PCR problem

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Mon Aug 21 10:08:37 EST 2000


>Riyaz Andrabi wrote:
>>
>> Hi
>> I used forward and reverse primers derived from conserved sequences
>> available in literature to hook out a particular gene.A single
>> amplified product of 1.4kb using both the primers together as well as
>> using the reverse primer alone was obtained, but no amplication was
>> obtained using the forward primer alone.I want to know whether it is
>> possible to hook out the full gene by using the single primer or can it
>> be a part of the gene or what?Thanks
>>

The gene I am working with was originally pulled out by PCR using a
strategy like you outline above, i.e. using conserved sequence from the
literature.  With one of the primers, we too got an amplified product (ours
was small, about 200 base pairs).  I never tried using this primer alone,
but I got this same fragment whenever this primer was used with almost any
other primer.  What was amplified between the primer was indeed authentic
sequence for our gene of interest.  Now that I have authentic sequence for
the entire gene, I know that this primer's 3' end had about 6 bases of
complimentary sequence on the opposite strand that must have led to the
amplification (and of course after just one or two mispriming events,
amplification of that product could become quite efficient).  I'm not sure
how often this happens, but given that in experiments like this, primers
are degenerate, etc., maybe it happens quite often amount.  Of course
products like these are not useless, as they can give you more information
about your gene and allow you to design better primers for your next go
around.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


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