RT-PCR/ qPCR Flashcards
Why is RT-PCR needed?
RNA is highly unstable and degraded by RNAse enzymes
What does RT-PCR do?
Convert RNA into cDNA
What is an oligo dT primer?
Most common method and binds to the polyA tail of all mature mRNAs
reverse transcriptase enzyme produced cDNA from the RNA template
What is the benefit of using random priming in RT-PCR?
Increases the cDNA yield in an RT-PCR reaction and allows an amplification of mRNAs lacking a polyA tail
What are random primers?
Bind throughout an RNA molecule
What is the benefit of using specific primer?
Will bind to only RNA od interest - specific
but can be poor yield
What primers do modern RNA to cDNA kits use?
Combine both oligo(dT) and random primers
What is one step RT-PCR?
DNA polymerase in the same tube as the reverse transcriptase reaction - rapid and is applicable when one or few targets are amplified
Benefits of one step RT-PCR?
Reduces risk of contamination
What is two step RT-PCR?
PCR is carried out after reverse transcriptase - can be done in multiple different tubes
Highly sensitive and better yield as can use random priming
Increase his of contamination and errors
What are the uses of one step RT-PCR in molecular cloning?
Isolate gene sequences for use in molecular cloning
- won’t contain introns so removes the need for splicing
What are the uses of two step RT-PCR in molecular cloning?
Add restriction sites for use in molecular cloning
