Dear All,
I would like to ask some help in second strand synthesis. I’m currently working on a project which require the conversion of a single stranded RNA virus genome (i.e. a norovirus) into a double stranded DNA. I’m using Superscript IV RT (Invitrogen) and random hexamers (50µM) for reverse transcription on total RNA from faeces (extracted by TriZol method), which works properly (I checked the presence of the virus by SYBR-Green based qPCR after RT). Then I used a Second strand synthesis kit (Invitrogen, Cat. No.: A48570) according to the manufacturer’s protocol. About the kit: “This Second Strand cDNA Synthesis Kit uses nick translational replacement of the mRNA to synthesize the second strand cDNA. The second strand cDNA synthesis is catalyzed by E. coli DNA polymerase I in combination with E. coli RNase H and E. coli DNA ligase. E.coli RNase H inserts nicks into the RNA, providing 3' OH-primers for DNA polymerase I. The 5'-3' exonuclease activity of E. coli DNA polymerase I removes the RNA strand in the direction of synthesis, while its polymerase activity replaces the RNA with deoxyribonucleotides” E. coli DNA ligase links the gaps to complete the ds cDNA strand Adding the whole amount of RT product into the second strand synthesis mix, incubated in 16 degrees Celsius for an hour as described in the manufacturers protocol. But then the following SYBR-Green based qPCR shows the absence of the virus. I think something in the second strand synthesis enzyme mix (DNA pol I maybe) is breaking down my cDNA. But why? Can anyone help me? What should I do to generate a second strand DNA rather than destroying my cDNA?
OK, you'll need to talk with a colleague about how to trouble-shoot your protocol. But the whole idea is that you are turning unstable, single-stranded RNA into a much more stable double-stranded DNA molecule.
You are not destroying your cDNA molecule. You are changing it from the RNA paired with cDNA double-stranded molecule into a double-stranded molecule with two cDNA strands. You replace the RNA, not the cDNA.
Take a read through the protocol book to get a better understanding of what is happening during the RT steps.
There are LOTS of places that can go wrong. What do your qPCR controls look like?
Possibly you're not losing your DNA. It's a matter of expectations.
Say, you have 100 viruses in your sample. You do 100 1st Strand and 2nd strand reactions. Number of virus genomes = 100. Now, if you do PCR amplification then it's easier to see it in a gel.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA