PCR Mutagenesis

the End jgraham at bronze.ucs.indiana.edu
Fri Nov 13 10:06:31 EST 1992


In <mxdq3j#@lynx.unm.edu> bhjelle at carina.unm.edu () writes:

>I encountered an impressively high level of mutagenesis (14 
>errors out of 411bp) in amplifying a segment of DNA from
>a plasmid clone. All mutations involved T or A in some
>manner. 11 were point mutations (transitions and
>transversions), but 3 were 1 bp insertions or deletions.

>Brian

Very interesting and disturbing. We routinely generate templates for in vitro
transcription via PCR using a protocol (Higuchi et al. NAR 16 (15) p.7351) 
recomended by a well-known colleague.

This procedure uses plasmid targets at 200-400 ng/ rxn. with dNTPs at 0.175 mM
and a mere 16 cycles. The low cycle number is suggested in order to 
reduce the chance of errors generated by Taq. I obtain about 1 to 5 ug of
500 bp fragments from target plasmids around 3 Kb.

In the NAR paper describing this technique, Krummel and associates report that
they have not seen a single base change in preparing 10 different transcription
templates in the 500 bp size range (p. 7359 line 14).

Are you using a significantly lower dNTP concentration ?


Jim
J. Graham
Biology/Chemistry Depts.
Indiana Univeristy -Bloomington
......................."Just saying NO to kits"................................



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