EVALUATION OF THE ALF DNA SEQUENCER FOR HIGH-SPEED SIZING OF SHORT TANDEM REPEAT ALLELES

DNA typing of short tandem repeat (STR) loci with automated real-time analysis of the fluorescent polymerase chain reaction (PCR) products h as given forensic DNA analysis a new dimension, In the present work, t he ALF DNA sequencer was evaluated for automated size determination of tetra-nucleotide STRs at high speed. Short gel plates, with a well-to -laser distance of 10 cm, allowed for the analysis of four STR loci (H UMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size-determination was done with two external allelic ladders f or each locus. Lane-to-lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR l oci. The accuracy of sizing and allele detection within and between di fferent gels was high (99.89%) for all four STR systems investigated a nd the gels could be reloaded without a decrease in accuracy of the al lele size estimation, This way, the throughput of the system was incre ased, which is of interest for linkage studies, gene mapping and popul ation diversity studies.