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| United States Patent | 6485944 |
| Link to this page | http://www.wikipatents.com/6485944.html |
| Inventor(s) | Church; George M. (Brookline, MA);
Mitra; Rob (Brookline, MA) |
| Abstract | Disclosed are improved methods of making and using immobilized arrays of
nucleic acids, particularly methods for producing replicas of such arrays.
Included are methods for producing high density arrays of nucleic acids
and replicas of such arrays, as well as methods for preserving the
resolution of arrays through rounds of replication. Also included are
methods which take advantage of the availability of replicas of arrays for
increased sensitivity in detection of sequences on arrays. Improved
methods of sequencing nucleic acids immobilized on arrays utilizing single
copies of arrays and methods taking further advantage of the availability
of replicas of arrays are disclosed. The improvements lead to higher
fidelity and longer read lengths of sequences immobilized on arrays.
Methods are also disclosed which improve the efficiency of multiplex PCR
using arrays of immobilized nucleic acids. |
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Title Information  |
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| Publication Date |
November 26, 2002 |
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| Filing Date |
March 12, 1999 |
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| Parent Case |
This application is a continuation-in-part of U.S. patent application Ser.
No. 09/143,014, filed Aug. 28, 1998; which claims the benefit of the
filing date of U.S. Provisional Application No. 60/061,511, filed Oct. 10,
1997; and U.S. Provisional Application No. 60/076,570, filed Mar. 2, 1998. |
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Title Information |
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References |
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U.S. References |
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| | Reference | Relevancy | Comments | Reference | Relevancy | Comments | 6274351
Peponnet
435/91.1
Aug,2001 |  Your vote accepted
[0 after 0 votes] | | 6017738
Morris
435/91.2
Jan,2000 | Your vote accepted
[0 after 0 votes] | | 5795714
Cantor
435/6
Aug,1998 | Your vote accepted
[0 after 0 votes] | | 5653939
Hollis
422/50
Aug,1997 | Your vote accepted
[0 after 0 votes] | | 5641658
Adams
Jun,1997 | Your vote accepted
[0 after 0 votes] | | 5631134
Cantor
435/6
May,1997 | Your vote accepted
[0 after 0 votes] | | 5616478
Chetverin
Apr,1997 | Your vote accepted
[0 after 0 votes] | | 5599695
Pease
435/91.1
Feb,1997 | Your vote accepted
[0 after 0 votes] | | 5593839
Hubbell
435/6
Jan,1997 | Your vote accepted
[0 after 0 votes] | | 5587128
Wilding
422/50
Dec,1996 | Your vote accepted
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Edwards
435/6
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[0 after 0 votes] | | 5578832
Trulson
250/458.1
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Hubbell
430/5
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[0 after 0 votes] | | 5556752
Lockhart
435/6
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[0 after 0 votes] | | 5552270
Khrapko
435/6
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[0 after 0 votes] | | 5545531
Rava
Aug,1996 | Your vote accepted
[0 after 0 votes] | | 5527681
Holmes
Jun,1996 | Your vote accepted
[0 after 0 votes] | | 5510270
Fodor
436/518
Apr,1996 | Your vote accepted
[0 after 0 votes] | | 5506350
Bittner
536/55.3
Apr,1996 | Your vote accepted
[0 after 0 votes] | | 5484702
Ludwig
Jan,1996 | Your vote accepted
[0 after 0 votes] | | 5445934
Fodor
435/6
Aug,1995 | Your vote accepted
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Utermohlen
435/6
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[0 after 0 votes] | | 5426180
Kool
536/25.3
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Fodor
435/6
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Uhlen
435/6
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Stapleton
435/6
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Warren, III
Jul,1994 | Your vote accepted
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Fodor
435/6
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Edwards
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Cheeseman
435/6
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Stapleton
435/288.3
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Augenlicht
435/6
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Melamede
435/6
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Palva
435/6
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Britten
435/285.1
May,1986 | Your vote accepted
[0 after 0 votes] | | 5451500
Stapleton
435/6
Dec,1969 | Your vote accepted
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Market Review |
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Technical Review |
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Claims |
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What is claimed is:
1. A method of amplifying a plurality of nucleic acids, said method
comprising the steps of:
a) creating an array of immobilized oligonucleotide primers, wherein the
primers are immobilized on a semi-solid support;
b) hybridizing amplification template to immobilized oligonucleotide
primers,
c) extending the immobilized oligonucleotide primers using a DNA polymerase
activity, and deoxynucleotide triphosphates to form immobilized nucleic
acid strands and then denaturing the amplification template from the
immobilized nucleic acid strands;
d) hybridizing non-immobilized oligonucleotide primers to the immobilized
nucleic acid strands and extending :the non-immobilized oligonucleotide
primers and denaturing the extended nonimmobilized oligonucleotide primers
from the immobilized nucleic acid strands, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules.
