FIG. 2 or the silicon wafer of FIG.
3. Other types of support surfaces suitable for the practice of the
invention include glass or plastic beads labelled with
uniquely-identifiable markers, such as defined-ratio combinations
of fluorescent dyes or specific combinations of other molecules.
Support media of these types are commercially available for
applications in combinatorial chemistry and biochemical
analysis.
[0174] In a first step of the method of this embodiment, the set 11
of oligonucleotide primers attached to the solid-support surfaces
12 to 19 inclusive is contacted with a solution containing a set 10
of the same oligonucleotide primers together with the sample 20 of
double-stranded DNA molecules to be analysed, a polymerase enzyme
21, for example Taq DNA polymerase, a mixture 22 of unlabelled
deoxynucleoside triphosphates comprising A-, G-, C- and
T-deoxynucleo side triphosphates, and a mixture 23 of magnesium
chloride and buffer salts, the solution being loaded into a
sealable container which is adapted to contain all of the
solid-support surfaces 12 to 19 inclusive. A wide variety of
containers are suitable for the practice of the invention, for
example microcentrifuge tubes may be used where RF tags are
employed as solid-supports, or alternatively a sealable multi-well
plate is also suitable. Where a single support surface subdivided
into zones is used in the practice of the invention, sealable
containers such as those employed for staining microscope slides
may be employed. After physical division of such support surfaces,
the individual zones may be transferred to microcentrifuge tubes
for subsequent steps of the method of the invention.
[0175] Apparatus designed for combinatorial chemical synthesis may
also be employed in practising the method of the invention. For
example, reactor blocks containing multiple reaction chambers are
suitable for use with RF tags or with individual zones of support
surfaces after physical division of a single support surface
subdivided into zones.
[0176] As will be understood by those skilled in the art of
biochemical analysis, a wide variety of containers other than those
mentioned here may also be employed in practising the method
without departing from the spirit of the invention.
[0177] The sealed container is then loaded into a heating block or
thermal cycler suitable for performing the Polymerase Chain
Reaction (PCR) and subjected to multiple amplification cycles each
comprising a denaturation step, an annealing step and an extension
step. During each amplification cycle, segments of double-stranded
DNA sequences in the sample to be analysed are amplified by PCR,
the section of each such sequence amplified being determined by the
presence and location of segments within each sequence possessing
complementarity to one or more pairs of oligonucleotide
primers.
[0178] After multiple cycles of amplification, the solution
contains amplified copies of DNA segments contained within the
sample to be analysed, formed by extension of members of the set 10
of oligonucleotide primers originally present in solution. In
addition, the set 11 of solid-support-bound oligonucleotide primers
also participates in the amplification process, so that each
support surface also bears covalently-bound amplified copies of DNA
segments contained within the sample to be analysed.
[0179] After completion of the PCR process, the solution is removed
and the support surfaces are washed under denaturing conditions in
order to remove all non-covalently-linked oligonucleotides and
polynucleotides. After this operation, the set of solid-support
surfaces carries a set 24 of single-stranded polynucleotides
derived by amplification of molecules of the sample DNA 20 and
covalently bound to the solid supports.
[0180] In a second step of the method of this embodiment, as
illustrated schematically in FIG. 5, the set 24 of
solid-support-bound single-stranded polynucleotides is subjected to
a further primer-dependent polymerase reaction in which the
sealable container accommodating the solid-supports is loaded with
a solution containing a single oligonucleotide primer 25 which may
be selected from the set 10 of oligonucleotide primers employed in
the first step of the method. Alternatively, an unrelated
oligonucleotide primer may be used. This oligonucleotide acts as a
primer for synthesis of a complementary strand on each of the set
24 of solid-support-bound single-stranded polynucleotides. The
solution also contains a polymerase enzyme 26, for example Taq DNA
polymerase, together with magnesium chloride and buffer salts, and
a mixture 27 of deoxy nucleoside triphosphates, comprising A-, G-,
C- and T-deoxynucleoside triphosphates, together with a labelled
molecule. The labelled molecule is one which may be incorporated
into the complementary strand formed on each polynucleotide during
the primer-dependent polymerase reaction. For example, a Uridine
derivative such as biotin-16-dUTP or aminoallyl-dUTP may be
included in the polymerase reaction mixture in order to incorporate
biotin labelled molecules or aminoallyl substituents suitable for
labelling in place of Thymidine units within the complementary
strand during the primer-dependent polymerase reaction.
[0181] Alternatively, the label may be attached to the 5'-terminus
of the oligonucleotide primer, as is commonly employed in DNA
sequencing. In this case fluorescent dyes such as those commonly
known as FAM, JOE, TAMRA and ROX, which are commercially available
from Applied Biosystems, may be incorporated into the
oligonucleotide primer during synthesis. Similarly, a 5'-biotin
label may be incorporated into the oligonucleotide during synthesis
by use of a biotin phosphoramidite. Radioactive labels may also be
used where appropriate. Where the label is attached to the
oligonucleotide, the subsequent polymerase-mediated reaction may be
omitted and replaced by a simple hybridisation process in which the
labelled oligonucleotide serves as a probe for detection of
complementary sequences contained in the solid-support-bound
single-stranded polynucleotides. However, greater sensitivity may
be obtained by incorporating multiple labels into the second strand
by performing the polymerase reaction in the presence of labelled
nucleotide triphosphates. Alternatively, different labels may be
incorporated into the oligonucleotide primer and the nucleotide
triphosphates, allowing determination of the ratio of 5'-terminal
label to labels incorporated during second-strand synthesis. This
may be used, for example, to obtain estimates of the chain length
and base composition of the second strand. Where suitable detection
means are available, multiple fluorescent dyes or other labels may
be used for this purpose, such that each of the four nucleotide
triphosphates contains a different dye, allowing the labels
associated with each nucleotide to be determined separately. Where
appropriate, multiple sets of reactions may be performed using the
same combinations of oligonucleotides but with different labelled
nucleotides in each reaction, providing further levels of
nucleotide-specific information.
