Validation of STR typing by capillary electrophoresis.
With the use of capillary electrophoresis (CE), high-resolution electrophoretic separation of short tandem repeat (STR) loci can be achieved in a semiautomated fashion. Laser-induced detection of fluorescently labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by capillary 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. Alleles were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demonstrate that allele sizes were reproducible, with standard deviations typically less than 0.12 bases for fragments up to 317 bases in length (largest allele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all alleles that are the same length should fall within the measurement error window of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were also 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 component comprised > or = 5% 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 PM+DQA1, and/or D1S80) were amplified using AmpFlSTR Profiler Plus and COfiler and were evaluated using the ABI Prism 310. Most samples yielded typable results. Compared with previously determined results for other loci, there were no discrepancies as to the inclusion or exclusion of suspects or victims. CE thus provides efficient separation, resolution, sensitivity and precision, and the analytical software provides reliable genotyping of STR loci. The analytical conditions described are suitable for typing samples such as reference and evidentiary samples from forensic casework.
Moretti
T R
TR
FBI Laboratory, FBI Academy, Quantico, VA 22135, USA.
Baumstark
A L
AL
Defenbaugh
D A
DA
Keys
K M
KM
Brown
A L
AL
Budowle
B
B
D016172:DNA Fingerprinting / Q000379:methods*; D019075:Electrophoresis, Capillary; D005554:Forensic Medicine / Q000379:methods; D005784:Gene Amplification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012984:Software; D013050:Spectrometry, Fluorescence; D020080:Tandem Repeat Sequences