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second strand synthesis from cDNA

second strand synthesis from cDNA - Real-Time PCR and Quantitative PCR Forum

second strand synthesis from cDNA - Real-Time PCR and Quantitative PCR Forum


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Old 02-08-2007, 06:48 PM
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Default second strand synthesis from cDNA



Hi,

I am in the process of sequencing a ssRNA viral genome. I was thinking about using a more 'shotgun'-like approach, as opposed to primer walking, as the genome is ~12kb.
The method I was considering was creating cDNA using reverse transcriptase, then somehow creating the second strand so dsDNA is present. Then I thought I'd digest it into ~1kb fragments, ligate into a cloning vector which can be grown up in bacteria and the insert PCR amplified for sequencing.
My issue at hand is how to go about getting the seconds strand from the cDNA. At this point, I'm leaning towards a product from QIAGEN called REPLI-g, which is a whole genome replication kit. I then planned to gel purify the higher molecular weight product, then cut that with restriction enzymes and so on.
Is this a better approach than, say, using RNase H to nick the RNA strand in the RNA-cDNA duplex, and then use E. coli DNA polymerase 1 to create the second DNA strand? This is where I'm having a hard time making up my mind.
The kit seems to be fast and effective.
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