[0182] The primer-dependent polymerase reaction may be driven to
completion by subjecting the solution to thermal cycling as
described in the foregoing description of the first step of the
method of this embodiment. In this second step, however, the
polymerisation products are formed in solution rather than on the
solid-support surface, the set 24 of solid-support-bound
single-stranded polynucleotides serving as the templates for
second-strand synthesis. The resulting solution contains labelled
polynucleotides complementary to the solid-support-bound
polynucleotides which are then allowed to hybridise together to
form double-stranded polynucleotides in which one strand is bound
to the solid-support surface. After washing to remove unbound
material, the quantity of label attached to each support surface is
then determined using standard procedures for quantitation of
fluorescent or luminescent molecules or, in the case of a
radioactive label, radiometry or autoradiography. Where
appropriate, support surfaces divided into zones such as those
illustrated in FIG. 2 and FIG. 3 may first be physically divided
before quantitation of the labelled molecules attached to each
zone. The divided zones may then be quantitated individually, for
example by elution of the labelled molecule prior to
spectrophotometric, spectrofluorimetric or radiometric
determination of the eluate. The process of elution and
quantitation is illustrated schematically in FIG. 6, in which a
double-stranded polynucleotide 29 attached to an individual support
zone is heated in an appropriate buffer solution in order to
dissociate the polynucleotide into a single-stranded support-bound
polynucleotide 30 and the labelled complementary strand 31, which
is released into solution. After removal of the solid support, the
concentration in solution of the labelled molecules is determined
by spectrometry, spectrofluorimetry or radiometry as
appropriate.
[0183] As will be apparent to those skilled in the art of DNA
sequence analysis, the method of the invention as described above
may be modified in a variety of ways without departing from the
spirit of the invention. For example, the set of
solid-support-bound polynucleotides produced in the first step of
the method may be applied directly in other techniques for which
such supported molecules are employed, as in the preparation and
application of DNA micro-arrays for detection of genetic sequences
by hybridisation. Similarly, the amplification of genetic sequence
information from sample molecules by PCR may be performed in two
stages, using only the solution-phase primers initially in order to
obtain a preliminary degree of amplification before introducing the
solid-supported primers. Where appropriate, a purification or
concentration step may be included after such initial amplification
in order to remove residual sample DNA or selectively carry forward
specific amplification products for incorporation into the
solid-support-bound polynucleotides by further cycles of PCR.
Similarly, other detection strategies may be adopted in order to
eliminate the requirement for labelling, for example by using
mass-spectrometric or electrochemical techniques to characterise
the polynucleotides produced.
[0184] The method of the invention as applied to a single support
surface divided into support zones is further illustrated
schematically in FIG. 7, which summarises the steps involved in
production of single-stranded PCR products attached to the
solid-support zones and in FIG. 8, which summarises the subsequent
operations of complementary strand synthesis, separation of the
support into individual zones, and elution and quantitation of
labelled complementary strands.
[0185] Alternatively, each zone of the surface may be quantitated
individually without physical division of the support surface where
an appropriate instrumental technique for doing so is available,
for example by use of a laser scanner where fluorescently-labelled
molecules are to be determined. Individual support surfaces are
identified by reading the identification means from each surface,
whether these be attached or engraved indicia as in the surface
zones of FIG. 2 and FIG. 3 or coded RF tags as in FIG. 1.
[0186] Since the sequence of the 5'-terminus of each
solid-support-bound polynucleotide is known from the sequence of
the oligonucleotide initially applied to that support surface, and
the sequence of the 3'-terminus of each solid-support-bound
polynucleotide is known to be complementary to the single primer
oligonucleotide present in solution in the second step of the
method, the results from all support surfaces may be used to deduce
sequence information for the sample to be analysed. For example,
the sequences of the primer oligonucleotides may be selected so as
to demonstrate the presence or absence of a specific polynucleotide
in the analyte which is associated with a microbial infection or
other disease condition or of a genetic disorder.
[0187] The second step of the method may be repeated using the same
set of support-bound polynucleotides with a different primer
oligonucleotide in solution in order to obtain further sequence
information. This procedure may be applied iteratively so as to
examine all possible combinations of the chosen set of primer
oligonucleotides. In the example illustrated schematically in FIG.
4, where a set of eight primer oligonucleotides is employed, there
are 64 possible combinations of the primer oligonucleotides which
may be examined in this manner.
[0188] A particular advantage of the process of this embodiment is
that it has the capacity to provide verification of results, since,
if double-stranded template DNA is being analysed, each of the
possible pairs of oligonucleotides should produce two signals, one
for the combination of a solid-support-bound primer P.sub.1 with a
solution-phase primer P.sub.2, and a corresponding signal for the
complementary strand represented by the combination of
solid-support-bound primer P.sub.2 with solution-phase primer
P.sub.1.
[0189] As will be appreciated by those skilled in the art of DNA
analysis and combinatorial chemistry, much larger sets of primer
oligonucleotides than those depicted in this figurative example may
be employed in order to generate results from hundreds or thousands
of combinations of the primer oligonucleotides.
[0190] A fourth preferred embodiment of the apparatus of the
invention comprises an apparatus for analysis of DNA samples using
a set of 96 primer oligonucleotides, as illustrated in FIG. 9. In
this embodiment, the support surface is formed into wells, the base
of each well being planar and bearing an array of oligonucleotides.
