Low Copy Number DNA Research Articles

How to evaluate PCR assays for the detection of low-level DNA.

High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real-time PCR-based detection of low-level DNA. The model was tested against experimental data of diluted cell-free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell-free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real-time PCR-based detection of low-level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.

Generating Human DNA Profile(s) from Cell Phones for Forensic Investigation

The goals of this study were to investigate whether sufficient amount of human DNA can be recovered from a cell phone and if the identity of the single and/or multiple user(s) can be established by generating genetic profile(s). Cell phones were used to collect human DNA. Two Buccal swabs were used to collect Buccal cells as a DNA reference samples. Four soaked sterile cotton swabs were used to collect biological materials from each user’s cell phone. Sufficient quantity and quality of human genomic DNA was obtained from the cell phones using Quantifiler® Human DNA Quantitation Kit. More human DNA was collected from volunteers’ Buccal swabs (=74.56 ng) than from the surface of their cell phones (=0.23 ng). One nanogram (1 ng) of template DNA was used for buccal swabs and cell phones swabs, if available. However, when the DNA recovered from cell phone swabs was 0.00 to 500 pg, 10 μL of template DNA was used. In addition, the number of PCR cycles was increased from 28 to 32 in anticipation of Low Copy Number DNA. Genetic profiles were developed from the DNA on the cell phones and were compared with the reference DNA profiles from the buccal cells of the cell phone owners. Full Short Tandem Repeats (STR) DNA profiles for all 15 STR loci plus Amelogenin locus were typed for 36 Buccal swabs, reference DNA of cell phone owners. Complete STR profiles were generated from 33% (N=36) cell phones and were matched participants’. In addition, 28% of cell phones had partial DNA profiles matched with the cell phone owners. While, 33% of the cell phones had unknown partial or complete profiles. Four of the unknown partial profiles had more than two alleles at the STR loci indicating multiple unknown cell phone users. The Y chromosome was detected at the Amelogenin locus from the DNA profiles generated from two females’ cell phones identifying unknown male contributor(s). Our study demonstrates that DNA collected from the cell phones can be used as reliable physical evidence in forensic investigations.

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

Impacts of carbon nanotubes on biochemical reactions: insight into interaction between carbon nanotubes and DNA polymerase enzyme

Recently, the Polymerase Chain Reaction technique has begun to benefit from nanotechnology. In this paper, effects of carbon nanotubes in the Polymerase Chain Reaction were investigated by Electrophoresis, Circular Dichroism Spectrometry and Dynamic Light Scattering Techniques. The unique ability to amplify low copy number DNA within minutes has made in vitro Polymerase Chain Reaction (PCR) one of the most essential techniques in modern biology. In order to harness this technique to its full potential, certain obstacles, such as nonspecific by-products, low yield, and complexity of GC rich and long genomic DNA amplification need to be surmounted. Nanomaterial-assisted PCR, so-called nanoPCR, is a new area in biotechnology that introduces nanostructured materials into PCR reaction to obtain improved results. Nanomaterials have unique physical and chemical properties, such as high thermal conductivity, stability and high surface to volume ratios. The effects of nanomaterials in PCR depend on their size, shape, concentration, heat conductivity, electron transfer properties and surface modifications. Carbon nanotubes are predicted to bind major PCR components, such as primers, template and polymerase enzyme, via specific or non-specific interactions. In this paper, we demonstrate the interaction of carbon nanotubes with wild type DNA polymerase enzyme, and the effect of this interaction in PCR for the first time. According to the results, chiral properties of the wild type DNApolymerase enzyme has changed after incubation with amine functionalized multiwall carbon nanotubes, which confirms direct interaction between the enzyme and tubes. Furthermore, this interaction has been found to be temperature dependent via dynamic light scattering spectroscopy.

Utility of amplification enhancers in low copy number DNA analysis.

