i have designed degenerate primer and amplified product (700 bp) is cloned and sequenced. but now how can i proceed for for cloning of rest (9.0kb) of the the genome.....
Design primer for rest of the sequences (like you need atleast 20 primer set covers 700 bp each with specific restriction enzyme sites for ligation reaction) or you can browse NEB web site. They have one cloning vector , such a way that single shot of 9.6 kb gene can be cloned.
sir this is the problem to design primer without knowing the rest of sequences..
Dear
If you want sequence PVY, or any other potyvirus group visit at NCBI, U will get full length gene sequence for the particular virus group and design primers accordingly. If size is > 9.2 kb...design 3-7 primers covering all the 9 kb sequence, amplify clone and do the sequencing.....
Cloning the entire genome of a potyvirus from a single PCR is terribly complicated if not unrealistic as quite often bacteria are neglectant to grow when the whole PCR is cloned into them. The common approach is to assemble the genome from different PCR products incorporating an intron around the P3 area.
For more info check Perdajña et al 2012 Acta Virologica "Cloning of the complete infectious cDNA of the plum pox virus strain PPV-Rec" or even López-Moya & García 2000 Virus Research "Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment."
If your virus is very divergent from the 193 different potyviruses in the genebank, then you could consider sequencing starting from the poly-A tail...
Good luck!
Dear Meena
I recommend reading this
Nagata T, Inoue-Nagata AK. Simplified methods for the construction of RNA and
DNA virus infectious clones. Methods Mol Biol. 2015;1236:241-54. doi:
10.1007/978-1-4939-1743-3_18. PubMed PMID: 25287508.
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