Validation of STR typing by capillary electrophoresis

With the use of capillary electrophoresis (CE), high-resolution electrophor etic separation of short tandem repeat (STR) loci can be achieved in a semi automated fashion. Laser-induced detection of fluorescently labeled PCR pro ducts and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by c apillary electrophoresis were investigated using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). An internal size standard was used with each run to effectively normalize mobility differences among injections. Allele s were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demon strate that allele sizes were reproducible, with standard deviations typica lly less than 0.12 bases for fragments up to 317 bases in length (largest a llele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all all eles that are the same length should fall within the measurement error wind ow of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were als o accurately typed by the analytical software. Alleles differing in size by one base could be resolved in two-donor DNA mixtures in which the minor co mponent comprised greater than or equal to5% of the total DNA. Furthermore, the quantitative data format (i.e., peak amplitude) can in some instances assist in determining individual STR profiles in mixed samples. DNA samples from previously typed cases (typed for RFLP, AmpliType (TM) PM+DQAI, and/o r DIS80) were amplified using AmpFlSTR((R)) Profiler Plus (TM) and COfiler (TM) and were evaluated using the ABI Prism 310. Most samples yielded typab le results. Compared with previously determined results for other loci, the re were no discrepancies as to the inclusion or exclusion of suspects pr vi ctims. CE thus provides efficient separation, resolution, sensitivity and p recision, and the analytical software provides reliable genotyping of STR l oci. The analytical conditions described are suitable for typing samples su ch as reference and evidentiary samples from forensic casework.