Methods to amplify highly uniform and less error prone nucleic acid libraries

Patent metadata

Field	Value
Publication number	US-10662469-B2
Application number	US-201715723015-A
Country	US
Kind code	B2
Filing date	Oct 2, 2017
Priority date	Mar 30, 2015
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

The present invention relates to a kit and a method of linear amplification of a least one nucleic acid target in a sample, said method comprising: (a) contacting each target in the sample with a nucleic acid polymerase and a primer comprising a component preventing copying of the primer by the nucleic acid polymerase; and at least one nuclease blocking nucleotide; (b) generating a primer extension product; (c) preventing priming by the 3′-end of the primer extension product, and (d) repeating steps b) and c) at least once.

First claim

The invention claimed is:

1. A method of linear amplification of a least one nucleic acid target in a sample comprising:
(a) contacting each target in the sample with a nucleic acid polymerase and a primer comprising
a component preventing copying of the primer by the nucleic acid polymerase; and
at least one nuclease blocking nucleotide;

(b) generating a primer extension product;
(c) preventing priming by the 3′-end of the primer extension product by contacting the primer extension product with a non-extendable oligonucleotide complementary to a sequence comprising the 3′-end of the primer extension product, and
(d) repeating steps b) and c) at least once.

2. The method of claim 1 , wherein the component preventing copying of the primer by the nucleic acid polymerase is a precursor of a quadruplex-forming sequence.

3. The method of claim 1 , wherein the component preventing copying of the primer by the nucleic acid polymerase is a nucleic acid modification selected from modified base, modified nucleotide, or a modified backbone.

4. The method of claim 1 , further comprising a step of increasing the rate of step (b) by contacting the sample with a facilitating enzyme selected from recombinase, helicase, exonuclease and an endonuclease.

5. The method of claim 1 , wherein the nuclease blocking nucleotide is a phosphorothioate modification of one or more nucleotides in the primer.

6. The method of claim 1 , further comprising a preliminary step of ligating adaptors to one or more targets in the sample.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Godwin Brian Christopher

Classifications

• C12Q1/6855

  Ligating adaptors · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q2525/125

  incorporating agents resulting in resistance to degradation · CPC title