A semi-automated method for analysis of intron 13 and intron 22 dinucleotide repeat polymorphisms of the factor VIII gene.
A semi-automated method has been developed for the simultaneous analysis of two micro-satellite 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 total 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.
Yip
B
B
University Department of Medicine, Queen Mary Hospital, Hong Kong.
Chan
V
V
Chan
T K
TK
D000483:Alleles; D001331:Automation; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D018900:Dinucleotide Repeats; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes; D005838:Genotype; D006239:Haplotypes / Q000235:genetics; D006467:Hemophilia A / Q000097:blood / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D007962:Leukocytes / Q000737:chemistry; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012984:Software
D017931:DNA Primers; D005456:Fluorescent Dyes; D005169:Factor VIII