SEQUENCING USING CAPILLARY ELECTROPHORESIS OF SHORT TANDEM REPEAT ALLELES SEPARATED AND PURIFIED BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequen ce, number of repeats and microvariants within a short tandem repeat ( STR) locus. Also, PCR amplification of tetranucleotide repeat loci, us ed in DNA typing assays, often result in heteroduplex formation, addin g to the complexity of analysis. Sequencing reactions require single s pecific target DNA for reliable sequencing analysis. Alkylated poly(st yrene-divinylbenzene) columns at elevated temperature and gradient elu tion conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-p airing reverse-phase (IPRP) high performance liquid chromatography (HP LC), molecular biologists can separate and purify DNA fragments withou t alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by se paration of alleles of the short tandem repeat loci of TH01, vWA31, F1 3A01 and FES/FPS. Alleles differing in size range of 12 to 4 base pair s were separated by IPRP/HPLC and individual alleles were peak-capture d, then cycle-sequenced. These HPLC fractions required no additional s teps prior to cycle sequencing. Capillary electrophoresis (CE) was use d to sequence the alleles. Furthermore, CE offers advantages over trad itional slab methods via automation and higher applied voltages. Inter estingly, unlike traditional gel electrophoresis, samples were introdu ced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderi vatized fused silica capillary with an interior diameter of 50 mu m an d a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 2 20-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE seq uencing, the results confirmed the sequence and number of nucleotide r epeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 micro variant for a TH01 heterozygous individual previously typed as a 9, 9. 3/10 using slab gel electrophoresis. The techniques described can be a pplied to other DNA purification and isolation problems.