Andy Urquhart

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.

Variation in Short Tandem Repeat sequences —a survey of twelve microsatellite loci for use as forensic identification markers

Alleles at 12 Short Tandem Repeat loci have been sequenced to investigate candidate loci for a multiplex Short Tandem Repeat system for forensic identification, and for single-locus amplification of Short Tandem Repeat loci. Variation from the consensus sequence was found at 6 loci, while one locus, D21S11, was found to be complex in sequence. The presence of non-consensus alleles does not rule out loci for inclusion as forensic identification markers, but size differences between alleles of 1 base pair require very precise sizing. We suggest criteria for the suitability of Short Tandem Repeat loci as forensic identification markers, and propose a universal allele nomenclature for simple and compound Short Tandem Repeats. The effect of the repeat unit sequence of the evolution of Short Tandem Repeats is discussed.ZusammenfassungAllele an 12 Short-Tandem-Repeat Loci wurden sequenziert, um Kandidaten für ein Multiplex Short Tandem Repeat System für forensische Identifikationen und für Single-Locus Amplifikationen von Short-Tandem-Repeat Loci zu untersuchen. Abweichungen von der Konsensus-Sequenz wurden an 6 Loci gefunden, während ein Locus, D21S11, als Komplex in der Sequenz gefunden wurde. Die Anwesenheit von Non-Konsensus-Allelen schließt solche Loci nicht aus für die Einbeziehung als forensische Identifikationsmarker. Aber Größendifferenzen von einem Basenpaar zwischen Allelen erfordern eine sehr genaue Größenbestimmung. Wir empfehlen Kriterien für die Eignung von Short-Tandem-Repeat Loci als forensische Identifikationsmarker und schlagen eine universale Allelnomenklatur für einfache und komplexe Short-Tandem-Repeats vor. Die Auswirkung der Sequenz der Repeateinheit auf die Entwicklung von Short-Tandem-Repeats wird diskutiert.

A new method of STR interpretation using inferential logic -development of a criminal intelligence database

Abstract A short tandem repeat (STR) system consisting of seven multiplexed loci has recently been introduced in the UK to support a National strategy to create large DNA databases for criminal intelligence purposes. The process uses automated sequencers, employing dye-labelled primers. Identification of tetrameric loci such as HUMTH01 are straightforward. Sizing windows are estimated by running a series of control allelic ladders on several gels and ‘unknown’ samples are designated if they fall within a defined window. However, utilisation of complex STRs (eg. D21 S 11) characteristically have common variants which differ by just 2 bp. In addition, rare alleles are encountered which may differ by just 1 by from a common variant. To assist with the identification of alleles, we have introduced a series of allelic ladders, so that direct comparisons with ‘unknown’ samples can be made on the same gel. To designate an allele, it should be within 0.5 by of an allelic ladder marker. Not all alleles (in particular rare alleles) can be included within an allelic ladder, however their expected positions can be easily calculated by reference to existing alleles in the ladder. Measurement of band shift is also a useful diagnostic tool. A series of guidelines are described to enable reliable allelic identification. These guidelines can be converted into computer programmes, which form the basis of an expert system.

The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artefacts, casework studies and success rates.

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

A genetic basis for anomalous band patterns encountered during DNA STR profiling.

Since 1995 the Forensic Science Service (FSS) has carried out DNA profiling of reference samples for the UK National DNA Database and in forensic casework using two multiplex STR profiling systems. During this period, profiles with anomalous banding patterns, although comparatively rare, have been encountered regularly. The FSS has collected instances of triallelic patterns and aberrant diallelic patterns. A systematic examination of these patterns has provided insight into their underlying genetic cause. The triallelic patterns could be classified into two types based on the relative intensities of their component alleles. In the Type 1 pattern the alleles were of uneven intensity, whereas in the Type 2 pattern, all three alleles were of even intensity. Evidence is presented that the more frequent Type 1 pattern is the result of somatic mutation at a heterozygous locus, and the Type 2 pattern is the result of a localized chromosomal rearrangement at a heterozygous locus. Directly from the Type 1 pattern, it was possible to deduce the size difference between the progenitor and mutated allele. All mutational changes were found to be multiples of four nucleotides, suggesting the loss or addition of one or more tetrameric repeat units. Aberrant diallelic patterns were identified by analysts due to an unexpectedly large difference in intensity between alleles at a heterozygous locus. While some of these diallelic patterns are likely caused by the same genetic phenomena described above occurring at a homozygous locus, others are demonstrated to be caused by a mutation in the primer binding sequence, leading to a reduction in amplification efficiency of one allele. It is concluded that based on a visual inspection of a profile, it is possible to infer a likely genetic basis directly from the triallelic pattern. By contrast, the aberrant diallelic patterns can be due to any one of a number of possible genetic effects.

Sequence variability of the tetranucleotide repeat of the human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) locus.

Sequence analysis of polymerase chain reaction (PCR) amplified products from the presumed tetranucleotide repeat at the human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) locus shows far greater variability in both PCR product length and sequence than has been previously reported. Alleles differing in size by 1 bp exist, and accurate sizing is required if the locus is to be used to its full potential.

New reference allelic ladders to improve allelic designation in a multiplex STR system

Abstract This paper reports the composition of a new reference allelic ladder mixture for use with a multiplex DNA profiling system consisting of six short tandem repeat loci. The loci included in this mixture are HUMTH01, D21S11, D18S51, D8S1179, HUMVWAF31/A, HUMFIBRA/FGA and an amelogenin sex test. Sequence analysis of individual ladder alleles was carried out and allelic designations made in accordance with the recommendations of the International Society of Forensic Haemogenetics (1992; 1994). A series of rare alleles which increase the range of alleles previously reported were identified. By including some of the rare alleles into the ladder marker system, we have significantly improved the ability to identify new alleles in unknown samples.

Selection of STR loci for forensic identification systems

Short Tandem Repeat (STR) profiling is rapidly growing as a method of individual identification for forensic and other purposes. Several multiplex STR systems are available (e.g. Kimpton et al, 1993), offering matching probabilities of about 10-4. The quadruplex STR system developed in our laboratory and presently in use in forensic casework gives a matching probability of this order in three British populations (Kimpton et al, 1993). We have investigated numerous STR loci for use in further multiplex STR systems. Here we discuss the criteria for locus selection, with particular reference to the repeat sequence at the loci under investigation.