Primer premier: program for design of degenerate primers from a protein sequence.

faced with the problem that only a partial protein sequence is known. Several procedures are commonly used involving the reverse translation of the protein sequence into a DNA sequence. These include synthesis of a short oligonucleotide sequence for screening libraries and of a primer pair for amplification of target sequence by polymerase chain reaction (PCR). However, due to redundancy in the genetic code, oligonucleotide design must account for ambiguous DNA bases. When a protein sequence is reverse-translated, the resulting DNA sequence frequently contains as high as 50% ambiguous bases. This requires that primers be designed from regions of lower degeneracy. Manual location of such primers is a tedious process and often fails due to the presence of secondary structures. Primer Premier automatically scans the target DNA sequences and designs primers in regions of low degeneracy that are free of secondary structures, including hairpins, dimers and false priming sites. These primers are checked for the overall percentage of ambiguous bases as well as the number of ambiguous bases occurring at the critical 3′ end. We report here successful amplification of a gene with the primers designed from its protein sequence using the Primer Premier primer design program. The surface antigen (HBsAg) protein sequence of the adw strain of hepatitis B virus (HBV) was reverse-translated using the program. The sequence was scanned, and primers were designed in regions of low degeneracy. Nonambiguous bases were substituted for ambiguous bases to produce a primer that contained no secondary structures (2). HBV genome cloned into pBR322-pAM6 (Catalog No. 45020; ATCC, Rockville, MD, USA) was used for positive control studies. Serum DNA isolated from HBsAg-positive patients according to protocol described by Nagaraju et al. (1) was amplified for the HBsAg sequence of viral genome by designing primers specific to this protein sequence. The primer sequences were HBs1: 5′-CAT GCC CTC CTC TAT GTC CTG-3′; HBsR1: 5′-GCG AGA GCC AGA CTG TAG GG-3′. Comparison with degenerate sequence is shown in Figure 1. Five hundred nanograms of plasmid and genomic DNA were used in amplification reactions in a 50-μL reaction volume containing 5 μL of 10× enzyme buffer (670 mM Tris buffer, 160 mM ammonium sulfate, 40 mM MgCl2 and 0.1% Tween 20), 0.25 mM each of dATP, dGTP, dCTP and dTTP, 1 μM each of primers HBs1 and HBsR1 and 2.0 U of Taq