Analysis of intron 13 microsatellite repeat polymorphism of the factor VIII gene by capillary electrophoresis.

To establish a rapid and automatic gene analysis method, we used capillary electrophoresis (CE) for the analysis of the intron 13 microsatellite repeat polymorphism (MRP) of the coagulation factor VIII gene for the diagnosis of hemophilia A. In the analysis of a 20-bp DNA ladder marker, the reproducibility evaluated using the relative standard deviation (RSD) of the relative migration time for one fragment of each fragment were less than 0.01%, whereas the RSDs of the actual migration time of each fragment were 0.1-0.3%. Thus, the appropriate internal standard should be mixed with the sample when CE resolves polymerase chain reaction (PCR) products. We next analyzed the intron 13 MRP evaluated with (CA)n repeats of the factor VIII gene using a 200-bp DNA fragment as the internal standard. The results showed that the PCR products from the intron 13 MRP could be resolved using CE, even with the repeat numbers of 20 and 21, which differ by only 2 bp. These results suggest that CE is a suitable method for analyzing PCR products for gene diagnosis.

Simultaneous analysis of genes by capillary electrophoresis with a laser-induced fluorescence detector using a stepwise field strength gradient.

A mixture of polymerase chain reaction (PCR) products, 100, 105, 300, 310, 485, and 500 base pair (bp) DNA fragments, was analyzed by capillary electrophoresis equipped with a laser-induced fluorescence detector (CE-LIF) using a stepwise gradient of electric field strength. The optimum condition for the analysis of PCR products was 0.5% methylcellulose and 160 V/cm from 0 to 10 min and 270 V/cm from 10 to 17 min. The length (bp) of DNA could be estimated from the relationship between the relative migration time and bp length. The relative standard deviation (R.S.D.) of DNA size (bp) was less than 3.5% and the difference from the true value was only 2.4 bp.

Rapid typing of variable number of tandem repeat locus in the human apolipoprotein B gene for DNA diagnosis of heart disease by polymerase chain reaction and capillary electrophoresis.

The apolipoprotein B (apoB) variable number of tandem repeat (VNTR) alleles containing larger repeat units is a risk factor for coronary heart disease. Capillary electrophoresis (CE) in entangled polymer solution was applied to the analysis of polymerase chain reaction (PCR) amplified apoB VNTR locus for DNA diagnosis of heart disease. The CE separation gives an excellent resolution of two alleles differing by one or two 16 bp repeat units in the DNA size range up to 600 bp with high speed. The apoB alleles differing in length by 2 or 4 repeat units are readily distinguishable by CE in the DNA size range from 600 to 1000 bp. The plate number achieved was 1 million plates per meter. CE combining with PCR provides an excellent technique for accurate determination of the number of repeat units of apoB VNTR alleles and differentiation of heterozygous from homozygous individuals. Using the CE technique, the apoB VNTR loci from some individuals in genotyping were examined towards precise DNA diagnosis for coronary heart disease.

High-resolution separation of PCR product and gene diagnosis by capillary gel electrophoresis.

High-resolution separation of a PCR product from a mixture of DNA restriction fragments was achieved using capillary gel electrophoresis. The capillary gel electrophoretic separation gives an excellent resolution of two fragments of the 500-bp PCR product and the 506-bp DNA fragment, which differs by only 6 bp, and the complete separation of a broader chain length range of DNA afragments up to 12 kbp within 20 min. The plate number of gel-filled capillary was achieved to be 2 million plates per meter. Capillary gel electrophoresis is applied to the gene diagnosis for heart disease through apolipoprotein E genotyping. The advantages and limitations of capillary gel electrophoresis in the application to PCR analysis and DNA diagnosis are discussed.