Method of nucleic acid amplification

• US 9,593,328 B2
• Filed: 01/20/2015
• Issued: 03/14/2017
• Est. Priority Date: 04/01/1997
• Status: Expired due to Fees

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Kawashima, Eric H., Farinelli, Laurent, Mayer, Pascal
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/601,013
• Publication Number:
  US 20150133320A1
• Time in Patent Office: 784 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 1/1
• 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 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.

Claims

1. A method for forming different modified nucleic acids in a plurality of samples from individuals, comprising:

providing a plurality of samples containing nucleic acids from individuals;

amplifying the nucleic acids in the plurality of samples, wherein each of a plurality of nucleic acids in each sample of the plurality of samples is amplified with at least two primers of a plurality of primers, thereby forming different modified nucleic acids, the at least two primers comprising the first primer comprising a first sequence portion identical to or complementary to a sequence of a third primer, a second sequence portion comprising a nucleic acid tag selected from a plurality of different nucleic acid tag sequences, and a third sequence portion identical to or complementary to a portion of its corresponding nucleic acid of the plurality of nucleic acids in said each sample of the plurality of samples;

wherein the nucleic acid tag sequence on the first primer of the at least two primers of the plurality of primers used for amplifying each sample of the plurality of samples is different;

the second primer comprising a fourth sequence portion identical to or complementary to a sequence of a fourth primer and a fifth sequence portion identical to or complementary to a portion of its corresponding nucleic acid of the plurality of nucleic acids in said each sample of the plurality of samples;

wherein at least the third primer or fourth primer is attached to a solid support for further amplification of the different modified nucleic acids;

wherein the fifth sequence portion of the second primer is used to complement an extension product of the first primer produced from the amplifying step, and the third sequence portion of the first primer is used to complement an extension product of the second primer produced from the amplifying step; and

wherein each of the modified nucleic acids amplified from one sample of the plurality of samples comprises a nucleic acid tag sequence of the plurality of different nucleic acid tag sequences that is different from a nucleic acid tag sequence of the plurality of different nucleic acid tag sequences in each of the modified nucleic acids amplified from other samples of the plurality of samples so that the modified nucleic acids amplified from one sample of the plurality of samples can be differentiated from the modified nucleic acids amplified from other samples of the plurality of samples.

2. The method of claim 1, wherein the first sequence portion includes the sequence of the third primer.

3. The method of claim 1, wherein the fifth sequence portion complements a sequence in the nucleic acid.

4. The method of claim 1, wherein the third sequence portion complements a sequence in the nucleic acid.

5. The method of claim 1, further comprising amplifying the different modified nucleic acid using said third and fourth primers, thereby forming a colony comprising amplified nucleic acid.

6. The method of claim 5, further comprising sequencing the nucleic acid in the colony.

7. The method of claim 6, wherein said sequencing the nucleic acid in the colony comprises providing a sequencing primer and incorporating at least one nucleotide into the sequencing primer.

8. The method of claim 7, wherein said sequencing the nucleic acid in the colony comprises adding a single nucleotide at a time to the sequencing primer.

9. The method of claim 6, wherein said sequencing the nucleic acid in the colony is performed by incorporating a single base at a time and using a mixture of labeled and unlabeled bases.

10. The method of claim 6, wherein said sequencing the nucleic acid in the colony comprises using labeled nucleotides, wherein different types of the labeled nucleotides are differently labeled.

11. The method of claim 1, further comprising:

combining multiple nucleic acids of the different modified nucleic acids, and

simultaneously amplifying multiple nucleic acids of the different modified nucleic acids on the solid support using said third and fourth primers, thereby forming multiple nucleic acid colonies comprising amplified nucleic acids.

12. The method of claim 11, further comprising simultaneously sequencing the nucleic acids in the colonies.

13. The method of claim 12, wherein said simultaneously sequencing the nucleic acids in the colonies comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.

14. The method of claim 12, wherein said simultaneously sequencing the nucleic acids in the colonies comprises adding a single nucleotide at a time to a sequencing primer.

15. The method of claim 12, wherein said simultaneously sequencing the nucleic acids in the colonies comprises using labeled nucleotides, wherein different types of the labeled nucleotides are differently labeled.