Search

Currently, a major focus of human genetics is the utilization of single-nucleotide polymorphisms for clinical diagnostics, whole-genome linkage disequilibrium screens to identify common disease genes such as Alzheimer disease, determination of the recent evolutionary history of a species, and the process of speciation. We have examined single-nucleotide extension coupled with high-performance liquid chromatography as a method to simultaneously genotype two SNPs occurring in the coding region of the HFE gene that produce clinical effects. This assay allows concurrent genotyping of the C282Y and H63D mutations in 11 min and is 100% concordant with current testing methods for both of these mutations.

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.