Referring to the drawings, FIG. 9 shows in plan view a 96-well
plate 32 in which each well 33 contains an array 34 of
oligonucleotides. A detailed plan view of an individual array is
shown in FIG. 10, in which the circular base 35 carries multiple
sub-zones 36 to each of which is attached an oligonucleotide. Each
oligonucleotide is attached to the surface by means of a stable
linker moiety covalently linked to the 5'-terminus of the
oligonucleotide and covalently linked to the support surface. The
stable linker moiety should be thermally stable in aqueous solution
at 95 deg. C. and hydrolytically stable between pH 4 and pH 11,
allowing the linkage of oligonucleotide to the surface to withstand
the conditions normally employed in practising techniques such as
the polymerase chain reaction (PCR). The linker moiety may
incorporate spacer molecules as part of the covalent linkage
between oligonucleotide and support surface, in order to reduce or
eliminate steric effects which may inhibit processes involving the
oligonucleotides, for example binding of oligonucleotides to an
enzyme molecule.
[0191] The identity of the oligonucleotide attached to each
position of the array is established at the time of manufacture, so
that individual identification means for each oligonucleotide are
not required, the position of the oligonucleotide within the array
serving to identify it uniquely. Additional positions may be
included in each array to provide loci for placement of control
substances and index markers for calibration and alignment
purposes, especially where automated systems are to be used in
quantitation and data acquisition. This is exemplified in FIG. 10,
which contains eight additional sub-zones 35 in addition to the
ninety-six zones required for placement of oligonucleotides.
[0192] The attachment of oligonucleotides to the planar base of
each well may be performed directly on the material of the 96-well
plate, for example by attaching suitable linker molecules to a
polystyrene or polypropylene surface as described, for example, in
the above-referenced patents U.S. Pat. No. 5,656,462 and WO
9932654. For ease of manufacture, however, it is preferable to
attach the oligonucleotides to a planar slice of support material
which may then be inserted into the base of each well. This permits
the mass-production of the body of the 96-well plate from plastic
materials suitable for use in an injection-moulding process while
allowing other materials to be used as the support surface for the
array which may then be fixed in position by an interference fit or
by welding or using a suitable adhesive. Preferred materials for
the planar slice are glass or a silicon oxide layer formed on a
silicon wafer, and the slice may be of any suitable shape, for
example circular, square, rectangular or hexagonal in form.
[0193] In a fifth preferred embodiment of the apparatus of the
invention, a 96-well plate of modular construction is provided.
This has three major components: a cover plate, a body divided into
96 cylindrical compartments opening on to both faces of the body,
and a baseplate carrying 96 oligonucleotide arrays. These
components may be combined to form a stack as illustrated in FIG.
11, in which the cover plate 37 overlies the body section 38, which
in turn is placed over the baseplate 39 carrying the arrays 40. The
assembly of the stack is shown in sectional view in FIG. 12, in
which the cover plate 37 overlies the body section 38, which in
turn is placed over the baseplate 39 carrying the arrays 40. O-ring
seals 45 and 46 are fitted to the upper and lower ends of each of
the cylindrical bores in the body component to form a fluid-tight
seal against the cover plate and the baseplate.
[0194] In use, as illustrated in FIG. 13, the baseplate and body
are fitted to a metal base 41 and held in place by spring clips 42
and 43. At this stage, each well is sealed at its lower end against
the baseplate and may be charged with solutions appropriate to the
analysis to be performed. Following this, as illustrated in FIG.
14, the cover plate 37 may be placed in position and held in place
by a metal pressure plate 44. This serves to seal the upper end of
each well against the cover plate. As illustrated in FIG. 15, where
thermal cycling is required, for example in order to perform
polymerase chain reactions on the arrays, the entire assembly may
now be compressed between two heated metal plates 47 and 48, the
upper plate serving to prevent condensation on the underside of the
cover plate. Where "hot start" conditions are required, the metal
base 41 may be placed on a heated surface prior to loading
solutions in the wells, following which the cover plate 37 and
pressure plate 44 may be fitted.
[0195] The internal arrangement of an individual well of the
assembled apparatus is illustrated in FIG. 16, in which the
baseplate 39 bearing the oligonucleotide array 40 is supported on
metal base 41, the cylindrical bore of the body component 38 is
sealed against the baseplate by O-ring seal 46 and by O-ring seal
45 against the cover plate 37 which is held in place by pressure
plate 44.
[0196] Where, as hereinbefore described, the process of the
invention is to be performed using an initial amplification step
using only solution-phase primers before introducing the
solid-support bound primers, this may be accomplished simply by
initially inverting the apparatus during the first step of the
process, so that the baseplate 39 bearing the oligonucleotide
arrays is uppermost. In this configuration, the solution in each
well is not in contact with the solid-support bound
oligonucleotides of the array surfaces, and PCR proceeds using only
solution-phase primers. After the initial amplification step, the
apparatus is inverted again, allowing the solid-support bound
primers to participate in subsequent cycles of PCR
amplification.
[0197] Preferred materials for the construction of the metal base
41 and pressure plate 44 are aluminium or stainless steel.
Preferred materials for the construction of the body 38 and cover
plate 37 are polypropylene, polytetrafluorethylene (PTFE),
polyether ether ketone (PEEK) or polystyrene. A preferred material
for the construction of the baseplate 39 is glass. Preferred
materials for the O-ring seals are fluorocarbon rubber or EPDM.
[0198] The arrays 40 may be formed directly on the surface of the
baseplate 39 using a commercial robotic arraying system.