One parameter that impacts the robustness and reliability of forensic DNA analyses is the amount of template DNA used in the polymerase chain reaction (PCR). With short tandem repeat (STR) typing, low copy number (LCN) DNA samples can present exaggerated stochastic effects during the PCR that result in heterozygote peak height imbalance, allele drop out, and increased stutter. Despite these effects, there has been little progress toward decreasing the formation of stutter products and heterozygote peak imbalance effects during PCR. In an attempt to develop a more robust system that is less refractory to stochastic effects, the PCR additives, betaine, DMSO, PEG, and PCRboost®, were investigated on low-quantity DNA samples. The effects of the additives were assessed by evaluating STR typing results. Of the four additives, the only positive effects were observed with betaine treatment. Betaine, at a final concentration of 1.25 mol/L, was found to improve the robustness of the amplification, specifically by decreasing stutter in a dual locus system. In contrast, the addition of 1.25 mol/L betaine to commercial STR amplification kits did not affect stutter ratios. However, the addition of betaine did lead to increased yield of PCR products in all commercial kits tested. The results support that betaine can improve amplification efficiency of LCN DNA samples.

The Successful Recovery of Low Copy Number and Degraded DNA from Bones Exposed to Seawater Suitable for Generating a DNA STR Profile

A universal method allowing for DNA profiling from bones exposed to seawater has not been reported yet. This study refers on the identification of a body immersed in seawater for 8months. The biological material for identification was the mandibular body, usually characterized by low success rates of DNA analysis. Initially, two extraction protocols were performed with negative results: one used for bones immersed in fresh water and a silica-column procedure. A third protocol was performed, which combined the extraction of a higher amount of bone powder, the use of multi-silica-based extraction columns followed by a concentration step. This protocol allowed to obtain low copy number DNA and to generate a 12-loci STR profile by combining conventional STR typing and mini-STR technologies. This protocol could be suitable when human bones have been exposed to severe environmental conditions, and the available nuclear DNA is highly degraded and in low copy number.

Reduction of stutter ratios in short tandem repeat loci typing of low copy number DNA samples

Increased height of stutter peaks is a phenomenon with low copy number (LCN) short tandem repeat (STR) typing that can impact interpretation. An alternative strategy of lowering the annealing/extension temperature (LT) at 56 °C was designed to attempt to decrease the heights of stutter peaks. STR typing results were generated in terms of stutter ratios using LT-PCR conditions and compared with data obtained using standard (STD) PCR conditions. DNA samples ranging from 100 to 25 pg were amplified using reagents contained in the AmpFℓSTR Identifiler PCR Amplification or AmpFℓSTR Identifiler Plus PCR Amplification kits with 32 or 34 PCR cycles. Stutter ratios decreased by an average of 14.7%, 14.9% and 18.1% at 100, 50 and 25 pg of template DNA under LT conditions compared with those of STD conditions in the Identifiler Kit amplified samples. The LT conditions also decreased average stutter ratios by 13.3% compared with those of STD conditions in the Identifiler Plus Kit amplified samples. The overall PCR efficiency obtained with STD and LT conditions with the two STR kits was comparable in terms of the number of detected alleles, peak heights and peak height ratios. These results support the hypothesis that a lower temperature annealing/extension step reduces the likelihood of slippage during PCR by enhancing the stability of the DNA polymerase/template DNA complex or the stability of the generated duplex than the conditions of the standard extension step. This stability in turn would result in lower stutter ratios.

Ultralocalized thermal reactions in subnanoliter droplets-in-air

Miniaturized laboratory-on-chip systems promise rapid, sensitive, and multiplexed detection of biological samples for medical diagnostics, drug discovery, and high-throughput screening. Within miniaturized laboratory-on-chips, static and dynamic droplets of fluids in different immiscible media have been used as individual vessels to perform biochemical reactions and confine the products. Approaches to perform localized heating of these individual subnanoliter droplets can allow for new applications that require parallel, time-, and space-multiplex reactions on a single integrated circuit. Our method positions droplets on an array of individual silicon microwave heaters on chip to precisely control the temperature of droplets-in-air, allowing us to perform biochemical reactions, including DNA melting and detection of single base mismatches. We also demonstrate that ssDNA probe molecules can be placed on heaters in solution, dried, and then rehydrated by ssDNA target molecules in droplets for hybridization and detection. This platform enables many applications in droplets including hybridization of low copy number DNA molecules, lysing of single cells, interrogation of ligand-receptor interactions, and rapid temperature cycling for amplification of DNA molecules.

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR® Identifiler® Plus for 28 cycles. Following amplification, samples were denatured at 99.9°C for 5min and incubated with one unit of DSN at 62°C in a 28μl volume for 1min. Nuclease activity was terminated through the addition of equal volume of 10mM EDTA and 95°C incubation for 2min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.

Whole genome amplification of degraded and nondegraded DNA for forensic purposes

Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to “recover” degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-012-0764-9) contains supplementary material, which is available to authorized users.