2. A method of amplifying a plurality of nucleic acids, said method
comprising the steps of:
a) creating an array of immobilized oligonucleotide primers,
b) hybridizing amplification template to immobilized oligonucleotide
primers,
c) extending the immobilized oligonucleotide primers using a DNA polymerase
activity, and deoxynucleotide triphosphates to form immobilized nucleic
acid strands and then denaturing the amplification template from the
immobilized nucleic acid strands;
d) hybridizing non-immobilized oligonucleotide primers to the immobilized
nucleic acid strands and extending the non-immobilized oligonucleotide
primers and denaturing the extended nonimmobilized oligonucleotide primers
from the immobilized nucleic acid strands, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules, wherein said
non-immobilized oligonucleotide primer comprises a pool of oligonucleotide
primers comprised of 5' and 3' sequence elements, said 5' sequence element
identical in all members of said pool, and said 3' sequence element
containing random sequences.
3. The method of claim 2 wherein said 5' sequence element comprises a
restriction endonuclease recognition sequence.
4. The method of either of claims 2 or 3 wherein said 5' element comprises
a transcriptional promoter sequence.
5. A method of amplifying a plurality of nucleic acids, said method
comprising the steps of:
a) creating an array of immobilized oligonucleotide primers,
b) hybridizing amplification template to immobilized oligonucleotide
primers,
c) extending the immobilized oligonucleotide primers using a DNA polymerase
activity, and deoxynucleotide triphosphates to form immobilized nucleic
acid strands and then denaturing the amplification template from the
immobilized nucleic acid strands;
d) hybridizing non-immobilized oligonucleotide primers to the immobilized
nucleic acid strands and extending the non-immobilized oligonucleotide
primers and denaturing the extended nonimmobilized oligonucleotide primers
from the immobilized nucleic acid strands, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules, wherein said
immobilized oligonucleotide primers of said array of step (a) are
heterogenous in nucleic acid sequence.
6. A method of amplifying a plurality of nucleic acids, said method
comprising the steps of:
a) creating an array of immobilized oligonucleotide primers,
b) hybridizing amplification template to immobilized oligonucleotide
primers,
c) extending the immobilized oligonucleotide primers using a DNA polymerase
activity, and deoxynucleotide triphosphates to form immobilized nucleic
acid strands and then denaturing the amplification template from the
immobilized nucleic acid strands;
d) hybridizing non-immobilized oligonucleotide primers to the immobilized
nucleic acid strands and extending the non-immobilized oligonucleotide
primers and denaturing the extended nonimmobilized oligonucleotide primers
from the immobilized nucleic acid strands, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules, wherein said
immobilized oligonucleotide primers are generated from genomic DNA.
7. A method of amplifying a plurality of nucleic acids, said method
comprising the steps of:
a) creating an array of immobilized oligonucleotide primers,
b) hybridizing amplification template to immobilized oligonucleotide
primers,
c) extending the immobilized oligonucleotide primers using a DNA polymerase
activity, and deoxynucleotide triphosphates to form immobilized nucleic
acid strands and then denaturing the amplification template from the
immobilized nucleic acid strands;
d) hybridizing non-immobilized oligonucleotide primers to the immobilized
nucleic acid strands and extending the non-immobilized oligonucleotide
primers and denaturing the extended nonimmobilized oligonucleotide primers
from the immobilized nucleic acid strands, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules, wherein the array,
template, non-immobilized primer, and polymerase are cast in a
polyacrylamide gel.
8. The method of claim 4 wherein the array, template, non-immobilized
primer, and polymerase are cast in a polyacrylamide gel.
9. The method of claim 5 wherein the array, template, non-immobilized
primer, and polymerase are cast in a polyacrylamide gel.
10. The method of claim 6 wherein the array, template, non-immobilized
primer, and polymerase are cast in a polyacrylamide gel.
11. The method of either of claims 1 or 2 wherein said immobilized
oligonucleotide primers of said array of step (a) are substantially pure
in nucleic acid sequence.
12. The method of claim 2 wherein said immobilized oligonucleotide primers
of the array of step (a) are heterogenous in nucleic acid sequence.
13. The method of claim 2 wherein said immobilized oligonucleotide primers
are generated from genomic DNA.
14. The method of claim 3 wherein said immobilized oligonucleotide primers
are generated from genomic DNA.
15. The method of claim 2 wherein the array, template, non-immobilized
primer, and polymerase are cast in a polyacrylamide gel.
16. The method of claim 3 wherein the immobilized oligonucleotide primers
are generated from genomic DNA.
17. The method of claim 1 wherein the non-immobilized oligonucleotide
primer comprises a pool of oligonucleotide primers comprised of 5' and 3'
sequence elements, said 5' sequence element identical in all members of
said pool, and said 3' sequence element containing random sequences.
