Method of nucleic acid amplification
First Claim
1. A process that can be used to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
- a) fragmenting nucleic acids in said sample;
b) linking adapter sequences to the nucleic acid fragments generated in step a);
c) binding said nucleic acid fragments to a solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments; and
d) identifying nucleic acid sequences within said amplified nucleic acid fragments.
7 Assignments
0 Petitions
Accused Products
Abstract
A nucleic acid molecule can be annealed to an appropriate immobilized primer. The primer can then be extended and the molecule and the primer can be separated from one another. The extended primer can then be annealed to another immobilized primer and the other primer can be extended. Both extended primers can then be separated from one another and can be used to provide further extended primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. These can be used for many different purposes, including sequencing, screening, diagnosis, in situ nucleic acid synthesis, monitoring gene expression, nucleic acid fingerprinting, etc.
79 Citations
26 Claims
-
1. A process that can be used to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
-
a) fragmenting nucleic acids in said sample; b) linking adapter sequences to the nucleic acid fragments generated in step a); c) binding said nucleic acid fragments to a solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments; and d) identifying nucleic acid sequences within said amplified nucleic acid fragments.
-
-
2. The process of claim 1 wherein there are a plurality of solid supports which each immobilizes primers which bind said nucleic acid fragments to the solid supports.
-
3. The process of claim 2 wherein the primers effect separation by interacting with or hybridizing to the adapter sequences.
-
4. The process of claim 2 wherein the solid supports are beads or a nylon or nitrocellulose membrane having an oligonucleotide capable of hybridizing to the adapter sequences attached to the nucleic acid fragments.
-
5. The process of claim 1 wherein sequence differences are identified in at least 10,000 nucleic acid fragments.
-
6. The process of claim 1 wherein the fragmenting is with restriction enzyme digestion.
-
7. The process of claim 1 wherein the nucleic acid fragments are amplified before or after step b of claim 2.
-
8. The process of claim 1 wherein the identifying of nucleic acid sequences in step (d) includes hybridizing sequencing primers to the amplified nucleic acid fragments.
-
9. The process of claim 8 wherein the sequencing primers are extended following hybridization.
-
10. The process of claim 1 wherein the adapter sequences are operatively linked to the nucleic acid fragments in step (b).
-
11. The process of claim 1 wherein the adapter sequences are ligated to the nucleic acid fragments in step (b).
-
12. The process of claim 9 wherein the sequencing primers are extended using a DNA polymerase.
-
13. The process of claim 1 wherein the identifying of nucleic acid sequences in step (d) includes monitoring the sequential addition of nucleotides to a primer.
-
14. The process of claim 1 wherein sequence differences are identified in over 1,000 nucleic acid fragments or over 1,000,000 nucleic acid fragments.
-
15. A process to identify in a nucleic acid sample the presence or absence of a nucleic add sequence difference with respect to a reference sequence, the process comprising:
-
a) fragmenting nucleic acids in said sample; b) linking adapter sequences to the nucleic acid fragments generated in step a); c) binding said nucleic acid fragments to a solid support by hybridizing the adapter sequences to complementary oligonucleotides attached to said solid support to form bound nucleic acid fragments, and amplifying said bound nucleic acid fragments to produce bound and amplified fragments, whereby there is enrichment of subsets of the nucleic acid fragments; and d) identifying the presence or absence of nucleic acid sequence differences within said bound and amplified fragments with respect to the reference sequence by hybridizing sequencing primers to the bound and amplified fragments and extending the sequencing primers.
-
-
16. The process of claim 15 wherein said complementary oligonucleotides act as primers in amplifying said solid support bound nucleic acid fragments.
-
17. The process of claim 15 wherein there are a plurality of solid supports which each immobilizes primers.
-
18. The process of claim 17 wherein the primers are oligonucleotides which bind the nucleic acid fragments to the solid support by hybridizing to the adapter sequences.
-
19. The process of claim 15 wherein the solid support is beads or a nylon or nitrocellulose membrane.
-
20. The process of claim 15 wherein the enrichment is of subsets such that the nucleic acid fragments in the subsets have particular lengths.
-
21. The process of claim 15 wherein sequence differences are identified in at least 10,000 nucleic acid fragments.
-
22. The process of claim 15 wherein the fragmenting is with restriction enzyme digestion.
-
23. The process of claim 15 wherein the nucleic acid fragments are amplified before or after step b).
-
24. The process of claim 15 wherein the adapter sequences are ligated to the nucleic acid fragments in step b).
-
25. The process of claim 15 wherein the adapter sequences are operatively linked to the nucleic acid fragments in step b).
-
26. The process of claim 22 wherein the nucleic acid fragments are size-fractionated.
Specification