Degenerate PCR.. A complication

Alejandro Abuin aabuin at bcm.tmc.edu
Wed Feb 16 14:04:20 EST 1994


     Recently I asked for some ideas on how to differentiate between
different "subspecies" from a pool of PCR products of the SAME
SIZE.... that is, all the products are of the same size,  but sevreal
different (i.e. divergent in sequence) "subspecies" are represented.
    
     First, thanks to all who answered...

    Several of the ideas suggested were:

        1. cloning the pool as a whole and determine the number of
crosshybridizing subsets within the pool

        2. use a number of restriction enzymes until you find one that
cuts within one "subspecies" but not others

        3. Use internal probes to any known "subspecies" to eliminate them
and pursue any non-hybridizing, supposedly new ones

        4. Heteroduplex mobility assay to differentiate between "subspecies"

        5. Do the PCR on genomic DNA, not cDNA (the distance between the
primers on the cDNA is small, so they might lie within adjacent exons with
size variations between different genes)

        All these have good and bad points....

        Anyway, thanks netters

        Don't hesitate to follow up on this if you want to...


      Alex




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