Alternatively, the arrays may be manufactured individually on
plates of glass or surface-oxidised silicon wafers as hereinbefore
described and fixed to the baseplate 39 by welding or by using a
suitable adhesive, in which case the baseplate may be manufactured
from a plastic material such as polypropylene or polystyrene. The
plates carrying the arrays may be disc-shaped, as depicted in FIG.
10, or may equally be rectangular, square or hexagonal in form.
Where silicon wafers are used for construction of arrays, a square
or rectangular form may be preferred to facilitate handling of
diced wafer sections by use of standard microfabrication and
handling equipment.
[0199] This embodiment of the apparatus of the invention allows all
possible combinations of 96 oligonucleotides to be generated in a
single analysis. By analysing the results from multiple
experiments, information may be deduced as to the sequence of DNA
molecules of the sample, particularly by using appropriate computer
programs to interpret the data generated. The 96-well layout may be
further extended to encompass 384-well or 1536-well formats,
enabling many more combinations of primers to be generated.
[0200] A particularly useful aspect of the procedures of the
invention is the ability to discriminate between populations of DNA
molecules even where little is known about the genetic sequences
involved. This can be achieved by choosing primers from sets of
oligonucleotides which are selected randomly, with the proviso that
their (G+C) content should be between 60% and 70% and that they
should not have self-complementary ends. These criteria ensure that
the oligonucleotides will prime efficiently during the PCR process.
Such random sets of oligonucleotides are commonly used in the
technique of Random Amplified Polymorphic DNA (RAPD) production by
PCR, and are commercially available. Examples of typical 10-base
primer sets are given in FIG. 17. The nucleotide sequences given in
this diagram are for illustrative purposes only and do not
represent the sequences of actual biological materials.
[0201] In a second preferred embodiment of the process of the
invention, individual support surfaces are functionalised with a
mixture of two or more oligonucleotides in combination prior to
practising the method hereinbefore described. This allows results
to be obtained for one or more sub-sets of the set of
oligonucleotides. This may be useful, for example, as a preliminary
screening method prior to refining the method in subsequent
experiments by using individual support-bound oligonucleotides as
in the hereinbefore-described method.
[0202] In a third preferred embodiment of the process of the
invention, the solution used during the second step of the method,
in which complementary strand synthesis is performed, contains a
mixture of two or more oligonucleotides in combination. The method
is otherwise practised as hereinbefore described. This allows
results to be obtained for one or more sub-sets of the set of
oligonucleotides. This may be useful, for example, as a preliminary
screening method prior to refining the method in subsequent
experiments by using individual support-bound oligonucleotides as
in the hereinbefore-described method.
[0203] In a fourth preferred embodiment of the process of the
invention, individual support surfaces are functionalised with a
mixture of two or more oligonucleotides in combination and the
solution used during the second step of the method, in which
complementary strand synthesis is performed, contains a mixture of
two or more oligonucleotides in combination. The method is
otherwise practised as hereinbefore described. This allows results
to be obtained for one or more sub-sets of the set of
oligonucleotides. This may be useful, for example, as a preliminary
screening method prior to refining the method in subsequent
experiments by using individual support-bound oligonucleotides as
in the hereinbefore-described method.
[0204] In a fifth preferred embodiment of the process of the
invention, solutions of two or more different oligonucleotides are
applied separately to individual solid-supports or solid-support
zones during the second step of the method, in which complementary
strand synthesis is performed. The method is otherwise practised as
hereinbefore described. This requires separate containment means
for the individual solid-supports or solid-support zones. Where the
individual solid-supports are separate entities, for example RF
tags as illustrated in FIG. 1, or a single support surface which
has been physically divided into individual support zones as
hereinbefore described, this may be accomplished by placing each
support entity in a separate tube or other container prior to
performing the second step of the method as hereinbefore described.
In the case of a single support surface subdivided into zones which
have not been physically divided, this may be accomplished by
applying a suitable gasket to the support surface to allow
different solutions to be applied to each zone without
cross-contamination.
[0205] In a sixth preferred embodiment of the process of the
invention, solutions of two or more different DNA samples are
applied separately to individual solid-supports or solid-support
zones during the first step of the method, in which PCR
amplification products are formed on the solid-supports. The method
is otherwise practised as hereinbefore described. This requires
separate containment means for the individual solid-supports or
solid-support zones. Where the individual solid-supports are
separate entities, for example RF tags as illustrated in FIG. 1,
this may be accomplished by placing each support in a separate tube
or other container prior to performing the method as hereinbefore
described. In the case of a single support surface subdivided into
zones, this may be accomplished by applying a suitable gasket to
the support surface to allow different solutions to be applied to
each zone without cross-contamination.
[0206] In a seventh preferred embodiment of the process of the
invention, solutions of labelled complementary strands eluted from
one support surface are applied to another support surface and
allowed to hybridise thereto. Following this operation, the support
surface is washed to remove unbound material and hybridised
polynucleotide is eluted by washing under more stringent conditions
and quantitated by the appropriate method. This procedure may be
repeated for all possible combinations of such eluted complementary
strands and support surfaces and the data thereby obtained applied
in various ways, for example in the construction of a linkage map
for the DNA sample of the analyte.
[0207] As will be apparent to those skilled in the arts of
biochemical analysis and DNA technology, a large number of
different combinations of oligonucleotides may be selected from the
total number of possible oligonucleotides of any given
chain-length, and each combination will give different results for
any given sample of DNA which may be subjected to analysis by
practising the invention. Very large volumes of data may therefore
be generated by performing multiple analyses of a sample using
different combinations of oligonucleotides in each case. As will
also be apparent to those skilled in the art of biochemical
analysis, the statistical probability of finding a match between
any given oligonucleotide and a sub-sequence of a component DNA
molecule within a given sample is a function of the length of the
oligonucleotide, and this parameter may be varied as required in
order to obtain informative results from the methods of the
invention.
