Validation of Short Tandem Repeats (STRs) for Forensic Usage: Performance Testing of Fluorescent Multiplex STR Systems and Analysis of Authentic and Simulated Forensic Samples

Abstract

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.

Variations in short tandem repeats deduced on the basis of the number of repeats and the relationship of these variations with longevity

“…Sample preparation and genotyping DNA was extracted from 3 ml of peripheral blood by the Chelex 100 procedure (Moretti et al 2001). Polymerase chain reaction (PCR) was performed using the AmpFLSTR Profiler plus PCR Amplification Kit (Perkin Elmer, Foster City, CA, USA) as per the manufacturer's recommendations; the PCR reaction volume was 50 μl, and a 9600 Perkin Elmer thermal cycler was used.…”

“…As a reliable assay performed using commercial multiplex kits, the procedure was carried out at half-atomized condition in accordance to the international standards, and the results were assessed by using standard software; therefore, error due to artificial performance and judgment was remarkably reduced (Budowle and Sprecher 2001;Buse et al 2003;Moretti et al 2001). CODIS STR loci were chosen in this study because of their reliability for genotypic analysis.…”

La electroforesis capilar como una nueva estrategia en la medicina y el diagnóstico clínico

“…La EC puede llevar a cabo el análisis de 15 marcadores genéticos tipo microsatélite marcados con fluorescencia, garantizando la máxima precisión y fiabilidad en pruebas de identificación humana (Figura 2), aun a partir de muestras degradadas o con poco ADN 45 . Estos marcadores, según las pautas de la Agencia de Investigación Federal de los Estados Unidos de Norteamérica (FBI), constituye el conjunto de secuencias de ADN denominado CODIS (del inglés, Combined DNA Index System) que permite identificar singularmente a una persona de otra en aproximadamente 5 h, requiriendo una cantidad mínima de material genético (0,125 ng) 46 . Estas pruebas son empleadas en medicina forense para la identificación de agresores sexuales, asesinos, restos humanos encontrados en accidentes como incendios, desastres naturales y guerras 47 ; la identificación de la relación biológica entre personas perdidas con el fin de demostrar parentesco (empleo que actualmente ha tomado auge entre los migrantes latinos en Estados Unidos de Norteamérica) y pruebas de paternidad, la demostración de linajes reales, así como en los análisis antropológicos y de migraciones 48 .…”

Comparison of short tandem repeat and variable number tandem repeat genetic markers for quantitative determination of allogeneic bone marrow transplant engraftment

“…Stutter peaks of tetranucleotide STRs are 4 bp shorter than the main peak and usually have a peak area between 5% and 10% of the main peak area. 7 Informative alleles for STR analysis are chosen to avoid stutter peak positions if possible. In cases where an informative allele in a stutter position must be used for post-transplant analysis, contribution from the stutter peak is compensated for by subtraction of a percentage of the main peak area.…”

“…STRs, which are standard tools for genotyping in parentage testing 6 and forensic human identity testing, 7 have recently been used for the quantitative analysis of BMT engraftment. [8][9][10][11] STRs provide an excellent tool for this purpose because of their high degree of polymorphism and relatively short length.…”