Visualizing Uncertainty: Anomalous Images in Science and Law

Challenges to the reliability of scientific evidence are increasingly common. Where DNA is concerned, this distrust has been most often associated with problems in forensic laboratories such as inconsistent quality standards, cross-contamination, sample mix-ups and even the falsification of results. Yet, the recent emphasis on the scientific analysis of low quantity DNA samples — referred to, variably, as degraded, low copy number (LCN) or trace DNA — has reoriented theoretical and analytical attention to the ‘visual’ as the privileged source of knowledge to discriminate these otherwise undetectable ‘non-conformities’. Paradoxically, the uncertainty rendered through the visual profile emerges as a result of the technological innovation purporting to improve results by increasing sensitivity and detection. This study traces uncertainty as it is exemplified through ambiguous visual constructs produced in the forensic laboratory, the translation into statistical probabilities, and the subsequent controversies such imagery has provoked in legal domains.

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

BackgroundThe consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification.MethodsA total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in.ResultsPerforming DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification.ConclusionsThe loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited.

Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system

A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used.This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out.The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

총기 흔적흔에서의 low copy number(LCN) DNA 검출에 관한 연구

유전자 감식은 다양한 범죄현장에서 발견되는 생체시료의 분석을 통해 신원을 식별하는 중요한 법과학적 수사과정으로 자리 잡았다. 최근에는 범인이 사용했던 펜, 뺑소니 차량에서의 핸들, 기어, 각종 버튼스위치 등에 남겨져 있는 touch evidence-type sample로 알려져 있는 low copy number (LCN) DNA에서의 A-STR분석을 위해 의뢰되는 감정물들이 증가하는 추세에 있다. 본 연구에서는 총기의 뭉개진 지문 등에 남겨져 있는 touch evidence-type의 LCN DNA를 추출하고 유전자형의 분석 성공률을 확인하고 자 하였다. 4종류의 총기(M16, K1A, COLT 45 권총, M29 리볼버)를 각각 격발한 후 총기별로 4곳의 부위에서 시료를 채취한 다음 LCN DNA의 추출을 위해 Microkit과 <TEX>$Prepfiler^{TM}$</TEX> 등 2종류의 시약을 이용하여 DNA 검출량과 유전자형 분석 성공률을 비교 분석하였다. 분석결과 <TEX>$Prepfiler^{TM}$</TEX>가 Microkit에 비해 평균 1.7배 DNA검출량이 많았으며, 유전자형 분석 성공률에 있어서도 Microkit은 0%인데 비해 <TEX>$Prepfiler^{TM}$</TEX>에서는 평균 24.9%의 성공률을 보였으며, K1A의 손잡이 부위에서 50.6%의 성공률을 나타냈다. Genetic Identification has become an important forensic investigation method which discerns identity through analysis of physical samples discovered in various crime scenes. Recently more samples are being requested to undergo A-STR analysis of low copy number (LCN) DNA, which is known as touch evidence-type sample and left on various objects such as a pen briefly used by the criminal, the gear of the car used for driving, the handle, and various buttons inside a car. This research attempted to extract the LCN DNA of the touch evidencetype left on crushed fingerprints on firearms, etc. and examine the genotyping success rate. Four types of firearms (M16, K1A, COLT 45 Pistol, M29 Revolver) were fired individually and physical samples were gathered from four parts of each firearm. Subsequently, in order to extract the LCN DNA, Microkit and <TEX>$Prepfiler^{TM}$</TEX> were used to compare and analyze the quantity of DNA extracted and the genotyping success rate. Analysis results showed that the quantity of DNA extracted by <TEX>$Prepfiler^{TM}$</TEX> was on average 1.7 times higher than that of Microkit, and in genotype analysis success rate <TEX>$Prepfiler^{TM}$</TEX> also demonstrated 24.9% on average in contrast to 0% for Microkit. In regards to the grip part of the K1A, <TEX>$Prepfiler^{TM}$</TEX>'s success rate was as high as 50.6%.

