The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR® Profiler Plus™ and AmpFlSTR COfiler™ (Applied Biosystems, Foster City, CA), GenePrint™ PowerPlex™ (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism® 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO® II Fluorescent Imaging Device, and the FluorImager™ were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.

Allele distributions for 13 tetrameric short tandem repeat (STR) loci, CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11, were determined in African American, United States Caucasian, Hispanic, Bahamian, Jamaican, and Trinidadian sample populations. There was little evidence for departures from Hardy-Weinberg expectations (HWE) in any of the populations. Based on the exact test, the loci that departed significantly from HWE are: D21S11 (p = 0.010, Bahamians); CSF1PO (p = 0.014, Trinidadians); TPOX (p = 0.011, Jamaicans and p = 0.035, U.S. Caucasians); and D16S539 (p = 0.043, Bahamians). After employing the Bonferroni correction for the number of loci analyzed (i.e., 13 loci per database), these observations are not likely to be significant. There is little evidence for association of alleles between the loci in these databases. The allelic frequency data are similar to other comparable data within the same major population group.

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 ≥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.

A presumptive reagent for dilute blood detection other than luminol is fluorescein. The sensitivity of fluorescein approaches the sensitivity of detection levels of luminol. The fluorescein detection method offers the advantages of working in a lighted environment, and the reaction persists longer than luminol. A series of diluted bloodstains, ranging from neat to 1:1,000,000, was placed on a variety of substrates. Three sets were made per substrate. One set was exposed to fluorescin, one set was exposed to luminol, and one set served as an uncontaminated control. The fluorescein signal persisted longer than luminol. However, background staining for fluorescein was observed on some substrates within 30 s to 1 min, and no background staining was observed for luminol. Stains on non-absorbent surfaces were detectable at 1:100,000 dilutions, and stains on absorbent surfaces were detectable usually at no more than 1:100. The sensitivity of detection of fluorescein was comparable to that of luminol in this study. In all cases, where sufficient DNA was recovered, typeable results at all 13 core CODIS STR loci were obtained from treated bloodstains and controls. The results from STR typing indicate that there was no evidence of DNA degradation.

A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.