Method of nucleic acid amplification

• US 20080286795A1
• Filed: 04/15/2008
• Published: 11/20/2008
• Est. Priority Date: 04/01/1997
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Farinelli, Laurent, Mayer, Pascal, Kawashima, Eric H.
• Granted Patent: US 8,476,044 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

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 provided 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.

Claims

1-39. -39. (canceled)

40. A method of simultaneously amplifying a population of random nucleic acid molecules of unknown sequence, comprising:

(a) attaching known ends to a population of random nucleic acids to form a plurality of nucleic acid targets each comprising a first part having a sequence capable of annealing to a first amplification primer, a second part having a sequence complementary to a sequence capable of annealing to a second amplification primer, and a third part comprising a random unknown sequence located between the first and second parts;

(b) dispersing the plurality of nucleic acid targets such that individual target molecules are separated from each other; and

(c) performing an amplification reaction using first and second amplification primers, wherein the first amplification primer anneals to the first part of said target, the second amplification primer anneals to a sequence complementary to the second part of said target and wherein at least one of the amplification primers is immobilised, such that the plurality of nucleic acid targets is simultaneously amplified to produce immobilized amplified nucleic acid molecules.

41. The method according to claim 40, further comprising the additional steps of:

(d) hybridizing primers to the immobilized amplified nucleic acid molecules;

(e) extending the primers by addition of one or more labeled nucleotides; and

(f) detecting the one or more labeled nucleotides incorporated during primer extension.

42. The method according to claim 41, wherein steps (e) and (f) are repeated one or more times.

43. The method according to claim 42, wherein a label attached to a nucleotide incorporated during primer extension in part (e) and detected in step (f) is removed after step (f).

44. The method according to claim 40, wherein the plurality of target sequences is amplified on a single planar solid support to provide a plurality of colonies, wherein each colony comprises a plurality of nucleic acid molecules comprising the same sequence.

45. The method according to claim 44, wherein the nucleic acid molecules present in 1000 or more different colonies having different nucleic acid molecules are simultaneously sequenced to determine at least a partial sequence of nucleic acid molecules in each colony.

46. The method according to claim 44, wherein the nucleic acid molecules present in 1,000,000 or more different colonies having different nucleic acid molecules are simultaneously sequenced to determine at least a partial sequence of nucleic acid molecules in each colony.

47. The method according to claim 41, wherein unincorporated nucleotides are removed after step (e).

48. The method according to claim 41, wherein step (f) uses a charge-coupled-device camera coupled to a magnifying device.