Colin P. Kimpton

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.

The validation of short tandem repeat (STR) loci for use in forensic casework.

A quadruplex reaction has been developed which amplifies the short tandem repeat (STR) loci HUMVWA31/A, HUMTHO1, HUMF13A1 and HUMFES/FPS. Detection of the PCR products employs denaturing poly-acrylamide gels coupled with fluorescent-based technology. This system has been evaluated for use in routine forensic casework and has been shown to be both robust and reproducible. The quadruplex reaction is as sensitive as the commercially available HLA DQα Amplitype typing system and can be used on both degraded and aged material. The problems of environmental contamination have been shown to be limited provided strict procedural practices are followed — i.e. physical separation of sample extraction and amplified products; the use of dedicated equipment such as pipettes; the separation of amplification preparation area. The ability of the system to detect mixtures and the successful analysis of case stains has shown that this system is well suited as a tool for forensic investigation.ZusammenfassungEine Quadruplex-Reaktion wurde entwickelt, die die Short tandem repeat loci (STR) HUMVWA31/1, HUMTHO1, HUMF13A1 und HUMFES/FPS amplifiziert. Zum Nachweis der PCR-Produkte werden denaturierende Polyacrylamid-Gele benutzt, in Kombination mit einer Fluoreszenz-basierten Technologie. Dieses System wurde fÜr den Gebrauch in der routinemäßigen Fallbearbeitung evaluiert und hat sich als robust und reproduzierbar erwiesen. Die Quadruplex-Reaktion ist genau so empfindlich wie das kommerzielle HLADQα-Ampli-type Typisierungssystem und kann sowohl an degradiertem als auch an gealtertem Material verwendet werden. Die Probleme einer umgebungsbedingten Kontamination haben sich als begrenzt erwiesen, vorausgesetzt, daß strikte prozedurale Praktiken benutzt werden, d. h. physische Trennung der Extraktion und der amplifizierten Produkte-, Benutzung persönlicher Einrichtungen, wie Pipetten; Abtrennung des Bereichs der Amplifikationsvorbereitung; Benutzung der Laminar Flow-Einrichtung für die Vorbereitung der Amplifikation. Die FÄhigkeit des Systems, Mischungen zu erkennen und die erfolgreiche Analyse von Spurenfällen haben gezeigt, daß dieses System gut geeignet ist als ein Werkzeug für forensische Untersuchung.

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups.

Development of guidelines to designate alleles using an STR multiplex system

We have carried out extensive validation of a multiplex system of 6 STR loci and the Amelogenin sex test to investigate the following characteristics: relative peak areas of heterozygote peaks; non-specific artefacts; stutters caused by slippage of the Taq enzyme; pull-up (an electronic artefact often associated with software). The purpose of this paper is to demonstrate how interpretation guidelines can be formulated by experimentation and analysis using casework samples. Automation allows data relating to band position (in base pairs), peak area and peak height to be easily collected and manipulated in spreadsheets or computer programs. This paves the way to designing expert systems to carry out the interpretative process.

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.

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.

STATISTICAL ANALYSIS OF DATA FOR THREE BRITISH ETHNIC GROUPS FROM A NEW STR MULTIPLEX

Data have been collected from 602 Caucasians, 190 Afro-Caribbeans and 257 Asians of Indo/Pakistani descent who have been profiled using a new six locus short tandem repeat (STR) multiplex. The data have been analysed by conventional significance testing methods: the exact test, homozygosity, and conventional goodness of fit to Hardy-Weinberg proportions. Frequency tables are given and the expected performance in British forensic casework is discussed.

High-throughput analysis using AmpFlSTR® Identifiler® with the Applied Biosystems 3500xl Genetic Analyser

High throughput analysis of buccal scrape reference samples using the Identifiler system on the Applied Biosystems 3500xl Genetic Analyser is described. This platform is much more sensitive than previous platforms, e.g. 3130xl. The range of signal detection is also much greater and this means that the system is more tolerant to a wide range of input template concentrations. DNA quantity is not a limiting factor in the analysis of buccal scrapes, hence the entire analytical procedure (process) was designed around a target input of 1.5ng, to minimise low level DNA profiles and associated events of allele dropout. A universal stochastic threshold/limit of detection (LOD) of 300relative fluorescent units (rfu) was applied across all loci. This level is twice that of comparable systems such as SGM Plus analysed on 3130xl instruments. After analysis of dropout probabilities, heterozygous balance, drop-in and stutter characteristics, rule-sets were programmed into the FSS i-cubed software. The process has a success rate >95%. No discordances or mis-designations occurred when applied to a validation set of more than 1000 samples.