Primer extension using modified nucleotides
View Patent ↗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.
1. A method for inhibiting template independent primer extension, comprising: employing a primer extension reaction using i) a polymerase, ii) a template, iii) a plurality of primers having a palindromic nucleotide sequence, by:
hybridizing a first primer of the plurality of primers to the template so as to form a primer-template duplex;
hybridizing a second of the plurality of primers to a third of the plurality of primers so as to form a primer-primer duplex, and each of the plurality of primers comprise one or more modified nucleotides at or near the 3′ or 5′ end of the palindromic nucleotide sequence that inhibit(s) template independent primer extension of the primer-primer duplex, wherein the polymerase extends the primer of the primer-template duplex and the one or more modified nucleotides of the plurality of primers is modified at the 2′ position and the first, second, and third of the plurality of primers have the same nucleotide sequence.
2. A method according to claim 1 , wherein the primer extension reaction is a single nucleotide primer extension reaction.
3. A method according to claim 1 , wherein the one or more modified nucleotides comprises an alkoxy group at the 2′ position of the nucleotide.
4. A method according to claim 1 , wherein the one or more modified nucleotide comprises a methoxy group at the 2′ position of the nucleotide.
5. A method according to claim 1 , wherein the primers are bound to a solid support.
6. A method according to claim 5 , wherein the solid support is an array.
7. A method according to claim 6 , wherein the array is an addressable array.
8. A method according to claim 6 , wherein the array is a virtual array.
9. A method according to claim 6 , 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.
10. A method according to claim 1 , wherein the primer extension reaction utilizes one or more chain terminating nucleotides.
11. A method according to claim 10 , wherein the one or more chain terminating nucleotides are selected from the group consisting of dideoxynucleotides and acyclonucleotides.
12. A method according to claim 1 , wherein the primer extension reaction utilizes at least one labeled nucleotide.
13. A method according to claim 1 , wherein the primer has a detectable moiety.
14. A method according to claim 13 , 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.
15. A method according to claim 1 , wherein the one or more modified nucleotides is at least one nucleotide from the 5′ end of the primer.
16. A method according to claim 1 , wherein the one or more modified nucleotides is one, two, or three nucleotides from the 5′ end of the palindromic nucleotide sequence.