Dirty PCR!

txpljfg at UABCVSR.CVSR.UAB.EDU txpljfg at UABCVSR.CVSR.UAB.EDU
Thu Apr 21 08:57:25 EST 1994


It is not unusual to clone out gene fragments that are unrelated to the
desired product when the hybridization conditions are not as stringent
as they should be, or when the primers contain significant homology to
another target sequence.  Once the primers hybridize, you will get
amplification of whatever is between them regardless of whether or not
it is the product you desire.  To avoid this problem, you must optimize
the reaction conditions to increase the specificity of your primers



> I have recently obtained two clones of the heavy chain variable region (Vh) of 
> human immunoglobulin gene from a human hybridoma cell line through a standard 
> procedure: RNA extraction, cDNA synthesis, PCR with published primers and 
> cloning into a common-used vector. However, after sequencing, both vector and 
> PCR primers are correct in  the two clones, somehow sequences of the inserts 
> have no homology at all to any human Vh gene fragments and are also totally 
> different from each other, though they differ to each other only three nucleic 
> acids in size. It is too hard to understand it for us! We would like to know 
> any similar experinces and suggestions for better understanding. Thank you in 
> advance.
> 
> Jian_fu Wang
> MPI for molecular biology
> Berlin
> 
> --------------------------------------------------------
> Jian_Fu Wang, Max-Planck Institut für molekulare Genetik
> Ihnestrasse 73, D-14169 Berlin, Germany
> e-mail: Wang_J at MPIMG-Berlin-Dahlem.MPG.DE
> 

==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
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