METHOD OF NUCLEIC ACID AMPLIFICATION

• US 20150133320A1
• Filed: 01/20/2015
• Published: 05/14/2015
• Est. Priority Date: 04/01/1997
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Kawashima, Eric H., Mayer, Pascal, Farinelli, Laurent
• Granted Patent: US 9,593,328 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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. (canceled)

2. A method for preparing a nucleic acid related to a sample from an individual for amplification on a solid support, comprising:

providing the nucleic acid;

amplifying the nucleic acid with at least two primers to form a modified nucleic acid, 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 that identifies a source of the nucleic acid, and a third sequence portion identical to or complementary to a portion of the nucleic acid;

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 the nucleic acid;

wherein the third primer and fourth primer are used to further amplify the modified nucleic acid on a solid support; and

wherein the fifth sequence portion of the second primer is configured to complement an extension product of the first primer on the nucleic acid, and the third sequence portion is configured to complement an extension product of the second primer on the nucleic acid.

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

4. The method of claim 2, wherein the fifth sequence portion complements a sequence in the nucleic acid.

5. The method of claim 2, wherein the third sequence portion complements a sequence in the nucleic acid.

6. The method of claim 2, further comprising providing the solid support, and amplifying the modified nucleic acid using third and fourth primers attached to the solid support to form a colony.

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

8. The method of claim 2, wherein the sequencing comprises providing a primer and incorporating at least one nucleotide into the primer.

9. The method of claim 2, wherein the sequencing comprises adding a single nucleotide at a time to the primer.

10. The method of claim 2, wherein sequencing is performed by incorporating a single base at a time and comprises using a mixture of labeled and unlabeled bases.

11. The method of claim 2, wherein the sequencing comprises using labeled nucleotides, wherein different types of nucleotides are differently labeled.

12. The method of claim 2, wherein sequencing comprises determining a number of bases incorporated by primer extension after a step.

13. The method of claim 2, further comprising simultaneously amplifying multiple nucleic acids from the sample using at least two primers for each of the multiple nucleic acids, whereby for the amplification of each of the multiple nucleic acids, one of the primers comprises the first sequence portion, one of the primers comprises the fourth sequence portion, and one of the primers comprises the second sequence portion.

14. The method of claim 2, further comprising amplifying multiple additional nucleic acids from multiple additional samples using at least two primers for each of the multiple nucleic acids to form multiple modified nucleic acids, whereby for the amplification of each of the multiple nucleic acids one of the primers comprises the first sequence portion, one of the primers comprises the fourth sequence portion, and one of the primers comprises a tag sequence portion that identifies a source of the nucleic acid being amplified.

15. The method of claim 13, further comprising:

combining multiple nucleic acids of the multiple modified nucleic acids,

providing the solid support, and

simultaneously amplifying multiple nucleic acids of the multiple modified nucleic acids on the same solid support using said third and fourth primers attached to the solid support to form multiple nucleic acid colonies.

16. The method of claim 15, further comprising simultaneously sequencing the nucleic acids in the colonies.

17. The method of claim 16, wherein said simultaneously sequencing comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.

18. The method of claim 16, wherein said simultaneously sequencing comprises adding a single nucleotide at a time to the sequencing primer.

19. The method of claim 16, wherein said simultaneously sequencing comprises using labeled nucleotides, wherein different types of nucleotides are differently labeled.

20. A method for amplifying multiple nucleic acids from multiple samples on a solid support, comprising:

amplifying the multiple nucleic acids with at least two primers to form multiple modified nucleic acids, for amplifying each nucleic acid the at least two primers comprising

a first primer comprising a sequence portion identical to or complementary to a sequence of a third primer, a nucleic acid tag sequence portion that identifies a source of the nucleic acid, and a sequence portion identical to or complementary to a portion of the nucleic acid;

a second primer comprising a sequence portion identical to or complementary to a sequence of a fourth primer and a sequence portion identical to or complementary to a portion of the nucleic acid;

wherein the second primer complements an extension product of the first primer on the nucleic acid, and the first primer complements an extension product of the second primer on the nucleic acid;

simultaneously amplifying multiple nucleic acids of the multiple modified nucleic acids on the same solid support using the third and fourth primers to form multiple colonies.

21. The method of claim 20, further comprising simultaneously sequencing the colonies using nucleic acid sequencing on the solid support.

22. The method of claim 21, wherein the sequencing comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.

23. The method of claim 21, wherein the sequencing comprises adding a single nucleotide at a time to the sequencing primer.

24. The method of claim 21, wherein the sequencing comprises using labeled nucleotides, wherein different types of nucleotides are differently labeled.

25. The method of claim 21, wherein sequencing comprises determining a number of bases incorporated by primer extension after a step.