[0208] A particular advantage of the technique of the invention is
the statistical improbability of a particular combination of two
sub-sequences occurring more than once in a given population of DNA
molecules. This is in contrast to the relatively high probability
of random occurrences of a single sub-sequence in a given
population of DNA molecules. Taking the example of a sub-sequence
composed of 10 nucleotides, the total possible number of such
sequences is approximately 1 million. This is a relatively small
number in relation to the size of the human genome, for example,
which contains three thousand million base-pairs. Random
occurrences of a particular 10 nucleotide sequence might therefore
be expected to occur relatively frequently within the human genome.
In contrast, the number of possible combinations of two different
10-nucleotide sequences is approximately five hundred million
million million. This number is much larger than the number of
base-pairs in the human genome, and the chances of a particular
combination of two 10-nucleotide sequences occurring more than once
in the population of DNA molecules comprising the human genome are
correspondingly reduced.
[0209] The technique of the invention may therefore be used to
implement highly-specific DNA-based detection procedures for a
particular genetic material with a low probability of obtaining
spurious results due to the random occurrence of matching DNA
sequences in the sample.
[0210] An example of the application of the process of the
invention to discriminate between two different DNA molecules is
illustrated in FIG. 18. The nucleotide sequences given in this
diagram are for illustrative purposes only and do not represent the
sequences of actual biological materials.
[0211] Referring to the diagram, if it is desired to discriminate
between the two double-stranded 100-base-pair DNA sequences,
sequence X and sequence Y, this may be achieved using a set of
oligonucleotide primers of appropriate sequence. The diagram
illustrates a set of eight 15-base oligonucleotide primers A to H
inclusive. The 8.times.8 matrix tables in the lower part of the
diagram represent the results which would be obtained by
application of the process of the invention using this set of
primers and the two DNA sample molecules X and Y. In each table,
the columns represent the oligonucleotides attached to the
solid-support during the first step of the process, in which
sequence information from the sample molecules is transcribed into
solid-supported primer-extension product. Conversely, the rows of
each table represent the solution-phase oligonucleotides used
during the second step of the process, during which labelled
second-strands complementary to the support-bound polynucleotides
are produced.
[0212] The presence of a "+" symbol in each table indicates a
combination which gives rise to incorporation of labelled molecules
into the product, whereas the presence of a "-" symbol denotes the
absence of labelled molecules.
[0213] As indicated by the sections highlighted in bold type,
sequence X contains two sequences of nucleotides which match those
of two of the eight primers. A match for primer C is found in the
"top" strand of sequence X, reading from left to right, while a
match for primer B is found in the "bottom" strand, in which the
5'-terminus is at the right-hand end, and the sequence therefore
reads from right to left. Reference to the matrix table for
sequence X shows that a positive result is obtained for this
combination of primers, which may be generated in two different
ways, depending on which of the two primers is attached to the
solid support.
[0214] Similarly, sequence Y contains matching sequences for primer
A in the "top" strand, reading from left to right, and for primer H
in the "bottom" strand, reading from right to left. Reference to
the matrix table for sequence Y again shows that a positive result
is obtained for both of the ways in which this combination of
primers may be generated.
[0215] As will be apparent to those skilled in the art of
electronic logic circuitry, the matrix tables shown in the figure
are analogous to the "truth tables" used in analysis of
semiconductor logic devices, and the process of the invention may
be regarded as providing the functionality of an "AND" logic gate
for DNA sequences. As such, the outcome of a particular experiment
may be predicted in advance if the nucleotide sequences of the
sample molecules and the primers are known, allowing analytical
procedures for specific applications to be designed "in-silico",
using computer modelling techniques.
[0216] As will be appreciated by those skilled in the art of DNA
analysis and combinatorial chemistry, the operations involved in
practising the methods of the invention may readily be automated by
using standard items of laboratory equipment designed for use in
combinatorial chemical synthesis and high-throughput screening.
These may include but are not restricted to thermal cyclers,
automated pipettors, diluters and dispensers, robotic systems,
two-dimensional (X-Y) positioning systems and three-dimensional
(X-Y-Z) motion systems, multi-tube racks, multi-compartment
reaction blocks and multi-well plate systems, plate readers, auto
sampling apparatus, spectrophotometers and spectrofluorimeters
including flow-through spectrophotometers and spectrofluorimeters.
In addition, equipment designed for specific applications in
oligonucleotide synthesis may also be adapted for the practice of
the invention. Such equipment may include but is not restricted to
automated synthesisers such as those described in my U.S. Pat. No.
4,728,502 entitled "Apparatus for the chemical synthesis of
oligonucleotides" and in my GB patent 2,347,141 entitled
"Combinatorial synthesiser".
[0217] The foregoing description illustrates embodiments of the
invention. However, the invention is not restricted to the specific
embodiments described, and many variations of the invention will be
apparent to those skilled in the arts of biochemical analysis and
DNA technology without departing from the spirit of the invention.
For example, a wide variety of enzymes and enzymatic processes
could be used in the practice of the invention in place of the
polymerase chain reaction, including reverse transcriptase in order
to practise the technique of RT-PCR, or DNA ligase in order to
practise the ligase chain reaction.
[0218] Similarly, although the subdivided support surfaces
hereinbefore described are arranged in a rectangular format, other
formats, for example formats employing a hexagonal or triangular
layout of sub-zones, would be equally effective.
[0219] Accordingly, the scope of the invention should not be
determined by reference to the foregoing description, but solely by
reference to the appended claims along with their full scope of
equivalents.
