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DNA isolation in sexual assault cases is complicated by the presence of large numbers of female epithelial cells, which are often in vast excess when compared to spermatozoa. The two-step differential extraction has been a standard method for isolating the spermatozoa; however, new techniques in forensic science allow the use of precision techniques for capturing spermatozoa. We have used an immuno-magnetic bead-based technique for sperm cell separation. We identified two antibodies that were specific to spermatozoa: SP17 polyclonal antibody and SP10 Intra Acrosomal Protein monoclonal antibody. These were conjugated to Dynabeads® M-450 Epoxy beads and used to isolate the spermatozoa in samples that exhibited the characteristic of sexual assault samples. Using these antibodies, we achieved separation of spermatozoa in the samples with sperm concentration 104/ml and 103/ml; STR analysis produced full male profiles. Mixed profiles were obtained with reduced levels of spermatozoa. Our preliminary data illustrate the potential to apply antibody-based capture to sexual assault cases.

Short tandem repeat (STR) profiling has been routinely used in kinship testing since the introduction of commercial kits in the mid-1990s. While 15 to 23 STR loci normally give definitive results in simple kinship testing, additional loci are sometimes required to resolve complex cases. The SureID 23comp Human Identification Kit, recently released by Health Gene Technologies (China), multiplexes amelogenin and 22 autosomal STRs, 17 of which are non-CODIS STRs. This enables the profiling of 38–40 loci when used in conjunction with widely used commercial kits. In this study, the kit was evaluated for kinship applications as a supplementary STR kit following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples. Performance was comparable with other commercial kits demonstrating: repeatability and reproducibility; precision (maximum s.d. 0.1048 nt); accuracy, all alleles were within ±0.41 nt compared to the actual sizes; heterozygous peak balances at all loci >68%; stutter ratios ranged from 3.8% to 16.15%; full profiles were generated with 125 pg DNA (95.12% of alleles at 62 pg),; and we found 100% concordance over 5 common STRs with the GlobalFiler kit.

Mitochondrial DNA analysis has earned its place in the field of genetics for providing a new window into understanding population ancestry. Its ability to produce results from minimal or decayed samples where nucleus DNA profiling proves are difficult is an additional benefit to forensic genetics. A total of 150 whole blood samples were collected on FTA paper, from Arabs population in United Arab Emirates, including inhabitants from rural regions. Both extraction of whole blood samples using PrepFiler® as well as direct amplification of mtDNA control region from purified 1.2 mm disc of FTA card stained with blood were attempted in this study. Quantity of the amplified control region was estimated using gel electrophoresis. Three sets of primers were used afterward to sequence purified products of amplified control region of mtDNA using Sanger sequencing method. 150 mtGenome sequences were obtained for UAE Arabs population, generated using MPS technology – Ion™ 5S. Concordance with control region sequences obtained using Sanger Sequencing approach was investigated. Resulted haplotypes were compared against worldwide mtDNA database (EMPOP) and estimated haplotypes frequency is shown in this study. As a forensic lab, non-probative challenging bone samples were tested to highlight the potentials value of using MPS technology – Ion™ 5S. This study shows the first mtGenome data generated for UAE Arabs population which helps extending the current available mtDNA control region database. As a result, this study shows great value of implementing MPS in the routine work in forensic genetics at Dubai Police.

A set of 87 reference samples collected from the population of Saudi Arabia were sequenced using the ForenSeq™DNA Signature Prep Kit on a MiSeq FGx™. The FASTQ files contain the sequences of the SE33 STR, but are not reported by the ForenSeq™ Universal Analysis Software (UAS). The STRait Razor software was used to recover and to report SE33 sequence‐based data for the Saudi population. Ninety-six sequence-based alleles were recovered, most of which had previously reported motif patterns. Two unreported motif patterns found in three alleles and seven novel allele sequences were reported. We also reported a single discordance between the sequence-based data and the CE data that was due to the presence of a common TTTT deletion. SE33 had 130% more sequence-based alleles; the highest number of observed sequence variants were in alleles 27.2 and 30.2, which each had 7 sequence variants. The statistical parameters emphasize the usefulness of using the sequence-based data.

The SNPforID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single-base-extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP-7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP-4™ on ABI 310 PRISM® and polymers POP-4™, POP-6™ and POP-7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP-6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP-7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.

Blowfly species of the family Calliphoridae can be used in forensic investigations to estimate the minimum post-mortem interval (PMI¬min). Lucilia caesar and Lucilia illustris (Diptera: Calliphoridae) are closely related and phenotypically similar, making reliable identification difficult, especially if specimens are in poor condition. To identify potential markers to genetically distinguish these species five complete mitochondrial genomes were sequenced: three for L. caesar (KM657111- KM657113) and two mitochondrial genomes for L. illustris (KM657109, KM 657110). The ND6 gene contained the most species-specific SNPs (1.71%), followed by the ND5 gene (1.68%) and then the COI gene (1.56%), identifying ND6 and ND5 as valuable loci for differentiating L. Caesar and L. illustris specimens.

