A METHOD FOR POINT MUTATION ANALYSIS THAT LINKS SSCP AND DYE PRIMER FLUORESCENT SEQUENCING

A method is presented for mutation detection directly from single-stra nd conformational polymorphism (SSCP) variants. This approach is based on: (i) amplification of the exons to be analysed by SSCP using the f orward primer with an eight-base tail io form a universal SSCP cassett e; (ii) excision from the gel of the shifted silver-stained bands; (ii i) reamplification of the eluted DNAs using, as the forward primer, a 26-base universal adaptor primer corresponding to the 18-base -21M13 s equence plus the eight nucleotides of the universal SSCP cassette; and (iv) direct sequencing of the purified products using the standard -2 1M13 fluorescent primer. This procedure presents several advantages in cluding the avoidance of a cloning step which leads to significant tim e reduction, while maintaining comparable accuracy at relatively low c osts. In conclusion, the presence of the universal SSCP cassette and s ubsequent reamplification with the same adaptor primer for direct sequ encing makes the method universal for scanning and identification of g ene alterations. (C) 1998 Academic Press.