PCR artefacts??

Dr. R.J. Beynon beynonrj at uxb.liv.ac.uk
Wed Feb 3 09:15:30 EST 1993


WE are using PCR to amplify segments of DNA, and then sequencing from an 
internal primer, all the way up to our other amplifying primer:

                                      seq primer   
                                          -->.................
    5'----------------------------------------------------^----....3'
        --->                                               <--
       amp primer 1                                        amp primer 2

We are finding a high occurence of a 'wrong' base, immediately
next to (3') the amp primer 2 (indicated by ^) on diagram. In other
words, this implies that the first base incorporated during amplification
is wrong - and when it happens is always a T incorporated instead of a C.

Is this a common observation of an artefact (explanation a),
 or is explanation b (real, not artefact) more likely.

                        
We'd really like to know, because 'b' is *very* interesting to us.
Needless to say, we're doing other experiments, but I wonder if anyone
else had come across this too?

Rob Beynon, Proteolysis Group, Biochemistry, Liverpool, UK
-Doing molecular biology without a license!:-)



More information about the Methods mailing list