The following links may help you:-

1. http://www.ncbi.nlm.nih.gov/pubmed/22527869

2. http://www.ncbi.nlm.nih.gov/pubmed/23504105

3. http://link.springer.com/article/10.1007%2Fs00705-013-1784-6

Dear Shikha,

I would say that the choice of the method depends on the situation you’re in. If you’re dealing with a potyvirus, which its full genome can be accessed on GenBank, I encourage you to go for Genome walking. In that case you may need to perform a 5’RACE to get the 5’end sequence.

If you’re dealing with a potyvirus that has no record on GenBank, you can start genome walking using Potyvirus degenerate primers, then cover the gap using specific primers. We found in our case that RT-PCR yielded better results using purified virus than using total RNA, especially for amplicons longer than 1500 bp. Alternatively, You could think of exploiting the benefit of Next generation sequencing (NGS).

Thank you for suggestions. By purified virus you meant cDNA with specific primers??

I meant virus extracted from its host. You get it by doing a virus extraction/extraction

Hi Shikha, if you explain more, I will help you more directly. Send me direct mail, if necessary.

Hi shikha

If you want to go NGS way Truseq stranded Ribozero-plant would be good for library preparation then select the platform you would want to use for sequencing depending on the number of samples you have and the coverage you would want to achieve.

Why don't you start from its polyA using a simple oligodT and as recommended above just walk along the geneome designing new oligos for each strech until reaching its 5' end? You might want to do various simultaneous sequencing reactions because potyviruses exist in cuasi-species and you will need to compare places with heterogeneities to retrieve the consensus sequence. Hope it helps!

Hi Shikha

first will u let me know the procedure to clone full length potyvirus? is in fragments or at single time amplified product to clone...which vector u hv used????