James W. Schumm

Development and Population Study of an Eight-Locus Short Tandem Repeat (STR) Multiplex System

Amplification of short tandem repeat (STR) loci has become a useful tool for human identification applications. To improve throughput and efficiency for such uses, the polymorphic STR loci CSF1PO, TPOX, TH01, vWA, D16S539, D7S820, D13S317, D5S818, F13A01, FESFPS, F13B, and LPL have been evaluated, developed, and configured into fluorescently labeled multiplex systems. Eight of these STR loci were combined to generate the PowerPlex System, a two-color multiplex system that supports rapid, accurate, reliable analysis and designation of alleles. The remaining four loci comprise the FFFL System, a one-color multiplex system. The PowerPlex System may be evaluated alternatively as two one-color, four-locus multiplex systems, CTTv Multiplex and GammaSTR Multiplex. The products of multiplex amplification may be analyzed with a variety of fluorescence detection instruments. Determination of genotypes of over 200 individuals from each of three different population/ethnic groups revealed independence of inheritance of the loci and allowed calculation of matching probability, typical paternity index, and power of exclusion for each multiplex.

Chinese population data on three tetrameric short tandem repeat loci--HUMTHO1, TPOX, and CSF1PO--derived using multiplex PCR and manual typing.

Allele and genotype frequencies for three tetrameric short tandem repeat (STR) loci were determined in a Chinese sample population using multiplex polymerase chain reaction (PCR), electrophoresis of the PCR products in DNA sequencing gels and subsequent detection by silver staining. The loci are HUMTHO1, TPOX, and CSF1PO. All loci meet Hardy-Weinberg expectations. In addition, there is no evidence for association of alleles among the three loci. The allelic frequency data can be used in human identity testing to estimate the frequency of a multiple STR locus DNA profile in the Chinese population.

Validation and Implementation of the PowerPlex® 16 BIO System STR Multiplex for Forensic Casework

The PowerPlex 16 BIO multiplex short tandem repeat (STR) system contains the 13 CODIS loci (FGA, TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, CSF1PO, D16S539, D7S820, D13S317, and DS5S818), plus two pentanucleotide repeat loci (Penta D and Penta E) and the sex-identifying locus. Amelogenin. The PowerPlex 16 BIO System is optimized for use with the Hitachi FMBIO gel imaging systems. A consortium of seven independent laboratories collaborated to perform the studies defined by the FBI standards for performing a developmental validation, including the evaluation of sample concordance, percent stutter determination, nonprobative casework, precision, sensitivity, mixture determination, effect of substrates, the impact of environmental insults, and species specificity. All samples tested for concordance were consistent except for one sample from the Virginia Division of Forensic Science database that displayed discordance at D13S317, a locus whose primer sequence was altered. Stutter values were comparable to those of other STR multiplex systems, the precision was comparable to other multiplexes analyzed by gel electrophoresis, the DNA profiles were unchanged by the substrate upon which the blood samples were placed, and the nonprobative casework samples re-typed for the PowerPlex 16 BIO System were consistent with previous typing results. When greater than 0.125 ng of DNA was placed into the PowerPlex 16 BIO System amplification reaction, a full profile was generated by all laboratories. The mixture study results were comparable to those reported for other multiplex systems, the environmental study demonstrated a loss of larger molecular weight loci when samples were incubated at elevated temperatures for a prolonged period of time, and the only notable cross species hybridization was observed with primate DNA samples. This extensive validation work performed demonstrates that the PowerPlex 16 BIO System provides STR data of a quality comparable with other PowerPlex STR multiplex kits as well as other widely used STR multiplexes and is thus suitable for evidentiary casework analysis as well as database sample profiling.

Validation of Multiplex Polymorphic STR Amplification Sets Developed for Personal Identification Applications

Polymorphic short tandem repeat (STR) loci, which typically consist of variations in the number of 3-7 base pair repeats present at a site, provide an effective means of personal identification. Typing can be accomplished by amplification of genomic DNA using the polymerase chain reaction (PCR) and locus-specific primers, separation of amplified alleles using gel electrophoresis and their display using silver staining or fluorescent detection. Primers for several STR loci can be combined in a single multiplex reaction so typing of multiple loci can be accomplished rapidly and with less DNA than required if each locus were analyzed separately. Before such muliplex systems are used in forensic or paternity applications, it is desirable that they undergo testing for their reliability. This study evaluates the performance of two STR triplex systems, one containing the loci HUMCSF1PO, HUMTPOX, and HUMTH01, and the other containing HUMHPRTB, HUMFESFPS, and HUMVWFA31. Protocols for amplification of these two triplexes, and their corresponding monoplexes, were evaluated for sensitivity of detection, resistance to changes in the annealing temperature of the amplification protocol, and the ability to identify the minority contributor in amplification of mixed samples. In addition, five laboratories determined the alleles of twenty DNA samples, each extracted by one of four different extraction methods. The results illustrate that the two STR triplex systems and the monoplex systems contained within them can be used with as little as 0.25 ng of DNA template. Both triplexes amplified with 100% success using the Perkin Elmer Model 480 thermal cycler. With the GeneAmp 9600 System, the CTT triplex amplified with 100% success and the HFv triplex in 95.6% of attempts. These experiments meet many requirements for use in validation of DNA typing systems for forensic cases and paternity identification.