Touch samples by their nature do not contain large amounts of biological material, especially compared to biological fluids such as blood, semen or saliva. Consequently, more careful recovery and storage are desirable in order to maximise the amount of DNA available. The main aim of the work presented is to determine whether detergent-based wetting agents increased DNA yield from touch samples when compared to water as a wetting agent. The results show that the use of the detergent-based lysis buffer led to greater DNA recovery from the fingerprints than when distilled water was used. The use of the lysis buffer in the touch DNA sample collection improved DNA recovery and stability over 24 h post-collection.

The commercial PowerPlex® Fusion kit is an autosomal STR multiplex kit that has high discrimination power and is more informative in forensic, paternity, and relationship-testing cases. Key features of this multiplex system are the possibility to direct amplify FTA™ card punches as well as non-FTA cards and commonly used swabs; optimised inhibitor tolerance and high sensitivity generating full profiles from amount as little as 100 pg of human DNA. This study focused on the optimization of performance variables such as FTA™ punch sizes, reduced reaction volumes, and FTA™ purification reagent aiming to increase the analytical sensitivity, decrease the sample consumption, and cost effectiveness. LOD and LOQ values demonstrated high sensitivity of the PowerPlex® Fusion system. In addition, population databases of Brunei Malay and Chinese from the Brunei Darussalam were established, and parameters of forensic importance were calculated. Overall, the forensic parameters indicated an enhanced utility of the PowerPlex® Fusion kit for forensic evidence analysis and paternity testing in Brunei Malay and Chinese populations.

When profiling a reference dataset of 500 DNA samples for the population of Saudi Arabia, using the GlobalFiler® PCR amplification kit, six unusual alleles were detected. At the SE33 locus, four novel alleles were found: 2, 14.3, 20.3, and 38; two alleles, at the D1S1656 locus: 7 and 8, had been previously reported, but no published sequence data was available. The D1S1656 alleles were sequenced using ForenSeq™ DNA Signature Prep with the MiSeq FGx System (Illumina, USA). As the SE33 is not reported by available Massively Parallel Sequencing (MPS) systems, samples that exhibited the unreported alleles were sequenced using BigDye™ Terminator v3.1 Cycle Sequencing Kit. Here we present the sequence and structure of the previously uncharacterized alleles.

This study profiled 1047 volunteers using the GlobalFiler™ Kit from the four main ethnic groups in Ghana: the Akan (282), Mole-Dagomba and Northern ethnic minority (253), Ewe (250) and Ga-Dangbe (262), representing about 98.4% ethnic coverage of the Ghanaian population. The polymorphic nature of the African population was demonstrated during the profiling of the reference dataset with the occurrence of some rare triplet variant alleles. At the TPOX locus, three tri-allelic variants were found with the allelic patterns 6-8-10, 8-9-10 and 9-10-11. In addition, tri-allelic patterns were seen at D5S818 (11-13-14) and D3S1358 (15-16-17). Although the TPOX tri-allelic pattern has been reported in other populations including African, the D5S818; 11-13-14 has never been reported in an African population. It has however, been reported once with a frequency of 1 in 69,600 in a convicted offender case in the USA. The D3S1358; 15-16-17 pattern has not been reported in an African population. In conclusion, the occurrence of rare alleles is illustrative of the genetic diversity within the Ghanaian population. This was further illustrated by the presence of three individuals that were heterozygous at all 21 STR loci.

Highlights:\ud * GlobalFiler® PCR amplification kit was used to generate a reference dataset for the population of Saudi Arabia.\ud * SE33, D12S391, and D1S1656 in this kit are more informative for the population of Saudi Arabia than any locus in the AmpFlSTR® Identifiler® PCR amplification kit.

Differentiating male lineages using non-recombining Y-chromosomal genetic markers is highly informative for tracing human migration and for forensic studies. Recently, it has been shown that the level of male lineage resolution can be enhanced by analysing rapidly mutating (RM) Y-STRs. The aim of this study was to develop an allelic frequency database for Qatari population to evaluate the resolution power of 13 RM Y-STRs. The overall haplotype diversity (HD) was 100%. It was found that the markers which contributed the most toward high HD were DYF399S1 and DYF403S1a/b. Together with their value for paternal male relative differentiation, these RM Y-STRs will be a valuable asset for forensic casework. AMOVA test was performed between Qatari population in comparison to Gulf countries, Middle East, and several worldwide population data sets. FST values were also calculated. Geography was found to account considerably for the pattern of population sub structuring. The RM Y-STR markers showed remarkable haplotype resolution power in the Qatari population, high gene diversity and sufficient robustness for a diverse range of applications.

