Hot start reverse transcription by primer design

Abstract

The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims a benefit of priority under 35 U.S.C. §119(e) from U.S. application Ser. No. 60 / 699,967, Jul. 15, 2005, the contents of which is incorporated herein by reference. FIELD [0002] The present teachings relate to methods of synthesizing nucleic acids by primer extension. INTRODUCTION [0003] The integrity of primer-mediated methods of synthesizing nucleic acids can be compromised by non-specific hybridization of primer to inappropriate target polynucleotides. The analysis of nucleic acids is benefited by approaches by approaches that minimize the generation of mis-extension products. For example, ‘hot start’ approaches have been employed in PCR, where inhibition of polymerase activity has been achieved. For example, U.S. Pat. No. 5,338,671 describes the use of antibodies specific for a thermostable DNA polymerase to inhibit the DNA polymerase activity at low temperatures. Chemical treatment with citraconic anhydride is...

AI Technical Summary

Benefits of technology

[0004] The present teachings provide a method for reducing the formation of primer extension products at a low temperature but allowing the formation of primer extension products at a high temperature comprising; forming a reaction mixture at the low temperature below about 27C, wherein the reaction mixture comprises a target polynucleotide, a primer extending enzyme, and a hot start primer, wherein the hot start primer comprises a loop and a self-complementary stem, wherein a target-specific region of the self-complementary stem comprises a sequence of at least six nucleotides that are complementary with the target polynucleotide, wherein the target-specific region of the self-complementary stem is substantially hybridized with a quencher region in the sel

Problems solved by technology

The integrity of primer-mediated methods of synthesizing nucleic acids can be compromised by non-specific hybridization of primer to inappropriate target polynucleotides.

The application of such hot start approaches to reverse transcription have proven challenging.

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