Degenerate Primer Design
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Article Summary: Vinay Singh, Sita Naik, “Primer Premier: A program for design of degenerate primers from a protein sequence”, Biotechniques, January 1998

  • Introduction:
    Researchers pursuing the cloning of novel gene are often faced with the problem that only a partial protein sequence is known.
    When a protein sequence is reverse translated, the resulting DNA sequence frequently contains as high as 50% ambiguous bases.
    Primer Premier automatically scans the target DNA sequence and designs primers in regions of low degeneracy that are free of secondary structures.
    Primers are checked for the overall percentage of ambiguous bases as well as the number of ambiguous bases occurring at the critical 3’ end.
  • Experimental Results:
    The surface antigen (HBsAg) protein sequence of the adw strain of hepatitis B was reverse translated.
    Primer Premier was used to design degenerate primers.
    HBV genome was cloned into pBR322-pAM6 and serum DNA was isolated from HBsAg positive patients.
    Virus DNA from both plasmids as well as clinical serum samples were successfully amplified.
  • Conclusion:
    The technique demonstrated in this paper allows design of primers with a high probability of successful amplification by reducing both the degeneracy and secondary structures of the primers.
    The technique can be used to design primers for PCR, sequencing or hybridization from a known protein sequence of even small regions.

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