How to seq. a single-primer PCR product

Bryan L. Ford fordb at bcc.orst.edu
Mon Jul 6 13:12:35 EST 1998


Brian Sydnor Burnes wrote:

> I have PCR products generated from inverse PCR
> primed off the inverted repeats of a Tn5 insertion.  Only one primer was
> used, so the ends of the products are inverted repeats like the
> transposon.  How can I directly sequence these products without getting
> overlapping forward and reverse reads?  Sequence an asymetric digest?
> Ligate (blunt) into a sequencing vector?  

Brian:

You are on the right track. I would either T-A clone and sequence with
vector-specific primers. Or you could digest by trial-and-error until
you found an RE that cuts once in a fairly medial location and then
proceed to sequence the gel-isolated fragments with your single primer.
Check your inverted repeats to make certain they can/do not act as
sequencing primers themselves-- if they do then cycle sequence without a
primer (high cycle numbers) after the digest and isolation.

(You probably know that one suggestion you received will not work: You
will not be able to resolve the two ends with fluorescently
differentiated but otherwise identical primers-- since there is nothing
to discriminate one side from the other, both labels will prime from
both ends.)

-Bryan



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