Isothermal amplification using oligocation-conjugated primer sequences

Patent metadata

Field	Value
Publication number	US-2016281153-A1
Application number	US-201615177149-A
Country	US
Kind code	A1
Filing date	Jun 8, 2016
Priority date	Sep 16, 2013
Publication date	Sep 29, 2016
Grant date	—

Abstract

Provided herein are methods and kits for isothermal nucleic acid amplifications that use an oligocation-oligonucleotide conjugate primer for amplifying a target nucleic acid to generate amplicons. Isothermal DNA amplification methods that employ a strand displacing DNA polymerase and polyamine-oligonucleotide conjugate primer are also provided.

First claim

1 . A kit for isothermal DNA amplification comprising:
a DNA polymerase having strand displacement activity; and an oligocation-oligonucleotide conjugate primer, wherein the oligocation-oligonucleotide conjugate primer comprises an oligocation moiety covalently conjugated to the 5′ end of an oligonucleotide sequence.

2 . The kit of claim 1 , wherein the nucleotide sequence is a random oligonucleotide sequence.

3 . The kit of claim 1 , wherein the oligocation moiety comprises at least one spermine moiety.

4 . The kit of claim 1 , wherein the oligocation-oligonucleotide conjugate primer consists of oligocation-oligonucleotide conjugate sequences having structure IV, wherein integer value of n is 1 or 2.

5 . The kit of claim 1 , wherein the DNA polymerase is a Phi29 DNA polymerase.

6 . A kit for isothermal DNA amplification comprising:
an oligocation-oligonucleotide conjugate primer; a DNA polymerase having a strand displacement activity; a dNTP mixture; and an oligocation-oligonucleotide conjugate primer, wherein the oligocation-oligonucleotide conjugate primer comprises an oligocation moiety covalently conjugated to the 5′ end of an oligonucleotide sequence and wherein the oligocation moiety comprises at least one lysine moiety.

7 . The kit of claim 6 , wherein the oligonucleotide sequence is a random oligonucleotide sequence or a partially constrained oligonucleotide sequence.

8 . The kit of claim 6 , wherein the oligonucleotide sequence comprises a nucleotide analogue.

9 . The kit of claim 6 , wherein the oligonucleotide sequence comprises a phosphorothioate linkage between a 3′ terminal nucleotide and a nucleotide that is adjacent to the 3′ terminal nucleotide.

10 . The kit of claim 9 , wherein the oligonucleotide sequence is NNNNN*N.

11 . The kit of claim 6 , wherein the oligocation-oligonucleotide conjugate primer comprises a random pentamer sequence or a random hexamer sequence.

12 . A kit for isothermal DNA amplification comprising:
an oligocation-oligonucleotide conjugate primer; a DNA polymerase having a strand displacement activity; a dNTP mixture; and an oligocation-oligonucleotide conjugate primer, wherein the oligocation-oligonucleotide conjugate primer comprises an oligocation moiety covalently conjugated to the 5′ end of an oligonucleotide sequence and wherein the oligocation moiety comprises peptides having cationic side chains or a cationic polyamine.

13 . The kit of claim 12 , wherein the oligonucleotide sequence is a random oligonucleotide sequence or a partially constrained oligonucleotide sequence.

14 . The kit of claim 12 , wherein the oligonucleotide sequence comprises a nucleotide analogue.

15 . The kit of claim 12 , wherein the oligonucleotide sequence comprises a phosphorothioate linkage between a 3′ terminal nucleotide and a nucleotide that is adjacent to the 3′ terminal nucleotide.

16 . The kit of claim 15 , wherein the oligonucleotide sequence is NNNNN*N.

17 . The kit of claim 12 , wherein the oligocation-oligonucleotide conjugate primer comprises a random pentamer sequence or a random hexamer sequence.

18 . The kit of claim 12 , wherein the oligocation-oligonucleotide conjugate primer comprises a single spermine unit covalently conjugated to the 5′ terminal nucleotide of the oligonucleotide sequence.

19 . The kit of claim 12 , wherein the oligocation-oligonucleotide conjugate primer comprises two or three spermine moieties covalently coupled to the 5′ terminal nucleotide of the oligonucleotide sequence.

20 . The kit of claim 12 , wherein the DNA polymerase comprises a Phi29 DNA polymerase.

Assignees

• Gen Electric

Inventors

• Heller Ryan Charles
• Nelson John Richard

Classifications

• C12Q1/6865

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title