RAPD PCR sequencing
horton at molbio.cbs.umn.edu
Fri Jul 24 13:21:13 EST 1992
The following scheme might be worth a try:
Make a set of new primers identical to your RAPD primers EXCEPT
extend the 3' end by one base: add A to one, C to another, and G to the
In 75% of randomly chosen cases, the next base past the primer
should be different at the two ends of the PCR product (if its A at one
end, there's only a 25% chance that it will be A at the other end, too,
etc.). Unless the next base past the primer is the same at both ends, one
of the extended RAPD primers should prime uniquely at only one end, so
you should be able to sequence with it.
Note that you only have to make three such primers (i.e., any
product that has a T at only one end by default has one of the other
bases uniquely at the other end.).
The 25% of products that you couldn't sequence this way you might
have to go ahead and clone, but its better than cloning all of them.
Alternatively, you could make a set of 15 primers with permutations of two
base additions which would allow you to directly seqeunce all but 1/16th
of randomly chosen products...
Bob Horton /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS || with nine women pregnant you get a baby a month"
1479 Gortner Ave. /||\ -Werner von Braun. Disclaimer:"Bob who?"
St. Paul, MN 55108 ^^ horton at molbio.cbs.umn.edu/(612) 624-3790
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