One step RT-PCR methods, Enzyme mixes and kits for use in practicing the same

In the Public Domain since

9 Oct 2013

Description

In 1999, Zhao Ningyue et al., patented enzyme compositions, kits and methods for one-step Reverse Transcription Polymerase Chain Reaction (RT-PCR). This method combines the reverse transcription of RNA into cDNA (complementary DNA) by the enzyme reverse transcriptase and then amplification of specific DNA targets by Polymerase Chain Reaction (PCR) using DNA Polymerases. In the one-step method, the reverse transcriptase is included in the same tube as the PCR. The two-step method involves creating cDNA in a separate reverse transcription reaction and then adding some of this cDNA to the PCR, which is more time-consuming.

RT-PCR is used to generate cDNA templates for cloning and sequencing, to distinguish exons from introns, and can be used clinically to diagnose genetic diseases and monitor drug therapy. It is also used for detection and study of expressed genes both qualitatively and quantitatively in combination with qPCR (known as RT-qPCR). RT-qPCR is particularly useful in detecting RNA viruses and is used in the gold-standard tests for SARS-CoV-2 virus as well as other major pathogens such as HIV and Hepatitis B.

Further Reading:

RT-PCR on Wikipedia

Quantitative RT-PCR: One-step or Two-step RT? On Bitesize Bio

Abstract

Enzyme compositions, kits comprising the same and methods for their use in one-step RT-PCR are provided. The subject enzyme compositions at least include a mutant thermostable DNA polymerase and a mutant reverse transcriptase. In preferred embodiments, the mutant thermostable DNA polymerase is an N-terminal deletion mutant of Taq polymerase and the mutant reverse transcriptase is a point mutation mutant of MMLV-RT. The subject kits, in addition to the above described mutant thermostable DNA polymerase and mutant reverse transcriptase, at least include one of, and usually both of, dNTPs and a buffer composition, where the subject kits may further include additional reagents, including nucleic acids, a thermostabilizing agent, a glycine based osmolyte and the like. In practicing the subject methods, a reaction mix that at least includes template RNA, the above described mutant polymerase and reverse transcriptase, dNTPs, buffer, and nucleic acid primers is prepared. The resultant reaction mixture is maintained at a first set of reverse transcription conditions and then a second set of PCR conditions, whereby amplified amounts of DNA from a template RNA(s) are produced.

Category

Others > COVID-19, Molecular Techniques

Related Inventors

unknown

Source

US Patent No 6,300,073

Patent Title: One step RT-PCR methods, Enzyme mixes and kits for use in practicing the same

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