Reverse transcription polymerase chain reaction
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Overview
In molecular biology, reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process.
Polymerase chain reaction itself is the process used to amplify specific parts of a DNA molecule, via the temperature-mediated enzyme DNA polymerase.
Reverse transcription PCR is not to be confused with real-time polymerase chain reaction which is also marketed as RT-PCR.
Processes
In the first step of RT-PCR, called the "first strand reaction," complementary DNA is made from a messenger RNA template using dNTPs and an RNA-dependent DNA polymerase (reverse transcriptase) through the process of reverse transcription. RT-PCR exploits a characteristic of mature mRNAs known as the 3' polyadenylated region, commonly called the poly(A) tail, as a common binding site for poly(T) DNA primers. These primers will anneal to the 3' end of every mRNA in the solution, allowing 5'->3' synthesis of complementary DNA by the reverse transcriptase enzyme.cDna can also be prepared from mRNA by using gene specific primer or random hexamer primers.
After the reverse transcriptase reaction is complete, and complementary DNA has been generated from the original single-stranded mRNA, standard polymerase chain reaction, termed the "second strand reaction," is initiated. If the initial mRNA templates were derived from the same tissue, subsequent PCR reactions can be used to probe the cDNA library that was created by reverse transcription. Primers can be designed to amplify target genes being expressed in the source tissue. Quantitative real-time PCR can then be used to compare levels of gene expression.
- A thermostable DNA polymerase and the upstream and downstream DNA primers are added.
- The reaction is heated to temperatures above 37°C to facilitate sequence specific binding of DNA primers to the cDNA
- Further heating allow the thermostable DNA polymerase to make double-stranded DNA from the primer bound cDNA.
- The reaction is heated to approximately 95°C to separate the two DNA strands
- The reaction is cooled enabling the primers to bind again and the cycle repeats.
After approximately 30 cycles, millions of copies of the sequence of interest are generated. The original RNA template is degraded by RNase H, leaving pure cDNA (plus spare primers).
This process can be simplified into a single step process by the use of wax beads containing the required enzymes for the second stage of the process which are melted, releasing their contents, on heating for primer annealing in the second strand reaction.
Usage of reverse transcription polymerase chain reaction
The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique, where a very low copy number of RNA molecules can be detected. Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases and, quantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Northern blot is used to study the RNA's gene expression further.