In molecular biology, reverse transcription polymerase chain reaction ( eng. Reverse transcription polymerase chain reaction ) is commonly referred to as RT-PCR . RT-PCR is a method for amplifying a specific ribonucleic acid fragment ( RNA ). [one]
The single-stranded RNA molecule is transformed into reverse transcription reactions (RT, RT, reverse transcription ) into complementary DNA ( cDNA ), and then the single-stranded DNA molecule is amplified using conventional PCR . RT-PCR should not be confused with real -time PCR ( eng. Real-time PCR, Q-PCR ), which is also sometimes incorrectly abbreviated as RT-PCR . [2]
Method Description
To transform an RNA sequence into complementary DNA, reverse transcriptase is used :
- 1. The reaction of the first chain:
- Complementary DNA ( cDNA ) is formed on the mRNA matrix of dNTP by the reverse transcriptase enzyme. The components of the reaction are mixed with DNA primer and buffer with reverse transcriptase for one hour at 37 ° C.
- 2. The reaction of the second chain:
- After the reverse transcription is completed and the cDNA is formed on the mRNA template, the following cycles are performed by the standard PCR method .
After 30 cycles of amplification, millions of copies of the desired sequence are formed. [2]
Application
RT-PCR is used to detect RNA molecules in a sample with a predetermined portion of the sequence complementary to the primer. Exponential amplification by RT-PCR is a sensitive technique by which a small number of RNA molecules can be detected.
Examples are RNA viruses, such as the human immunodeficiency virus (HIV), which is a retrovirus and therefore uses the HIV reverse transcriptase enzyme ( HIV-Reverse Transcriptase ) to synthesize viral DNA, which is then inserted into the host genome .
Also, RT-PCR is widely used to diagnose genetic diseases and semi-quantitative determination of specific RNA molecules in a cell or tissue as an indicator of the expression of the corresponding genes [3] .
See also
- Polymerase Chain Reaction (PCR)
- Real-time PCR
Notes
- ↑ Hunt M. (2006) Real time PCR tutorial - Copyright 2006, The Board of Trustees of the University of South Carolina
- ↑ 1 2 Glick, B., Pasternak, J. Molecular Biotechnology. Principles and application. - Moscow: World, 2002. - 589 p. - ISBN 5030033289 .
- ↑ Bustin SA. Journal of Molecular Endocrinology, 25 // Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. - 2000. - P. 169–193.
Literature
- Glick B., Pasternak J. Molecular biotechnology. Principles and application. - Moscow: World, 2002. - 589 p. - ISBN 5030033289 .