Jonathan Whitaker

An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA

By increasing the PCR amplification regime to 34 cycles, we have demonstrated that it is possible routinely to analyse <100 pg DNA. The success rate was not improved (without impairing quality) by increasing cycle number further. Compared to amplification of 1 ng DNA at 28 cycles, it was shown that increased imbalance of heterozygotes occurred, along with an increase in the size (peak area) of stutters. The analysis of mixtures by peak area measurement becomes increasingly difficult as the sample size is reduced. Laboratory-based contamination cannot be completely avoided, even when analysis is carried out under stringent conditions of cleanliness. A set of guidelines that utilises duplication of results to interpret profiles originating from picogram levels of DNA is introduced. We demonstrate that the duplication guideline is robust by applying a statistical theory that models three key parameters - namely the incidence of allele drop-out, laboratory contamination and stutter. The advantage of the model is that the critical levels for each parameter can be calculated. This information may be used (for example) to determine levels of contamination that can be tolerated within the strategy employed. In addition we demonstrate that interpreting one banded loci, where allele dropout could have occurred, using LR=1/2f(a) was conservative provided that the band was low in peak area. Furthermore, we demonstrate that an apparent mis-match between crime-stain and a suspect DNA profile does not necessarily result in an exclusion. The method used is complex, yet can be converted into an expert system. We envisage this to be the next step.

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.

Analysis and interpretation of mixed forensic stains using DNA STR profiling

The use of multiplex PCR and fluorescent dye technology in the automated detection and analysis of short tandem repeat loci provides not only qualitative information about the profile--i.e. which alleles are present--but can also provide quantitative information on the relative intensities of the bands, and is therefore a measure of the amount of amplified DNA. The availability of this quantitative information allows for the interpretation of mixtures in a detailed way which has not been previously possible with many other human identification systems. In this paper we present a simple approach to the resolution and analysis of mixed STR profiles resulting from the testing of mixed biological stains in forensic casework and highlight factors which can affect it. This approach requires a detailed knowledge--gained through a mixture of experiments and validation studies--of the behaviour of each locus within the multiplex systems described. We summarise the available data from previously published experimental work and validation studies to examine the general principles underlying this approach.

A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis.

DNA STR profiles have been generated from 1 ng and low copy number (LCN) templates using 28 and 34 cycles of amplification, respectively. Characteristics which facilitate the interpretation of profiles, such as heterozygous balance, allelic dropout and stutter proportions have been quantified. We demonstrate that a reduction in DNA template coupled with an increase in amplification cycle number produces an increased rate of allelic dropout out which can be correlated to the peak areas of those alleles observed. In addition, the LCN conditions increase the degree of peak area asymmetry observed from heterozygotes and the size range of stutters. Analysis of the data allows us to develop sets of guidelines appropriate for interpreting both single and mixed DNA profiles.

Interpreting simple STR mixtures using allele peak areas

Although existing statistical models can interpret mixtures qualitatively based upon the alleles present, the use of automated sequencers opens the opportunity to take account of quantitative aspects embodied by the peak area. One step in understanding simple mixtures consisting of just two donors is to estimate the mixture ratio. This is relatively easy to do when four-allele mixtures are evident at a given locus. However, if the mixture consists of three or fewer alleles, the process it is not straightforward. We demonstrate that mixture estimates are consistent across all loci in a multiplex system. Once the mixture ratio is known, then the expected peak areas for any given combination of alleles can be estimated using a simple spreadsheet 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.

Response to low level DNA profiling

Dear Sir/Madam, DNA reviews: low level DNA profiling For. Sci. Med. Pathol. (2008) 4: 129-131. E. A. M. Graham. (1) The low copy number (LCN) technique as used by the Forensic Science Service (FSS) has been used in casework since 1999. The FSS has always engaged in a rigorous programme of research, development, and validation of any DNA profiling technique it uses which supplies DNA profile results to the Criminal Justice System (CJS). The LCN DNA profile technique was no exception to this process and its implementation into forensic casework followed the same process as other previous DNA profiling tests. Nevertheless in 2006 the validation of the technique came under scrutiny in the case of R-v-Hoey in relation to the Omagh bomb enquiry and other linked explosive devices in Northern Ireland. Even prior to the verdict and the judgement in this case, the Home Office had ordered an independent review of the science underlying the LCN approach. On April 11, 2008, the office of the UK Forensic Regulator (a department of the Home Office) issued a detailed independent review of the adequacy of the DNA LCN technique, which was produced by Professor Brian Caddy, Graham R. Taylor, and Dr Adrian M. T. Linacre. This review reported that the science supporting the delivery of low template DNA (LtDNA) analysis was sound, and that the three companies providing this service to the Criminal Justice System (the Forensic Science Service Ltd, LGC Forensics, and Orchid Cellmark Ltd) have validated their processes in accord with accepted scientific principles using both 28 (standard) and 34 (LCN) PCR cycles for extracts containing less than 200 pg of DNA. On May 7, 2008 the Forensic Regulator, Andrew Rennison, published a response to this review. The Regulator concludes: