problems with primer dimer!
Gys de Jongh
GysdeJongh at planet.nl
Tue Aug 28 15:50:19 EST 2001
"Babett Bartling" <babett.bartling at medizin.uni-halle.de> wrote in message
news:3B8A4C6B.3DEC2735 at medizin.uni-halle.de...
> Hope somebody can help me.
> We investigated gene expression using PCR without any problems.
> Since last week, we suddenly have a lot of primer dimers and problems
> with the right size band.
> How can we solve the problem with the excess of primer dimers??
here is my (bit sad) hypothesis :
your PCR worked perfectly . Like in most other PCR reactions primer dimers were
formed. Especially in those instances were there was little or no template. This
primer dimer product is just a good ds DNA. Something like
where P1 is the 5' beginning of primer 1 ; same for P2
it is formed even if P1 and P2 have only 1 or 2 bases overlap. The sad thing is
that this short sequence is a very good template. If you denature this ds primer
dimer DNA it will still be a perfect , ss , template. The ss strands couple will
still multiply log.
In your case the primers have just 1 overlapping base : G<=>C
So the following may have happened :
CCC AAA GCA CTC CGA CAC CAA G
CAT CCT GGC AGC GCT CAA ACA C
Very little of this , unlikely , annealing :
5'-CCC AAA GCA CTC CGA CAC CAA G-3'
3'-C ACA AAC TCG CGA CGG
Possible ds Primer dimer product _after_ the PCR reaction :
(note that this will still act as template after melting)
5'-CCC AAA GCA CTC CGA CAC CAA G TGT TTG AGC GCT GCC AGG ATG-3'
3'-GGG TTT CGT GAG GCT GTG GTT C ACA AAC TCG CGA CGG TCC TAC-5'
(Hope it lines up after mailing)
So I think that some were in the past you contaminated your primer set with a
primer dimer product of one of your previous PCR's :(
You could try to irradiate a aliquote of your primers with short UV . This will
destroy your primers _and_ the primer dimer product. As there will be much more
of your primers then of the primer dimer contamination your PCR will show an
increase in the long product you want and a decrease in the primer dimer product
you don't want. The obvious thing is of couse new primers (and fanatic filtertip
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