Reverse Transcriptase Polymerase Flashcards
What is Reverse Transcriptase Polymerase
also known as Reverse Transcriptase PCR, is a variation of the polymerase chain reaction that typically measures RNA expression levels
What does it do ?
In RT-PCR, complementary DNA (cDNA) is made by reverse transcribing of the RNA templates with the enzyme reverse transciptase.
How it works ?
First, the enzyme reverse transcriptase uses the mRNA template to produce a complementary single-stranded DNA strand called cDNA in a process known as reverse transcription.
Next, DNA polymerase is used to convert the single-stranded cDNA into double-stranded DNA. These DNA molecules can now be used as templates for a PCR reaction as described above.
The value of RT-PCR is that it can be used to determine if an mRNA species is present in a sample or to clone a cDNA sequence for a subsequent experiment.
Advantages of this technique
The method can do quantitative as well as qualitative analysis.
A small amount of RNA can be used for gene expression studies.
Post PCR processing such as agarose gel electrophoresis is not required here.
The method is simple, easy to use, rapid and cost-effective
Disadvanatges of
The method is extremely sensitive, even a small amount of DNA contamination can lead to false results.
The method is restricted for some of the assays as higher expertise and experimentation are required to develop new assays.
Application
T-PCR is commonly used in research methods to measure gene expression. For example, Lin et al. used qRT-PCR to measure expression of Gal genes in yeast cells.
Gene insertion
RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns, which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of reverse transcriptases) DNA sequence that would be directly translated into protein after transcription.
Cancer Detection :
Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and monitor response to therapy.
History
1977
