Excess PCR primers may dramatically affect SSCP efficiency

Excess PCR primers may dramatically affect SSCP efficiency.

SSCP analysis is one of the most commonly used techniques for screening of small genetic alterations and a very convenient alternative to direct sequencing. In this technique, PCR products are denatured and electrophoresed on non-denaturing polyacrylamide gels. Any change in the sequence theoretically causes a shift in the mobility of the analysed conformers. However, it is known (1) that certa...

Designing PCR Primers Painlessly†

PCR with arbitrary primers

Improved resolution of asymmetric-PCR SSCP products.

amide gel electrophoresis (PAGE) on a 180× 160× 1.5-mm 10% polyacrylamide gel. The separated DNA strands were subsequently visualized by staining with either EtdBr or silver (2). Separation of the DNA strands before electrophoresis using streptavidin paramagnetic beads consistently yielded single-stranded products upon SSCP analysis (Figure 1). Treatment of DNA strands with S1 nuclease before S...

Selecting Degenerate Multiplex PCR Primers

Single Nucleotide Polymorphism (SNP) Genotyping is an important molecular genetics technique in the early stages of producing results that will be useful in the medical field. One of the proposed methods for performing SNP Genotyping requires amplifying regions of DNA surrounding a large number of SNP loci. In order to automate a portion of this method and make the use of SNP Genotyping more wi...