A semi-automated method for analysis of intron 13 and intron 22 dinucleotide repeat polymorphisms of the factor VIII gene

Abstract

A semi-automated method has been developed for the simultaneous analysis of two microsatellite repeat polymorphisms located in intron 13 and 22 of the Factor VIII gene. The fluorescent dyes, 6-FAM- and HEX- phosphoramidites were used to 5'-end label the respective 5' primers of these two microsatellite repeats and a multiplex polymerase chain reaction (PCR) devised for amplification. The PCR product was loaded onto the gel with DNA size marker labelled with ROX. A rural of 24 samples could be analysed simultaneously on an automated DNA sequencer. The results were computed using a dedicated software, with assignment of PCR product size in basepair. This method compares well with the conventional manual procedure using radio-labelled primers, but at the same time overcomes many of the inherent disadvantages associated with the latter method.