Gillian Tully

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces

We have shown that there is a difference between individuals in their tendency to deposit DNA on an item when it is touched. While a good DNA shedder may leave behind a full DNA profile immediately after hand washing, poor DNA shedders may only do so when their hands have not been washed for a period of 6h. We have also demonstrated that transfer of DNA from one individual (A) to another (B) and subsequently to an object is possible under specific laboratory conditions using the AMPFISTR SGM Plus multiplex at both 28 and 34 PCR cycles. This is a form of secondary transfer. If a 30 min or 1h delay was introduced before contact of individual B with the object then at 34 cycles a mixture of profiles from both individuals was recovered. We have also determined that the quantity and quality of DNA profiles recovered is dependent upon the particular individuals involved in the transfer process. The findings reported here are preliminary and further investigations are underway in order to further add to understanding of the issues of DNA transfer and persistence.

DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing.

Sequence analysis of human mitochondrial DNA (mtDNA) is being used widely to characterize forensic biological specimens, particularly when there is insufficient nuclear DNA in samples for typing. Hair shafts, bones, teeth and other samples that are severely decomposed may be subjected to mtDNA analysis, e.g. [1–5]. Although many of the quality assurance, quality control and interpretational guidelines used for PCR-based nuclear DNA analyses apply to mtDNA analysis, there are some features of mtDNA that warrant specific consideration: (1) mtDNA is maternally inherited; (2) heteroplasmy; and (3) the greater sensitivity of detection of mtDNA typing. It is imperative that guidelines consider the features of mtDNA and that practices do not exceed the state-of-knowledge on mtDNA. In a effort to refine previously published guidelines [6] and to assist those currently using mtDNA protocols and those considering implementing mtDNA analysis, the DNA Commission of the ISFG met on 16th August 1999 in San Francisco to develop current guidelines. The following are the recommendations by the DNA Commission on the use of mtDNA analysis.

Integrated Microfluidic System for Rapid Forensic DNA Analysis: Sample Collection to DNA Profile

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Genotype versus phenotype: Human pigmentation

The natural range of hair and skin colour is a continuous spectrum, controlled by multiple genes in a complex fashion. Many of these genes are as yet unknown, but several key pigmentation genes have been characterised, in particular the melanocortin 1 receptor gene (MC1R). Here, the function and known mutations of MC1R and other human pigmentation genes including ASIP, MATP, SLC24A5, TYR, TYRP1 and OCA2 are outlined, and a forensic test based on MC1R SNPs presented. The forensic utility of this and potential future genetic tests for phenotypic traits are discussed, in the light of the extensive debate on the ethics of predicting phenotypic traits from crime scene samples.

Analysis of 6 VNTR loci by ‘multiplex’ PCR and automated fluorescent detection

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to ‘allelic dropout’ was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.

An automated instrument for human STR identification: Design, characterization, and experimental validation

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab‐on‐a‐chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a “sample‐to‐profile” analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500™, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.

Use of low copy number DNA in forensic inference

Since January 1999, the Forensic Science Service has routinely carried out low copy number (LCN) DNA profiling in casework. To support this initiative, research has been carried out to discover the characteristics and limitations of LCN DNA by studying a series of well-defined evidence types, such as latent fingermarks, and by measuring the propensity of donors to deposit DNA onto objects that they have touched. D 2003 Elsevier Science B.V. All rights reserved.

Consideration of the probative value of single donor 15-plex STR profiles in UK populations and its presentation in UK courts

The adoption of new 15 locus STR multiplex systems into UK forensic science would be facilitated by agreed guidelines for reporting the strength of DNA evidence using likelihood ratios. To facilitate such an agreement, we present an analysis of previously published UK allele frequencies for white Caucasian, Afro-Caribbean and Indo-Pakistani populations and investigate their effect on likelihood ratios for single donor profiles. We consider the implication of the five additional loci and suggest a procedure for reporting likelihood ratios for 15-plex STR profiles.