We have an issue to amplify 3' end of dsDNA virus genome (ASFV). Basically, we use Long-Range PCR to amplify whole viral genome. Everything was fine except 3'end region. We still have around 6 kb to get. We have already tried to design primers to amplify smaller PCR fragments and get closer, but still part of genome left. There is of course a possibility of gene deletion or insertion or whatever in 3'end. The question is to find a way to get this region for following sequencing.
If you the end then, by designing multiple primers go for the primer walking. Hope it will help to get complete sequence.
Formally Sequence is unknown. We have designed primers based on reference sequence in Genbank, but some changes are likely exist.
There are some great approaches here. However, if you want your answer to be complete and clear, I would suggest taking any sample you have that is enriched for the viral genome and sending it off for next-generation sequencing. Worst case scenario it would cost you about $3000 and no effort except the bioinformatics. In addition, it would show you the exact sequence for the entire virus and probably any heterogeneity in the virus.
I agree that this one is the easiest way to solve this issue. The problems is quiet difficult to send out any samples with viral content rather viral genome.
You may directly sequence the genomic DNA with primers binding to the known region, if you could prepare enough viral genome (>10ng/ul). Or, you could add dATPs to the 3' end with Terminal Deoxynuleotidyl Transferase (TDT), and then perform nest-PCR with specific primers and Archor-oligodT, Archor primer.
Thanks Bing. So, basically you a talking about RACE PCR isn't?
Well it could an option as well. Thanks. What about protocol for TDT? Is it tricky to use? I have no experience with it.
You could use tailing with terminal transferase; dGTP will only add about 20 residues.
Usinf oligo (dC) and nested primers shoudl give your sequence
I have experience sequencing viruses via NGS. The 5' and 3' ends are often under represented, and in many cases you may still miss a handful of nucleotides. As mentioned above, the TdT approach is likely your best best.
Hii, i would suggest another alternative which was quite successful while working with ss-RNA virus. I would suggest you to try a snap-cool method wherein u keep your DNA with the specific primer(s) and heat it for some time. Then you may directly keep in ice. this actually prevents the formation of secondary structures which normally hinders the PCR reactions at both the ends. (i dont know whether its the same thing that is happening with your ds-DNA!!). But, just give a try! all d best! :)
Thank's everybody! We will try to do our best to solve this issue based on yours comments and suggestions…
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