Primer Premier: Program for Design of Degenerate Primers from a Protein Sequence

Premier Biosoft International, Palo Alto, CA, USA and 1Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Lucknow, India
BioTechniques 24:318-319 (February 1998)

Researchers pursuing the cloning of novel genes are often 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-mL reaction volume containing 5 mL of 10´enzyme buffer (670 mM Tris buffer, 160 mM ammonium sulfate, 40 mM MgCl ₂ and 0.1% TweenÒ20), 0.25 mM each of dATP, dGTP, dCTP and dTTP, 1 mM each of primers HBs1 and HBsR1 and 2.0 U of Taq

Figure 1. Comparison between degenerate and nondegenerate primers.(A) sense primer; (B) anti-sense primer.

Figure 2. Band pattern of amplified product on 1.5% agarose gel. Lane M: pBR322 digested with Hinf1 set as marker lane. Lanes 1 and 2: 15 mL of amplified product of HBV plasmid DNA. Lane 3: negative control; PCR run without target DNA. Lanes 4 and 5: amplified DNA of HBsAg-positive sera of patients.

DNA Polymerase (Bangalore Genei Pvt. Ltd., Bangalore, India) overlaid with two drops of mineral oil. Reaction mixture was denatured for 7 min at 95°C and subjected to 28 cycles each with a profile of 60 s at 92°C, 80 s at 52°C and 105 s at 72°C in a DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA). The electrophoresis of the PCR product was performed on a 1.5% agarose gel. Figure 2 shows the banding pattern of the PCR products.

Thus, we have demonstrated that using degenerate primers for viral sequence of HBV, designed from the known protein sequence, it was possible to successfully amplify the virus DNA from both plasmid and clinical serum samples. This technique will allow design of primers with a high probability of successful amplification by reducing both the degeneracy and secondary structures of the primer. The technique can also be used for the design of primers for PCR sequencing or hybridization from a known protein sequence of even small regions. Information on Primer Premier is available at www.PremierBiosoft.com, by calling 415-856-2703 or from the author’s address.

REFERENCES
1.Nagaraju, K., S. Misra, S. Saraswat, N. Choudhary, B. Masih, V.Ramesh and S. Naik.1994. High prevalence of HBV infectivity in blood donors detected by dot blot hybridization assay. Vox Sang. 67:183-186.
2.Nomenclature Committee of the International Union of Biochemistry (NC-IUB). 1985. Nomenclature for incompletely specified bases in nucleic acid sequences. Eur. J. Biochem. 150:1-5.
Address correspondence to Dr. Sita Naik, Head, Department of Immunology, Sanjay Gandhi Post Graduate Institute
of Medical Sciences, Raibareli Road, Lucknow-226 014,India.Vol.