A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications

In forensic DNA profiling use is made of the well-known technique of PCR. When the amount of DNA is high, generally unambiguous profiles can be obtained, but for low copy number DNA stochastic effects can play a major role. In order to shed light on these stochastic effects, we present a simple model for the amplification process. According to the model, three possible things can happen to an individual single DNA strand in each complete cycle: successful amplification, no amplification, or amplification with the introduction of stutter. The model is developed in mathematical terms using a recursive approach: given the numbers of chains at a given cycle, the numbers in the next can be described using a multinomial probability distribution. A full set of recursive relations is derived for the expectations and (co)variances of the number of amplicon chains with no, 1 or 2 stutters. The exact mathematical solutions of this set are given, revealing the development of the expectations and (co)variances as function of the cycle number. The equations reveal that the expected number of amplicon chains without stutter grows exponentially with the cycle number, but for the chains with stutter the relation is more complex. The relative standard deviation on the numbers of chains (coefficient of variation) is inversely proportional to the square root of the expected number of DNA strands entering the amplification. As such, for high copy number DNA the stochastic effects can be ignored, but they play an important role at low concentrations. For the allelic peak, the coefficient of variation rapidly stabilizes after a few cycles, but for the chains with stutter the decrease is more slowly. Further, the ratio of the expected intensity of the stutter peak over that of the allelic peak increases linearly with the number of cycles. Stochastic models, like the one developed in the current paper, can be important in further developing interpretation rules in a Bayesian context.

Profiling the Dead: Generating Microsatellite Data from Fossil Bones of Extinct Megafauna—Protocols, Problems, and Prospects

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means by which the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the ‘shotgun’ reads, >60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36–70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600–5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research.

Simple method to prepare DNA templates from a slice of peanut cotyledonary tissue for Polymerase Chain Reaction

An efficient DNA extraction method was developed for peanut seed, where only 3-5 mg cotyledonary tissue was enough for more than 50 PCR reactions with a reaction volume of 15 μl. Both low copy number and high copy number DNA sequences were successfully amplified. Processing one seed sample only took about half an hour. Sampling had no significant effects on germination and development. The DNA extraction method makes it possible to identify transformants and conduct molecular marker studies prior to sowing, and thus may greatly hasten research progress.

Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpF lSTR ® SGM Plus™ multiplex

The characteristics of STR profiles produced from approximately 1 ng starting template using the AMPF lSTR ® SGM Plus™ multiplex and 28 PCR cycles, are well documented. However, the analysis of samples perceived as low in starting template (less than 100 pg), and referred to as low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the number of PCR amplification cycles from 28 to 34. This type of analysis has become known as low copy number, or LCN, DNA profiling. Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic ‘drop-in’ and allelic ‘drop-out’. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results. A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique. We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.

DcDegenerate Oligonucleotide Primed-PCR for Multilocus, Genome-wide Analysis From Limited Quantities of DNA

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples (<or= 0.100 ng). Experiments involved altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases. Increasing the degeneracy of the primer and the number of cycles that use a low annealing temperature should improve the nonspecific amplification of the DOP-PCR reaction. The addition of proofreading enzymes should allow for longer amplification products, increasing the genome coverage of the reaction. Low-input DNA quantities were examined for the primer and the cycle number studies using standard DOP-PCR parameters. The optimized DOP-PCR technique was then implemented for the polymerase study. All DOP-PCR products were amplified by using a multiplex microsatellite amplification kit to evaluate products from multiple chromosomes, followed by separation and detection by capillary electrophoresis. The 10 N primer, 12 nonspecific cycles, and the addition of the DeepVent proofreading enzyme all significantly increased the number of short tandem repeat alleles successfully amplified. All modifications also lowered the rate of allele drop-in, or sporadic additional allele occurrence, when compared with DOP-PCR results published earlier. Further, an average of > 0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results show that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples.

Review of low template DNA typing

Low copy number (LCN) DNA profiling has played a prominent part in the investigation of many high profile crimes. It has been accepted by a limited number of criminal justice systems throughout the world but due to issues with interpretation and reporting of results there have been questions raised about the technique. In the UK the method was suspended when its use in a trial was questioned, after DNA was found on explosive devices used in the detonation of a bomb that killed 29 people in a town centre. In particular the validation procedures leading to the introduction of LCN were at issue. LCN had also come under question in a second high profile case leading to a review being commissioned by the UK Home Office. The review had unprecedented access to the validation process that the three UK suppliers of low template DNA typing had performed prior to introducing the technique. The review published its findings and made 21 recommendations while recommending that the technique be reintroduced into the UK criminal justice system. This presentation will comment upon the background to the review, its findings and the future of low template DNA typing.