Chemically-enhanced primer compositions, methods and kits

Patent metadata

Field	Value
Publication number	US-9410192-B2
Application number	US-201314053571-A
Country	US
Kind code	B2
Filing date	Oct 14, 2013
Priority date	Oct 28, 2010
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

A composition is provided comprising a negatively charged group, an oligonucleotide sequence and at least none or one nuclease-resistant linkage group to form a chemically-enhanced primer. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the primer as well as a method of preparing DNA for sequencing and a method of sequencing DNA and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition under conditions in which excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded.

First claim

What is claimed is:

1. A method comprising the steps of:
a) amplifying DNA in a first reaction mixture comprising nuclease-sensitive amplification primers to form amplified DNA;
b) contacting the first reaction mixture of the amplifying step with a second reaction mixture comprising a nuclease and a chemically-enhanced primer, whereby the nuclease sensitive amplification primers are degraded by the nuclease and the chemically-enhanced primer comprising a negatively charged moiety (NCM) attached to a terminal 5′ end or within the primer sequence, the NCM being selected from a (Cn) spacer or branched (Cn) spacer, with n being any integer from 1 to 9 and when n equals 3 the NCM is a (C3)x spacer, with x being at least 5 in a linear arrangement or [(C3)x]z, with z equal to 2 or 3 in a branched arrangement;
c) inactivating the nuclease; and
d) reacting the amplified DNA in a sequencing reaction wherein the chemically-enhanced primer primes the sequencing reaction.

2. The method of claim 1 , further comprising:
a) obtaining sequencing results based on the sequencing reaction; and
b) determining a nucleotide base sequence of the amplified DNA based on the results.

3. The method of claim 1 , wherein the second reaction mixture further comprises a polymerase, deoxynucleotide triphosphates, dideoxynucleotide triphosphates and a dye-label.

4. The method of claim 1 , wherein the branched (C3)x can be a doubler or a trebler.

Assignees

• Life Technologies Corp

Inventors

• Lee Linda
• Woo Sam
• Ma Peter

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q2525/113

  incorporating modified backbone · CPC title

• C12Q2525/125

  incorporating agents resulting in resistance to degradation · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title