degenerated primers PCR HELP!!!!

Daniele Eusebe deusebe at
Mon Sep 18 11:36:53 EST 1995

I want to prepare a cloning probe by PCR amplification of an eukaryotic
gene fragment in one animal species using degenerated primers
corresponding to protein sequences conserved in several (distant) other
species. I am looking for advices concerning the following questions:
- Is it better to use degenerated primers with, for example, G or A at one
position or inosine at this position?
- How important can be the primer degeneracy in PCR?
- Is it necessary to increase total primer concentration to account for
the degeneracy? To what extent?
- If using inosine at ambiguous positions, how many significant
nucleotides are necessary in the primer ?
- Does inosine contributes to the stability (Tm) of the hybrid?
- How low can be the annealing and elongation temperatures?
Thank you in advance.

Daniele Eusebe (Ecole Normale Supérieure-INSERM U293-Paris-France)
e-mail: deusebe at

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