Validation of the Applied Biosystems Prism 377 automated sequencer for the forensic short tandem repeat analysis.

The Applied Biosystems (ABI) Prism 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well-to-read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51 degrees C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software. Complete resolution of 1 base differences was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2-fold higher than the 373A and the sensitivity was increased by one third.

Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification.

Short tandem repeat (STR) loci are routinely employed for individual identification. WE have examined the performance and reproducibility of a highly informative co-amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50-500 pg, partial profiles were produced, but no allelic drop-out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop-out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.

Automated short tandem repeat (STR) analysis in forensic casework--a strategy for the future.

Short tandem repeat (STR) loci are routinely analysed for forensic purposes in the UK. Because small regions of DNa are amplified, successful results are more likely to be obtained from highly degraded material where the DNA fragment length may be < 500 bp. The method is superceeding conventional analysis with single locus probes (SLPs). Dimeric STR loci display stutter artefacts, hence STRs used in casework are restricted to tri or tetrameric loci. Some STRs are complex repeats and have more alleles than simple repeats - for example the locus D21S11 has 21 alleles which differ in size by 2 bp because of the presence/absence of a hexanucleotide within the block of tetrameric repeats. These loci are of great potential interest because they combine increased discriminating power with reduced potential to stutter. Multiplexing 4 different loci with different dye labelled primers (i.e. carrying out polymerase chain reaction of 4 loci simultaneously) using the ABD 373A automated sequencer enables a large numbers of samples to be processed. In addition data aquisition and manipulation is automated so that minimum postelectrophoresis operator input is required. It is our aim to develop a system equivalent in power to that of 4 single locus probes. To achieve this we have developed an octoplex system consisting of 7 loci and a sex test (amelogenin locus) which has a probability of chance of association of 10(-9); the power of this system is equivalent to that achieved by 4 conventional SLPs.

Short tandem repeat typing of bodies from a mass disaster: high success rate and characteristic amplification patterns in highly degraded samples.

We have used a PCR-based DNA-typing method, involving the coamplification of four tetrameric short tandem repeat loci, in the analysis of a large number of severely degraded tissue samples taken from the scene of a mass disaster in which bodies were exposed to extreme thermal, physical and chemical insult. Analysis of the amplified DNA in a number of the samples revealed uniquely sized artifact PCR products resulting from the amplification of degraded genomic DNA as well as characteristic patterns in the amounts of PCR products generated from differently sized loci. This system has proved to be very reliable and robust, and we were successful in typing all of the four loci in 66% of the samples tested and at least one locus in 83% of the cases. A PCR-based sex test also proved to be very effective when applied to the degraded samples.

A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification.

Through the use of fluorescence-based polymerase chain reaction systems, a highly discriminating multiplex with the potential for individual identification has been developed. The use of multiple dye technology enabling loci with overlapping size ranges to be co-amplified has enabled us to successfully amplify seven tetranucleotide short tandem repeat loci within a single reaction resulting in a discriminating power in the region of 1 x 10(9). Three out of the seven loci employed exhibit alleles differing in size by only 2 bp as opposed to the conventional 4 bp, which results in such loci being more powerful in terms of distinguishing between samples, particularly when co-amplified in this manner. The size ranges of the loci contained within the system are such that windows still exist for the inclusion of additional loci at a later stage, which could increase the discriminating power of the system still further. In addition, further weight and utility is lent to the system through the incorporation of a simple and reliable sex test involving the amplification of a segment of the X-Y homologous gene Amelogenin.

Automated DNA profiling employing multiplex amplification of short tandem repeat loci.

We have employed automated fluorescence-based technology to detect amplified tri-, tetra-, and pentanucleotide short tandem repeat (STR) loci electrophoresed on denaturing polyacrylamide sequencing gels. The system described incorporates an internal size standard in each sample, allowing the STR-PCR products to be sized automatically with a high degree of precision. By utilizing different fluorescent dye markers for loci that have overlapping allele size ranges, we have developed three multiplex STR systems containing a total of 14 different loci. These multiplex systems were then used to evaluate the usefulness of the 14 loci for the identification of individuals. Allele frequency data were collected from a minimum of 50 individuals from each of three different racial groups: Caucasians, Afro-Caribbeans, and Asians. Of the resulting 42 locus population sets, deviation from Hardy-Weinberg equilibria was detected in only the STR HUMCYARO3-Caucasian data. The probabilities of two unrelated individuals matching by chance (pM) at all 14 loci in the three multiplex reactions was < 1 x 10(14). The combination of multiplex STR-PCR and automatic fluorescence-based detection is thus a rapid and powerful technique for individual identification.

Population genetics of four hypervariable loci.

Populations of white Caucasians, Afro-Caribbeans and Asians residing within the UK have been analysed at 4 different hypervariable loci. A computerised system was used to store and to analyse the data. Simulation experiments were carried out in order to determine whether there was any evidence for population stratification, which would lead to non-independence of allelic distributions.

Population genetics of four hypervariable loci

Populations of white Caucasians, Afro-Caribbeans and Asians residing within the UK have been analysed at 4 different hypervariable loci. A computerised system was used to store and to analysed the data. Simulation experiments were carried out in order to determine whether there was any evidence for population stratification, which would lead to non-independence of allelic distributions. (AU)