Sequence CWU 1
1
212110DNAArtificial Sequencechemically synthesized 1gtttcgctcc
10210DNAArtificial Sequencechemically synthesized 2tgatccctgg
10310DNAArtificial Sequencechemically synthesized 3catccccctg
10410DNAArtificial Sequencechemically synthesized 4ggactggagt
10510DNAArtificial Sequencechemically synthesized 5tgcgcccttc
10610DNAArtificial Sequencechemically synthesized 6cccaaggtcc
10710DNAArtificial Sequencechemically synthesized 7ggtgcgggaa
10810DNAArtificial Sequencechemically synthesized 8ccagatgcac
10910DNAArtificial Sequencechemically synthesized 9gtgacatgcc
101010DNAArtificial Sequencechemically synthesized 10tcagggaggt
101110DNAArtificial Sequencechemically synthesized 11cccggcataa
101210DNAArtificial Sequencechemically synthesized 12cccgttggga
101310DNAArtificial Sequencechemically synthesized 13tctccgcttg
101410DNAArtificial Sequencechemically synthesized 14ccgaacacgg
101510DNAArtificial Sequencechemically synthesized 15ctccatgggg
101610DNAArtificial Sequencechemically synthesized 16ggcacgtaag
101710DNAArtificial Sequencechemically synthesized 17acgtagcgtc
101810DNAArtificial Sequencechemically synthesized 18ctgttgctac
101910DNAArtificial Sequencechemically synthesized 19aagtccgctc
102010DNAArtificial Sequencechemically synthesized 20cccagtcact
102110DNAArtificial Sequencechemically synthesized 21gggccactca
102210DNAArtificial Sequencechemically synthesized 22ggagagactc
102310DNAArtificial Sequencechemically synthesized 23tccactcctg
102410DNAArtificial Sequencechemically synthesized 24cacagaggga
102510DNAArtificial Sequencechemically synthesized 25gggtttggca
102610DNAArtificial Sequencechemically synthesized 26gtggcatctc
102710DNAArtificial Sequencechemically synthesized 27catcgccgca
102810DNAArtificial Sequencechemically synthesized 28acagcctgct
102910DNAArtificial Sequencechemically synthesized 29ggctgcaatc
103010DNAArtificial Sequencechemically synthesized 30ggctgcgaca
103110DNAArtificial Sequencechemically synthesized 31caaagggcgg
103210DNAArtificial Sequencechemically synthesized 32ctgaaccgct
103310DNAArtificial Sequencechemically synthesized 33tctcgcctac
103410DNAArtificial Sequencechemically synthesized 34gtaggcctca
103510DNAArtificial Sequencechemically synthesized 35accgcatggg
103610DNAArtificial Sequencechemically synthesized 36ggcatcggct
103710DNAArtificial Sequencechemically synthesized 37agccgttcag
103810DNAArtificial Sequencechemically synthesized 38gggtccaaag
103910DNAArtificial Sequencechemically synthesized 39ctatcctgcc
104010DNAArtificial Sequencechemically synthesized 40gtcgtagcgg
104110DNAArtificial Sequencechemically synthesized 41actccacgtc
104210DNAArtificial Sequencechemically synthesized 42caccgcagtt
104310DNAArtificial Sequencechemically synthesized 43agccaggctg
104410DNAArtificial Sequencechemically synthesized 44ggcgtaagtc
104510DNAArtificial Sequencechemically synthesized 45gggtgcagtt
104610DNAArtificial Sequencechemically synthesized 46cacaccgtgt
104710DNAArtificial Sequencechemically synthesized 47gtcctcgtgt
104810DNAArtificial Sequencechemically synthesized 48acggttccac
104910DNAArtificial Sequencechemically synthesized 49gtcttgggca
105010DNAArtificial Sequencechemically synthesized 50gtcacctgct
105110DNAArtificial Sequencechemically synthesized 51tgctctgccc
105210DNAArtificial Sequencechemically synthesized 52ggtgacgcag
105310DNAArtificial Sequencechemically synthesized 53gtccacacgg
105410DNAArtificial Sequencechemically synthesized 54tgggggactc
105510DNAArtificial Sequencechemically synthesized 55ctgctgggac
105610DNAArtificial Sequencechemically synthesized 56aagacccctc
105710DNAArtificial Sequencechemically synthesized 57agatgcagcc
105810DNAArtificial Sequencechemically synthesized 58tcaccacggt
105910DNAArtificial Sequencechemically synthesized 59cttcacccga
106010DNAArtificial Sequencechemically synthesized 60caccaggtga
106110DNAArtificial Sequencechemically synthesized 61tcgttccgca
106210DNAArtificial Sequencechemically synthesized 62cctctcgaca
106310DNAArtificial Sequencechemically synthesized 63cataccgtgg
106410DNAArtificial Sequencechemically synthesized 64tgagcctcac
106510DNAArtificial Sequencechemically synthesized 65aagcccgagg
106610DNAArtificial Sequencechemically synthesized 66ccacgggaag
106710DNAArtificial Sequencechemically synthesized 67cagcactgac
106810DNAArtificial Sequencechemically synthesized 68cctccagtgt
106910DNAArtificial Sequencechemically synthesized 69tcccacgcaa
107010DNAArtificial Sequencechemically synthesized 70tcagagcgcc
107110DNAArtificial Sequencechemically synthesized 71caagggcaga
107210DNAArtificial Sequencechemically synthesized 72ggcaggctgt
107310DNAArtificial Sequencechemically synthesized 73aacggcgaca
107410DNAArtificial Sequencechemically synthesized 74cacccctgag
107510DNAArtificial Sequencechemically synthesized 75ccttcggaag
