Methods and kits for reducing non-specific nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-9353393-B2
Application number	US-201213446474-A
Country	US
Kind code	B2
Filing date	Apr 13, 2012
Priority date	Feb 15, 2012
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

Methods and kits for efficient amplification of nucleic acids are provided. The disclosure generally relates to methods and kits for nucleic acid amplification of target nucleic acids of interest. The methods described herein promote the synthesis of the target nucleic acid (i.e., template nucleic acid) by reducing the production of undesirable primer-dimer structures and chimeric nucleic acid products during the amplification process by using novel modified primers.

First claim

The invention claimed is:

1. A method for amplifying a nucleic acid comprising:
a) providing a nucleic acid template;
b) contacting the nucleic acid template with a reaction solution comprising a DNA polymerase, deoxyribonucleoside triphosphates, and a primer with a 3′ end and a 5′ end, wherein the primer comprises a plurality of nucleotide analogues, wherein one or more nucleotide analogues of the plurality of nucleotide analogues increases the melting temperature (T m ) of the primer, one or more nucleotide analogues of the plurality of nucleotide analogues prevents primer-dimer formation,
and at least two nucleotide analogues of the plurality of nucleotide analogues are not a 2-amino-locked nucleic acid (LNA) nucleotide or a 2-thio-LNA nucleotide, and wherein one of the nucleotide analogues is 2-amino-deoxyadenosine (2-amino-dA) and one of the nucleotide analogues is 2-thio-deoxythymidine (2-thio-dT), and the 2-amino-dA increases the T m of the primer and both the 2-amino-dA and the 2-thio-dT prevent primer-dimer formation, and wherein the primer is a hexamer having the general structure of (+N)(+N)(atN)(atN)(atN)*N, wherein N represents a nucleotide having a nucleobase of cytosine, guanine, adenine, or thymine, wherein (+N) is the 5′ end nucleotide and *N is the 3′ end nucleotide of the hexamer, and wherein “+” indicates an LNA that precedes a nucleotide base, (atN) represents a random mixture of 2-amino-dA, deoxycytidine (dC), deoxyguanosine (dG), and 2-thio-dT, and “*” represents a phosphorothioate linkage; and
c) amplifying the nucleic acid template.

2. The method of claim 1 , wherein amplifying the nucleic acid template is performed under isothermal conditions.

3. The method of claim 1 , wherein amplifying the nucleic acid template is performed under high stringency conditions.

4. The method of claim 1 , wherein the DNA polymerase is phi29 DNA polymerase.

5. The method of claim 1 , wherein amplifying the nucleic acid template comprises a rolling circle amplification (RCA) or a multiple displacement amplification (MDA).

6. The method of claim 1 , wherein the method permits amplification of a trace amount of the nucleic acid.

Assignees

• Nelson John Richard
• Grossmann Gregory Andrew
• Duthie Robert Scott
• Shah Sonali Jagdish
• Heller Ryan Charles
• Gen Electric

Inventors

• Nelson John Richard
• Grossmann Gregory Andrew
• Duthie Robert Scott
• Shah Sonali Jagdish
• Heller Ryan Charles

Classifications

• C12Q2525/113

  incorporating modified backbone · CPC title

• C12Q2527/107

  Temperature of melting, i.e. Tm · CPC title

• C12Q2525/204

  specific length of the oligonucleotides · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12P19/34 Primary

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