Investigation of Species Specificity Using Nine PCR-Based Human STR Systems

Several eukaryotic genomes contain polymorphic markers consisting of trimeric and tetrameric short tandem repeats (STR). Recent reports have demonstrated the variability of short tandem repeat (STR) polymorphisms at a variety of loci among several human population groups. Currently, there are nine commercially available STR PCR systems from Promega Corporation that may be utilized for human identification. We report here the analysis of 23 different species DNAs using these nine STR primer systems to assess their specificity for human euchromatin. The STR systems tested include, CSF1PO, TPOX, THO1, HPRTB, FESFPS, vWF and F13A01 as single systems and as triplex systems (CSF1PO/TPOX/THO1 and HPRTB/FESFPS/vWF). There were no STR PCR products observed for seventeen of the twenty-three species regardless of the STR system. Amplified STR fragments were detected in rhesus DNA for CSF1PO, TPOX and HPRTB systems. STR PCR products were detected for human, gorilla, chimpanzee, and orangutan DNAs using eight of the nine systems. FESFPS primers did not amplify DNA fragments from any of the species tested. Most of the STR PCR products detected from primate DNAs electrophoretically migrated outside of the human allelic ladder fragments and as a result, allele designations were not possible.

Allele Frequencies for Fourteen STR Loci of the PowerPlex™ 1.1 and 2.1 Multiplex Systems and Penta D Locus In Caucasians, African-Americans, Hospanics, and Other Populations of the United States of America and Brazil

Allele Frequencies for Fourteen STR Loci of the PowerPlex™ 1.1 and 2.1 Multiplex Systems and Penta D Locus In Caucasians, African-Americans, Hospanics, and Other Populations of the United States of America and Brazil

Detection of a primer-binding site polymorphism for the STR locus D16S539 using the Powerplex 1.1 system and validation of a degenerate primer to correct for the polymorphism.

Quality assurance samples submitted from the NCSBI as part of a contract with TBTG to outsource DNA Database samples showed unexpected discrepancies for the locus D16S539 when all other loci yielded identical results. Discrepancies observed included allele drop out and an imbalance in sister alleles with samples returned from TBTG. This led to a comprehensive review of the technical procedures used between the two laboratories to determine the cause of the discrepancies noted for the locus D16S539, since both laboratories were using the PowerPlex 1.1 typing kit from the Promega Corporation. The NCSBI and the TBTG utilize different extraction methods (organic extraction vs. FTA) and amplification conditions (AmpliTaq vs AmpliTaq Gold), respectively, so the exact cause of discrepancy observed was not immediately apparent. Experiments at the NCSBI associated the observed allele drop out and the imbalance of the sister alleles with the use of AmpliTaq Gold and a hot start procedure. Sequencing data revealed that a point mutation resides on the D16S539 primer-binding site that reaches polymorphic levels in African-American populations. This led to the development of a degenerate primer by the Promega Corporation to detect missing alleles when AmpliTaq Gold is used. The degenerate primer was then thoroughly tested to show its efficacy in detecting the true D16S539 profile when used.

TWGDAM VALIDATION OF A NINE-LOCUS AND A FOUR-LOCUS FLUORESCENT STR MULTIPLEX SYSTEM

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.

Buccal DNA samples for DNA typing: new collection and processing methods

With the recent expansion of DNA database laws in many states, there is a critical need for the rapid and simple collection of DNA samples and streamlined processing for downstream applications. The Buccal DNA Collector was developed to address the need for a reliable, practical alternative to blood collection that is compatible with high-throughput operations. The collection area consists of filter paper that is placed against the inside of the cheek, and the sample is taken by swiping the cheek several times while pulling the device out of the mouth. Using this method, DNA profiles have been obtained from samples stored for 2 years at room temperature. Cells are collected on all regions of the filter paper with the maximum DNA recovery from the tip. The processing of DNA for DNA typing is accomplished with BodeElute, a new product that prepares DNA for amplification in a single 30-min heating step. Extracted DNA samples were successfully amplified with four commonly used multiplex short tandem repeat (STR) amplification kits. These products provide simplified approaches for collecting and processing buccal cell samples.

An oligonucleotide probe to assay lysis and DNA hybridization of a diverse set of bacteria.

A computer bank of 16 S rRNA bacterial sequences was searched to determine a consensus sequence expected to hybridize with DNA from a wide variety of bacteria. An oligonucleotide probe, named a panprobe, containing this sequence was used to assay the degree of lysis of bacterial colonies on filter paper heated in a microwave oven and subsequently treated with NaOH. As determined by colony hybridization with the panprobe, lysis was achieved for 51 of 59 different species of bacteria tested. DNA, isolated from the eight bacteria not detected by colony hybridization, did hybridize with the panprobe in slot blot hybridizations.