107610DNAArtificial Sequencechemically synthesized 76aaggctcacc
107710DNAArtificial Sequencechemically synthesized 77agagccgtca
107810DNAArtificial Sequencechemically synthesized 78aggcagagca
107910DNAArtificial Sequencechemically synthesized 79agcagcgcac
108010DNAArtificial Sequencechemically synthesized 80caaacgtggg
108110DNAArtificial Sequencechemically synthesized 81aagtgcacgg
108210DNAArtificial Sequencechemically synthesized 82ccctactggt
108310DNAArtificial Sequencechemically synthesized 83ggcaggcaag
108410DNAArtificial Sequencechemically synthesized 84tcgcttctcc
108510DNAArtificial Sequencechemically synthesized 85aagaggccag
108610DNAArtificial Sequencechemically synthesized 86tgccgcactt
108710DNAArtificial Sequencechemically synthesized 87acgagcatgg
108810DNAArtificial Sequencechemically synthesized 88aagcccccca
108910DNAArtificial Sequencechemically synthesized 89tcgctggtgt
109010DNAArtificial Sequencechemically synthesized 90tcgggcatca
109110DNAArtificial Sequencechemically synthesized 91gggaacccgt
109210DNAArtificial Sequencechemically synthesized 92tcgctgcgga
109310DNAArtificial Sequencechemically synthesized 93aaggctgctg
109410DNAArtificial Sequencechemically synthesized 94gggggagatg
109510DNAArtificial Sequencechemically synthesized 95ctgtgtgctc
109610DNAArtificial Sequencechemically synthesized 96ggcgcgttag
109710DNAArtificial Sequencechemically synthesized 97gacgagcagg
109810DNAArtificial Sequencechemically synthesized 98ggctgccagt
109910DNAArtificial Sequencechemically synthesized 99tggagtcccc
1010010DNAArtificial Sequencechemically synthesized 100cccgtctacc
1010110DNAArtificial Sequencechemically synthesized 101gtagacccgt
1010210DNAArtificial Sequencechemically synthesized 102ccttgacgca
1010310DNAArtificial Sequencechemically synthesized 103ttcccccgct
1010410DNAArtificial Sequencechemically synthesized 104tccgctctgg
1010510DNAArtificial Sequencechemically synthesized 105ggagggtgtt
1010610DNAArtificial Sequencechemically synthesized 106gagtctcagg
1010710DNAArtificial Sequencechemically synthesized 107ttatcgcccc
1010810DNAArtificial Sequencechemically synthesized 108cccgattcgg
1010910DNAArtificial Sequencechemically synthesized 109tgcggctgag
1011010DNAArtificial Sequencechemically synthesized 110acgcacaacc
1011110DNAArtificial Sequencechemically synthesized 111actcctgcga
1011210DNAArtificial Sequencechemically synthesized 112gtcccgtggt
1011310DNAArtificial Sequencechemically synthesized 113ccacactacc
1011410DNAArtificial Sequencechemically synthesized 114cacccggatc
1011510DNAArtificial Sequencechemically synthesized 115tgtagcaggg
1011610DNAArtificial Sequencechemically synthesized 116gacaggaggt
1011710DNAArtificial Sequencechemically synthesized 117cagtgctgtg
1011810DNAArtificial Sequencechemically synthesized 118gtcagagtcc
1011910DNAArtificial Sequencechemically synthesized 119agcatggctc
1012010DNAArtificial Sequencechemically synthesized 120tggcgtcctt
1012110DNAArtificial Sequencechemically synthesized 121ttccccgcga
1012210DNAArtificial Sequencechemically synthesized 122gggtgtgtag
1012310DNAArtificial Sequencechemically synthesized 123aggatgccag
1012410DNAArtificial Sequencechemically synthesized 124aatgccgcag
1012510DNAArtificial Sequencechemically synthesized 125ggatgccact
1012610DNAArtificial Sequencechemically synthesized 126agacgatggg
1012710DNAArtificial Sequencechemically synthesized 127aagcctgcga
1012810DNAArtificial Sequencechemically synthesized 128gggtctcggt
1012910DNAArtificial Sequencechemically synthesized 129ggtcgatctg
1013010DNAArtificial Sequencechemically synthesized 130agtcgccctt
1013110DNAArtificial Sequencechemically synthesized 131caatcgggtc
1013210DNAArtificial Sequencechemically synthesized 132aagagggcgt
1013310DNAArtificial Sequencechemically synthesized 133ggttcctctg
1013410DNAArtificial Sequencechemically synthesized 134gaacgagggt
1013510DNAArtificial Sequencechemically synthesized 135tttgccccgt
1013610DNAArtificial Sequencechemically synthesized 136acggcgatga
1013710DNAArtificial Sequencechemically synthesized 137gactctaacc
1013810DNAArtificial Sequencechemically synthesized 138acgctgcgac
1013910DNAArtificial Sequencechemically synthesized 139tggtgcactc
1014010DNAArtificial Sequencechemically synthesized 140gacacagccc
1014110DNAArtificial Sequencechemically synthesized 141gtccatgcag
1014210DNAArtificial Sequencechemically synthesized 142aacggcggtc
1014310DNAArtificial Sequencechemically synthesized 143cttccaggac
1014410DNAArtificial Sequencechemically synthesized
144agccgggtaa 1014510DNAArtificial Sequencechemically synthesized
145tgatgccgct 1014610DNAArtificial Sequencechemically synthesized
146accgtgccgt 1014710DNAArtificial Sequencechemically synthesized
147tgaccaggca 1014810DNAArtificial Sequencechemically synthesized
