Methods of cDNA preparation

• US 20080145844A1
• Filed: 01/09/2007
• Published: 06/19/2008
• Est. Priority Date: 01/25/2006
• Status: Abandoned Application

• Current Assignee: Evrogen Joint Stock Company
• Original Assignee: Evrogen Joint Stock Company
• Inventors: Barsova, Ekaterina V., Lukyanov, Sergey A.
• Application Number:
  US11/651,852
• Publication Number:
  US 20080145844A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention provides an improved method for cDNA preparation. The method of the present invention comprises the following steps: (1) contacting mRNA with a cDNA synthesis primer which can anneal to RNA and a suitable enzyme which possesses reverse transcriptase activity under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA-cDNA intermediates; (2) contacting a mixture from step 1 with a deoxyribonucleotide adapter in the presence of Mn²⁺-ions, wherein said oligonucleotide adapter has a pre-selected arbitrary nucleotide sequence at its 5′-end, and a short dG stretch at its 3′-end. The 3′-end nucleotide of the adapter is a terminator nucleotide, e.g., a nucleotide with a modified 3′-OH group of a deoxyribose residue.

Claims

1. A method of first-strand cDNA preparation, comprising the steps of:

(1) annealing a cDNA synthesis primer to RNA templates and synthesizing first cDNA strands to form RNA-cDNA intermediates using an enzyme which possesses reverse transcriptase activity;

(2) contacting a reaction mixture from step (1) comprising the RNA-cDNA intermediates and the enzyme which possesses reverse transcriptase activity with a deoxyribooligonucleotide adapter and Mn²⁺-ions to permit a template switching reaction, wherein said adapter comprises;

a pre-selected arbitrary nucleotide sequence at its 5′

-end; and

a 3′

-end terminator deoxyribonucleotide.

2. The method according to claim 1, wherein step (2) follows step (1).

3. The method according to claim 1, wherein steps (1) and (2) are performed simultaneously.

4. The method according to claim 1, wherein the enzyme which possesses reverse transcriptase activity is a MMLV-related reverse transcriptase.