Primer extension using modified nucleotides

• US 20030190627A1
• Filed: 04/09/2002
• Published: 10/09/2003
• Est. Priority Date: 04/09/2002
• Status: Active Grant

• Current Assignee: Orchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Orchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Swenson, Rolf E., Zhao, Xiaodong, Gelfand, Craig A.
• Granted Patent: US 7,211,382 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides methods and compositions that reduce target-independent primer extension or enhance template dependent primer extension. The methods and compositions of the present invention are applicable not only in PCR, but also in nucleic acid sequencing, genotyping, and other applications employing extension of a primer in a target-dependent manner.

Claims

1. A method for inhibiting template independent primer extension, comprising:

employing a primer extension reaction using a primer having one or more modified nucleotides, under suitable conditions for primer extension, wherein the primer inhibits template independent primer extension.

5. A method according to claim 1, wherein the primer extension reaction is a single nucleotide primer extension reaction.

6. A method according to claim 1, wherein the one or more modified nucleotides is the third nucleotide from the 3′

end of the primer.

7. A method according to claim 1, wherein the one or more modified nucleotides is the fourth nucleotide from the 3′

end of the primer.

8. A method according to claim 1, wherein the one or more modified nucleotides is the fifth nucleotide from the 3′

end of the primer.

9. A method according to claim 1, wherein the one or more modified nucleotides comprises an alkoxy group at the 2′

position of the nucleotide.

10. A method according to claim 1, wherein the one or more modified nucleotide comprises a methoxy group at the 2′

position of the nucleotide.

11. A method according to claim 1, wherein the primer extension reaction utilizes a polymerization agent comprising DNA polymerase.

12. A method according to claim 1, wherein the primer is bound to a solid support.

13. A method according to claim 12, wherein the solid support is an array.

14. A method according to claim 13, wherein the array is an addressable array.

15. A method according to claim 13, wherein the array is a virtual array.

16. A method according to claim 13, wherein the primer comprises a unique tag which comprises a nucleic acid sequence that is capable of hybridizing with a complementary sequence at known positions on the array.

17. A method according to claim 1, wherein the primer extension reaction utilizes one or more chain terminating nucleotide.

18. A method according to claim 17, wherein the one or more chain terminating nucleotides are selected from the group consisting of dideoxynucleotides and acyclonucleotides.

19. A method according to claim 1, wherein the primer extension reaction utilizes at least one labeled nucleotide.

20. A method according to claim 1, wherein the primer has a detectable moiety.

22. A method according to claim 1, wherein a plurality of primers are employed in the primer extension reaction with a plurality of targets.

23. A method according to claim 20, wherein the detectable moiety is selected from the group consisting of mass moiety, radioactive moiety, bioluminescent moiety, chemiluminescent moiety, nucleic acid moiety, hapten moiety, enzyme moiety, and fluorescent moiety.

2. A method for inhibiting template independent primer extension, comprising:

employing a primer extension reaction using a primer having one or more modified nucleotides, under suitable conditions for primer extension, wherein each of the one or more modified nucleotides of the primer is modified at the 2′

position so that template independent primer extension is inhibited.

3. A method for identifying one or more nucleotide bases of a target nucleic acid sequence comprising the steps of:

a) hybridizing the target nucleic acid sequence with a primer having one or more modified nucleotides, wherein each of the one or more modified nucleotides of the primer is modified at the 2′

position so that template independent primer extension is inhibited;

b) extending the primer at the 3′

end with one or more nucleotide bases using the one or more nucleotide bases to be identified of the target as a template; and

c) detecting the extended primer, thereby identifying the one or more nucleotide bases of the target nucleotide sequence.

21. A method according to claim 3, wherein the target is an allele.

24. A method according to claim 3, wherein the target comprises a polymorphism.

25. A method according to claim 24, wherein the polymorphism is selected from the group consisting of a single nucleotide polymorphism, an insertion, a deletion, a re-arrangement, or a repetitive sequence.

4. A method of enhancing template dependent primer extension, comprising:

employing a primer extension reaction using a primer having one or more modified nucleotides under suitable conditions for primer extension, wherein the primer enhances template dependent primer extension.

26. A method for inhibiting template independent primer extension, comprising:

a) employing a primer extension reaction using two or more primers each of the two or more primers having a region of complementarity to each other, wherein each of the two or more primers has one or more modified nucleotides immediately adjacent to the region of complementarity and each of the two or more primers inhibits template independent primer extension;

b) hybridizing a target nucleic acid sequence to the two or more primers; and

c) extending the two or more primers at the 3′

end with one or more nucleotide bases using the target nucleic acid sequence as a template.