PCR then Sequence

Shiao Y. Wang sywang at whale.st.usm.edu
Tue Mar 15 01:41:11 EST 1994


I have a number of clones from a cDNA library (in lambda gt22A) I would
like to sequence. The approach I used before was to subclone into a
plasmid vector, CsCl purify and then sequence with Sequenase. Beautiful
results. Problem: very slow process. I would like to use cycle sequencing
in the future. To increase the amount of template, I'm thinking about
doing PCR w/ primers that flank the insert first and then sequence the PCR
product use internal primers. The inserts are less than 2 kb. 

My questions are:
1). Is fidelity of the Taq polymerase a concern? Since I won't be
sequencing a cloned PCR product, it's probably not. But is this approach
commonly accepted (e.g. by journal editors)?
2). If fidelity is a concern, would something like VENT be better?
3). Would it be better to do asymetric PCR to increase the amount of ssDNA
as template? If so, would it be even better to simply use one primer and
forget about exponential amplification and go with linear amplification to
generate template DNA for sequencing?

Thanks for sharing your experience. If I get responses, I'll
summarize and post the recommendations.



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