When performing PCR, why do some people use RNA instead of DNA?
When performing PCR, why do some people use RNA instead of DNA?
Last Updated on Saturday, 18 December 2010 04:40 Written by Administrator Saturday, 18 December 2010 04:40
Question by swtfantasiez: When performing PCR, why do some people use RNA instead of DNA?
What I mean is, why do scientists use RNA strands, change them into DNA using reverse transcriptase, and then use that DNA to be used in PCR instead of directly using DNA?
Best answer:
Answer by also known as “aka”
What ppl use as template in a PCR reaction is always DNA. When ppl use RNA in their PCR experiment, what they first have to do is to convert it into DNA using an enzyme called reverse transcriptase. Then they use this as template for the polymerase to do PCR. DNA polymerase does not recognize RNA as a template directly.
ppl that use RNA as starting material usually want to know something about whether a certain gene is expressed in a cell or how strongly it is expressed. By reverse transcribing the RNA and then doing PCR one can measure the amount of transcript in a cell or tissue.
The answer below makes a valid point about how to generate cDNAs. However, to detect transcript levels (eg by Northern Blot) you dont necessarily need the entire cDNA. It is sufficient to have a large enough stretch of an exon to use it as a probe. This exon can be PCRed up from genomic DNA as well.
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The previous answer is a good description of RT-PCR, but the reason I am most familiar with is the generation of cDNA. Recall that eukaryotic genes contain introns and exons. Introns are not made into mRNA, and thus are not part of the protein (they are mostly used for regulation of gene expression). Exons are made into mRNA, and are expressed in the protein.
If you want to clone a protein from a eukaryotic and express the purified protein (or mRNA) you must have cDNA. cDNA is made by taking the mRNA transcript of a gene (which, remember, does not contain the introns) and using reverse transcriptase (a viral enzyme), transcribe it into DNA.
This can be used for a lot of different functions. For instance, if you want to make tons and tons of a particular eukarotic protein, you can put this into E. coli. Recombinant insulin for diabetes is made this way…E. coli, being prokaryotic, has no ability to remove introns from a gene before making the protein, so a scientist most do that before it gives E. coli the DNA.
You can also use cDNA to make nucleic acids complimentary to mRNA. This way you can check levels of mRNA in cells and determine how much a gene is expressed. This is how microarrays and real time PCR is accomplished.
For more information, you can search the internet for cDNA and get some good sources. GOOD LUCK!