Primer Design Research Paper
Singh, Sita Naik, "Primer Premier: A program for design
of degenerate primers from a protein sequence", Biotechniques,
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 5 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.
The surface antigen (HBsAg) protein sequence of the
adw strain of hepatitis B was reverse translated.
Primer Premier 5 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.
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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