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Questions about the primer design software usage

Raymond Dalgleish ray at le.ac.uk
Thu Feb 26 13:53:06 EST 2004

By convention, primer sequences are reported in the 5' to 3' direction.
Normally, when a pair of primers is specified in a paper, the first (e.g.
Primer A) is the forward primer and the second (e.g. Primer B) is the
reverse. As long as you type in the two sequences into Primer3 in the 5' to
3' direction, the program should be able to locate the primers in the target

Things can go wrong if the primer sequences have been modified by the
inclusion of a "tail" at the 5' end to, perhaps, introduce a restriction
site. A single base change might have been deliberately introduced within
the body of each primer to introduce a particular restriction site. Many
primers will work well across species (e.g. human primers on chimpanzee
DNA). The primers that you are checking might have been designed with one
genome in mind but have been found to work with another, even though there
may be mismatches.

The fact that you get the same results with Primer3 and with Primer Premier
suggest that one of the explanations is probably correct.

Raymond Dalgleish
Department of Genetics
University of Leicester

""Jack Lee "" <wleemd at mail.edu.cn> wrote in message
news:20040222091712.F19127D394 at mercury.hgmp.mrc.ac.uk...
> I tried the "Primer3" software (web based edition) to test a pair of
primers I
> found on a paper, but always got the output:"specified left/right primer
not found
> in the sequence."
> What's the wrong here?
> I searched the web, and found an introduction to "Primer3", it mentioned
> and reverse primer, but in papers "forward and reverse primer" never
> Some papers only gave:
> "Primer A 5' agttca......ccagtatt 3'
>         B 5' ccagta......cagattat 3'"
> Some said:
> "Primer 5' agttca......ccagtatt
>         3' ccagta......cagattat
> So which is the forward primer, which is the reverse primer?
> I also tried the "Primer Premier", and experienced the same problem.
> ---


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