Method of nucleic acid amplification

• US 8,476,044 B2
• Filed: 04/16/2008
• Issued: 07/02/2013
• Est. Priority Date: 04/01/1997
• Status: Expired due to Fees

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Mayer, Pascal, Farinelli, Laurent, Kawashima, Eric H.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US12/148,133
• Publication Number:
  US 20080286795A1
• Time in Patent Office: 1,903 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/6.1, 435/6.11, 435/6.12, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• 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. A method of simultaneously sequencing a population of random nucleic acid molecules of unknown sequence, comprising:

(a) attaching known ends to said population of random nucleic acids thereby forming a plurality of nucleic acid targets each of said nucleic acid targets 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, wherein the known ends are attached to each of the random nucleic acids in the population;

(b) dispersing the plurality of nucleic acid targets such that individual target molecules of the plurality of nucleic acid targets 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 each of said nucleic acid targets, the second amplification primer anneals to a sequence complementary to the second part of each of said nucleic acid targets and wherein at least said first or said second amplification primers are immobilized at different locations on a single solid support, such that each of the plurality of nucleic acid targets is simultaneously amplified thereby producing 1000 or more different nucleic acid colonies of immobilized amplified nucleic acid molecules, a plurality of said immobilized amplified nucleic molecules in each of said colonies having the same sequence, and wherein the amplification reaction results in amplification of each of the random nucleic acids in the population; and

(d) simultaneously sequencing the immobilized amplified nucleic acid molecules present in said 1000 or more different nucleic acid colonies such that said population of random nucleic acid molecules of unknown sequence are simultaneously sequenced.

2. The method according to claim 1, wherein said sequencing comprises the steps of:

(d1) hybridizing primers to the immobilized amplified nucleic acid molecules;

(d2) extending the primers by addition of one or more labeled nucleotides, thereby producing incorporated labeled nucleotides; and

(d3) detecting said incorporated labeled nucleotides.

3. The method according to claim 2, wherein steps (d2) and (d3) are repeated one or more times.

4. The method according to claim 3, wherein a label attached to the one or more labeled nucleotides is removed after step (d3).

5. The method according to claim 2, wherein unincorporated nucleotides from said one or more labeled nucleotides are removed after step (d2).

6. The method according to claim 2, wherein step (d3) uses a charge-coupled-device camera coupled to a magnifying device.

7. The method according to claim 1, wherein the solid support comprises a planar solid support.

8. The method according to claim 7, wherein said 1,000 or more different nucleic acid colonies are 1,000,000 or more different nucleic acid colonies.