Method of nucleic acid amplification
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.
1. A method for amplifying a nucleic acid derived from a sample derived from an individual on a solid support, comprising:
providing the nucleic acid;
amplifying the nucleic acid with at least two primers by adding additional sequences to the nucleic acid thereby forming 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 the origin 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;
amplifying the modified nucleic acid using the third and fourth primers, at least one of the third and fourth primers attached to the solid support, thereby forming a nucleic acid colony comprising extended third and fourth primers, and
further amplifying the nucleic acids in the nucleic acid colony;
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.
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 sequencing the nucleic acid in the colony.
6. The method of claim 5 , wherein said sequencing the nucleic acid in the colony comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.
7. The method of claim 6 , wherein said sequencing the nucleic acid in the colony comprises adding a single nucleotide at a time to the sequencing primer.
8. The method of claim 5 , wherein said sequencing the nucleic acid in the colony is performed by incorporating a single base at a time and comprises using a mixture of labeled and unlabeled bases.
9. The method of claim 5 , wherein said sequencing the nucleic acid in the colony comprises using labeled nucleotides, wherein different types of the labeled nucleotides are differently labeled.
10. The method of claim 5 , wherein said sequencing the nucleic acid in the colony comprises a primer extension step followed by determining a number of bases incorporated by said primer extension step.
11. The method of claim 1 , further comprising simultaneously amplifying multiple nucleic acids derived from the sample using at least two primers for each of the multiple nucleic acids thereby forming 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 including the second sequence portion.
12. The method of claim 1 , further comprising amplifying multiple additional nucleic acids from multiple additional samples using at least two primers for each of the multiple nucleic acids thereby forming 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 the origin of each of the nucleic acids.
13. The method of claim 11 , further comprising:
combining multiple nucleic acids of the multiple modified nucleic acids on the solid support, and
simultaneously amplifying the 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 thereby forming multiple nucleic acid colonies.
14. The method of claim 13 , further comprising simultaneously sequencing the nucleic acids in the colonies.
15. The method of claim 14 , wherein said simultaneously sequencing the nucleic acids in the colonies comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.
16. The method of claim 14 , wherein said simultaneously sequencing the nucleic acids in the colonies comprises adding a single nucleotide at a time to the sequencing primer.
17. The method of claim 14 , wherein said simultaneously sequencing the nucleic acids in the colonies comprises using labeled nucleotides, wherein different types of the labeled nucleotides are differently labeled.
18. The method of claim 13 , wherein said multiple nucleic acid colonies comprise 1000 or more different nucleic acid colonies of immobilized amplified nucleic acid molecules.
19. The method of claim 1 , wherein said amplifying the modified nucleic acid comprises one or more extension rounds comprising extension of the third and fourth primers, followed by multiple additional rounds of amplifying sequences of the extended third and fourth primers in the nucleic acid colony.
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 by adding additional sequences to the nucleic acids thereby forming multiple modified nucleic acids, for amplifying each of the multiple nucleic acids, 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 the origin of each of the nucleic acids, and a sequence portion identical to or complementary to a portion of one of the nucleic acids;
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 one of the nucleic acids;
wherein the second primer complements an extension product of the first primer on one of the nucleic acids, and the first primer complements an extension product of the second primer on the nucleic acid;
providing the multiple modified nucleic acids on the solid support, wherein at least said third primer or said fourth primer is attached to the solid support;
simultaneously amplifying multiple nucleic acids of the multiple modified nucleic acids on the solid support using the third and fourth primers thereby forming multiple nucleic acid colonies comprising extended third and fourth primers; and
further amplifying the nucleic acids in the multiple nucleic acid 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 said sequencing the colonies comprises providing a sequencing primer and incorporating at least one nucleotide into the primer.
23. The method of claim 21 , wherein said sequencing the colonies comprises adding a single nucleotide at a time to the sequencing primer.
24. The method of claim 21 , wherein said sequencing the colonies comprises using labeled nucleotides, wherein different types of the labeled nucleotides are differently labeled.
25. The method of claim 21 , wherein said sequencing the colonies comprises a primer extension step followed by determining a number of bases incorporated by said primer extension step.
26. The method of claim 20 , wherein said multiple nucleic acid colonies comprise 1000 or more different nucleic acid colonies of immobilized amplified nucleic acid molecules.
27. The method of claim 20 , wherein said simultaneously amplifying the modified nucleic acid comprises one or more extension rounds comprising extension of the third and fourth primers, followed by multiple additional rounds of amplifying sequences of the extended third and fourth primers in the nucleic acid colonies.