Composition and methods for RT-PCR comprising an anionic polymer

Patent metadata

Field	Value
Publication number	US-9758812-B2
Application number	US-201214238801-A
Country	US
Kind code	B2
Filing date	Aug 13, 2012
Priority date	Aug 17, 2011
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

The present invention is in the fields of molecular biology. The present invention is directed to novel compositions, methods and kits useful for the generation of nucleic acids from an RNA template and further nucleic acid replication. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase-polymerase chain reaction.

First claim

The invention claimed is:

1. A composition suitable for the generation of nucleic acids from reverse transcription of an RNA template followed by polymerase chain reaction amplification, said composition comprising
an anionic polymer selected from the group consisting of sodium alginate, sodium carboxymethylcellulose, polyphloretin phosphate, poly-L-glutamate, polyacrylic acid, poly(propylacrylic acid) and poly(methyl-vinyl-ether-maelic anhydride),
one or more oligonucleotide primers that facilitate reverse transcription of a nucleic acid molecule complementary to at least a portion of the RNA template and replication of the nucleic acid molecule by polymerase chain reaction amplification,
one or more nucleotides or derivatives thereof, and
two or more different polymerases, at least one of which has reverse transcriptase activity and at least one of which has nucleic acid polymerase activity.

2. The composition of claim 1 further comprising one or more selected from the group consisting of a buffering agent, a salt, DMSO, glycerol, formamide, betaine, tetramethylammonium chloride, PEG, polyoxyethylene (20) sorbitan monolaurate, NP 40, extoine, polyoles, E. coli SSB protein, Phage T4 gene 32 protein, and BSA.

3. The composition of claim 1 , wherein the anionic polymer comprises a net charge of at least −1 at a pH used in RT-PCR and comprises an amount of counterions sufficient for the anionic polymer to maintain electric neutrality.

4. A kit for performing coupled RT-PCR, said kit comprising at least one reverse transcriptase, at least one DNA polymerase, one or more oligonucleotide primers that facilitate reverse transcription of a nucleic acid molecule complementary to at least a portion of an RNA template and replication of the nucleic acid molecule by polymerase chain reaction amplification, and at least one anionic polymer that is not a nucleic acid selected from the group consisting of sodium alginate, sodium carboxymethylcellulose, polyphloretin phosphate, poly-L-glutamate, polyacrylic acid, poly(propylacrylic acid) and poly(methyl-vinyl-ether-maelic anhydride).

5. The kit according to claim 4 , further comprising a mixture of two or more of dATP, dCTP, dGTP, and dTTP, and/or one or more additives useful in RT-PCR and/or a buffering agent.

Assignees

• Fang Nan
• Missel Andreas
• Qiagen Gmbh

Inventors

• Fang Nan
• Missel Andreas

Classifications

• C12Q2521/107

  RNA dependent DNA polymerase,(i.e. reverse transcriptase) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title