148cacggaccga 1014910DNAArtificial Sequencechemically synthesized
149tcgcagcgtt 1015010DNAArtificial Sequencechemically synthesized
150ctgcaatggg 1015110DNAArtificial Sequencechemically synthesized
151tttgcccgga 1015210DNAArtificial Sequencechemically synthesized
152agggaacgag 1015310DNAArtificial Sequencechemically synthesized
153ccacagcagt 1015410DNAArtificial Sequencechemically synthesized
154acccccgaag 1015510DNAArtificial Sequencechemically synthesized
155ggacccttac 1015610DNAArtificial Sequencechemically synthesized
156ggtgactgtg 1015710DNAArtificial Sequencechemically synthesized
157ctactgccgt 1015810DNAArtificial Sequencechemically synthesized
158ggactgcaga 1015910DNAArtificial Sequencechemically synthesized
159acggcgtatg 1016010DNAArtificial Sequencechemically synthesized
160aacggtgacc 1016110DNAArtificial Sequencechemically synthesized
161ctgcttaggg 1016210DNAArtificial Sequencechemically synthesized
162acgccagttc 1016310DNAArtificial Sequencechemically synthesized
163tggtcgcaga 1016410DNAArtificial Sequencechemically synthesized
164ggacaccact 1016510DNAArtificial Sequencechemically synthesized
165aagcggcctc 1016610DNAArtificial Sequencechemically synthesized
166tcggcggttc 1016710DNAArtificial Sequencechemically synthesized
167ggcttatgcc 1016810DNAArtificial Sequencechemically synthesized
168ctcgctatcc 1016910DNAArtificial Sequencechemically synthesized
169ggtgcacgtt 1017010DNAArtificial Sequencechemically synthesized
170acacacgctg 1017110DNAArtificial Sequencechemically synthesized
171ggtgaacgct 1017210DNAArtificial Sequencechemically synthesized
172ccaacgtcgt 1017310DNAArtificial Sequencechemically synthesized
173gatgccagac 1017410DNAArtificial Sequencechemically synthesized
174gtccgtatgg 1017510DNAArtificial Sequencechemically synthesized
175gaccaatgcc 1017610DNAArtificial Sequencechemically synthesized
176gggccaatgt 1017710DNAArtificial Sequencechemically synthesized
177gacgtggtga 1017810DNAArtificial Sequencechemically synthesized
178gtggagtcag 1017910DNAArtificial Sequencechemically synthesized
179tgagggtccc 1018010DNAArtificial Sequencechemically synthesized
180agccgtggaa 1018110DNAArtificial Sequencechemically synthesized
181aacgggcgtc 1018210DNAArtificial Sequencechemically synthesized
182ggcaaaccct 1018310DNAArtificial Sequencechemically synthesized
183acgagaggca 1018410DNAArtificial Sequencechemically synthesized
184cttggcacga 1018510DNAArtificial Sequencechemically synthesized
185tcttcggagg 1018610DNAArtificial Sequencechemically synthesized
186aaggcacgag 1018710DNAArtificial Sequencechemically synthesized
187cctcacgtcc 1018810DNAArtificial Sequencechemically synthesized
188tcgcggaacc 1018910DNAArtificial Sequencechemically synthesized
189ggcaaagctg 1019010DNAArtificial Sequencechemically synthesized
190cctgttccct 1019110DNAArtificial Sequencechemically synthesized
191gtgtcgagtc 1019210DNAArtificial Sequencechemically synthesized
192tcagcacagg 1019310DNAArtificial Sequencechemically synthesized
193tgtcctgcgt 1019410DNAArtificial Sequencechemically synthesized
194accacgcctt 1019510DNAArtificial Sequencechemically synthesized
195gagtcctcac 1019610DNAArtificial Sequencechemically synthesized
196aacccttccc 1019710DNAArtificial Sequencechemically synthesized
197agttccgcga 1019810DNAArtificial Sequencechemically synthesized
198gttgcgcagt 1019910DNAArtificial Sequencechemically synthesized
199tgacagcccc 1020010DNAArtificial Sequencechemically synthesized
200tctgcctgga 10201100DNAArtificial Sequencechemically synthesized
201agttcttccg tagctgattc gattcgatcg agctacgttc gatcgatacg
ctagctcata 60ctggccctag cttagcttac taacttagga ttagtagctc
100202100DNAArtificial Sequencechemically synthesized 202tcaagaaggc
atcgactaag ctaagctagc tcgatgcaag ctagctatgc gatcgagtat 60gaccgggatc
gaatcgaatg attgaatcct aatcatcgag 100203100DNAArtificial
Sequencechemically synthesized 203tgccatacga agttcagcat cgctatcgtg
tctagcatca tagctctacg actacagact 60ttagctacgt acgactgatg catccgacta
gctctagcta 100204100DNAArtificial Sequencechemically synthesized
204acggtatgct tcaagtcgta gcgatagcac agatcgtagt atcgagatgc
tgatgtctga 60aatcgatgca tgctgactac gtaggctgat cgagatcgat
10020515DNAArtificial Sequencechemically synthesized 205acgaagttca
gcatc 1520615DNAArtificial Sequencechemically synthesized
206gtagctgatt cgatt 1520715DNAArtificial Sequencechemically
synthesized 207attctggtat gctag 1520815DNAArtificial
Sequencechemically synthesized 208ataatccgta gctat
1520915DNAArtificial Sequencechemically synthesized 209taagctaggg
ccagt 1521015DNAArtificial Sequencechemically synthesized
210atgatccatg ttact 1521115DNAArtificial Sequencechemically
synthesized 211gatctttagc tagtc 1521215DNAArtificial
Sequencechemically synthesized 212agtcgtacgt agcta 15
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