18. The method of claim 17 wherein the 5' sequence element comprises a
restriction endonuclease recognition sequence.
19. The method of claims 17 wherein the 5' element comprises a
transcriptional promoter sequence.
20. The method of claim 1 wherein the immobilized oligonucleotide primers
of the array of step (a) are heterogenous in nucleic acid sequence.
21. The method of claim 1 wherein the immobilized oligonucleotide primers
are generated from genomic DNA.
22. The method of claim 1 wherein the array, template, non-immobilized
primer, and polymerase are cast in, a polyacrylamide gel.
23. The method claim 1 wherein the immobilized oligonucleotide primers of
the array of step (a) are substantially pure in nucleic acid sequence.
24. The method of claim 1 wherein the non-immobilized oligonucleotide
primers are heterogenous in nucleic acid sequence.
25. The method of claim 1 wherein the non-immobilized oligonucleotide
primers are substantially pure in nucleic acid sequence.
26. The method of claim 1 wherein the immobilized oligonucleotide primers
are heterogeneous with the non-immobilized oligonucleotide primers.
27. The method of claim 1 wherein the immobilized oligonucleotide primers
are substantially pure in nucleic acid sequence with the non-immobilized
oligonucleotide primers.
28. A method of amplifying a nucleic acid molecule, said method comprising
the steps of:
a) creating an array of immobilized oligonucleotide primers, wherein the
primers are immobilized on a semi-solid support;
b) hybridizing amplification template to an immobilized oligonucleotide
primer,
c) extending the immobilized oligonucleotide primer using a DNA polymerase
activity, and deoxynucleotide triphosphates to form an immobilized nucleic
acid strand and then denaturing the amplification template from the
immobilized nucleic acid strand;
d) hybridizing a non-immobilized oligonucleotide primer to the immobilized
nucleic acid strand and extending the non-immobilized oligonucleotide
primer and denaturing the extended nonimmobilized oligonucleotide primer
from the immobilized nucleic acid strand, and
e) repeating steps (b) and (c) and (d) for a defined number of cycles to
yield a plurality of amplified nucleic acid molecules. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The invention relates in general to the reproducible, mass-production of
nucleic acid arrays. The invention also relates to methods of sequencing
nucleic acids on arrays.
BACKGROUND OF THE INVENTION
Arrays of nucleic acid molecules are of enormous utility in facilitating
methods aimed at genomic characterization (such as polymorphism analysis
and high-throughput sequencing techniques), screening of clinical patients
or entire pedigrees for the risk of genetic disease, elucidation of
protein/DNA- or protein/protein interactions or the assay of candidate
pharmaceutical compounds for efficacy; however, such arrays are both
labor-intensive and costly to produce by conventional methods. Highly
ordered arrays of nucleic acid fragments are known in the art (Fodor et
al., U.S. Pat. No. 5,510,270; Lockhart et al., U.S. Pat. No. 5,556,752).
Chetverin and Kramer (WO 93/17126) are said to disclose a highly ordered
array which may be amplified.
U.S. Pat. No. 5,616,478 of Chetverin and Chetverina reportedly claims
methods of nucleic acid amplification, in which pools of nucleic acid
molecules are positioned on a support matrix to which they are not
covalently linked. Utermohlen (U.S. Pat. No. 5,437,976) is said to
disclose nucleic acid molecules randomly immobilized on a reusable matrix.
There is need in the art for improved methods of nucleic acid array design
and production. There is also a need in the art for methods with improved
resolution and/or sensitivity for detection of sequences on nucleic acid
arrays. There is also a need in the art for improved methods of sequencing
the molecules on nucleic acid arrays.
SUMMARY OF THE INVENTION
The invention provides a method of producing a high density array of
immobilized nucleic acid molecules, such method comprising the steps of:
1) creating an array of spots of a nucleic acid capture activity such that
the spots of said capture activity are separated by a distance greater
than the diameter of the spots, and the size of the spots is less than the
diameter of the excluded volume of the nucleic acid molecule to be
captured; 2) contacting the array of spots of nucleic acid capture
activity with an excess of nucleic acid molecules with an excluded volume
diameter greater than the diameter of the spots of nucleic acid capture
activity, resulting in an immobilized array of nucleic acid molecules in
which each spot of nucleic acid capture activity can bind only one nucleic
acid molecule with an excluded volume diameter greater than the size of
said spots of nucleic acid capture activity.
In a preferred embodiment of the invention, the nucleic acid capture
activity may be a hydrophobic compound, an oligonucleotide, an antibody or
fragment of an antibody, a protein, a peptide, an intercalator, biotin,
avidin, or streptavidin.
In another embodiment of the invention the immobilized array of spots of a
nucleic acid capture activity are arranged in a predetermined g | | |