The main focus in forensic genetics for two decades has been to improve the extraction of DNA from a wide variety of evidence and to make the profiling technology more sensitive and robust. In contrast, the recovery methods for biological material have seen little development. This study aims to improve the efficacy of the collection and storage processes, from crime scene to receipt at the laboratory. This study compared the use of ultrapure water as a wetting agent when collecting biological evidence using swabs with a detergent-based buffer. The results show that the stability post-collection greatly improved by using a newly developed buffer. When ultrapure water is used, DNA degradation was seen after 6 h at room temperature. However, the detergent-based buffer stabilized DNA for up to 48 h, even when the temperature was increased to 50 °C. The impact of these findings may be limited where crime scene evidence can be refrigerated until it reaches the laboratory. However, there are many situations/contexts where sample refrigeration is not possible and there is scope to improve the preservation of the genetic forensic evidence before it reaches the laboratory.

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single-copy nuclear recombination activation gene (RAG-1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature—24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

Mitochondrial DNA (mtDNA) has been used in forensic investigations for over 20 years and continues to be employed at times when conventional short tandem repeat typing is not possible. The key features of mtDNA that make it valuable in forensic investigations are its high copy number and maternal mode of inheritance. Another feature of mtDNA is that the distribution of different mutations across the global populations is not uniform, thereby allowing geographical ancestry of biological evidence to be inferred, which can be critical in forensic investigations. Finally, mtDNA can also be a valuable tool for species identification.

Forensic DNA profiling uses a series of commercial kits that co-amplify several loci in one reaction; the products of the PCR are fluorescently labelled and analysed using CE. Before CE, an aliquot of the PCR is mixed with formamide and an internal lane size standard. Using the SGM Plus amplification kit, we have developed two internal non-amplified controls of 80 bp and 380 bp that are labelled with ROX fluorescent dye and added to the PCR. Combined with two internal amplification controls of 90 bp and 410 bp, they provide additional controls for the PCR, electrokinetic injection, and CE and also function as an internal size standard.

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

After death DNA becomes progressively more fragmented as biological tissue degrades and this results in a decreasing ability to gain a complete STR profile. When extracting and profiling DNA from human remains understanding the likely persistence of DNA in different tissues is important. Studies in the UK have demonstrated that when using pigs as an experimental model, DNA up to 400bp will persist for up to 3 weeks in the summer. However, it is well known that DNA degradation, especially in muscle tissue that has not become dehydrated, is dependent to a large degree on temperature. To assess DNA persistence in more extreme environmental conditions pig carcasses were exposed to the environment in Thailand during June for 10 days, with samples being collected every 12h: muscle tissue was present for up to three days post-mortem. Extracted DNA could not be amplified after 36h exposure unless the muscle was collected from tissue that was in contact with the ground; DNA persisted for up to 72h in these samples.

A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518, DYS526ab, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. This study reflects the proof of concept to analyse all currently known RM Y-STRs simultaneously and describes the optimization of the multiplex assay. The RM-Yplex assay generated complete RM Y-STR profiles down to 62.5 pg of male template DNA, and from male-female DNA mixtures at all ratios tested. We herewith introduce and make available for widespread use in forensic and anthropological studies, an effective and sensitive single multiplex assay for simultaneous genotyping of 13 RM Y-STRs. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The mtDNA and Y chromosomes of the Malay population of peninsular Malaysia show high levels of diversity. Both the Malay and the indigenous Orang Asli populations showed a similar level of divergence between the different Y chromosome haplotypes.

East African athletes now dominate international distance running events from the 800 m to the marathon. Explanations for their phenomenal success have included optimal environmental conditions for developing distance running performance, psychological advantage and advantageous physiological characteristics. It is well established that genetics plays a role in determining inter-individual differences in exercise performance and adaptation to training stimuli. It is not known, however, to what extent inter-population differences (i.e. between ‘races’ and/or ethnic groups) in exercise performance can be attributed to genetics. There have been considerations that ‘black’ athletes are genetically adapted towards performance, given the concurrent success of athletes of West African ancestry in sprint events. However, the current notion of ‘race’ is not universally accepted, and genetic differences within and between populations are not clearly delineated by geographical or ethnic categorizations. Recent findings from mitochondrial DNA show that the populations from which Ethiopian athletes are drawn have not been isolated populations and are not genetically distinct from other Ethiopians. Y-chromosome analysis of the same population shows concurrent results, although some differences are present between athletes and the general Ethiopian population, suggesting an influence of the Y chromosome on athlete status in Ethiopia. It is concluded that there may be a role for genetics in the success of East African athletes; however, any genetic component to their success is unlikely to be limited to East Africans and is more likely to be found in other populations. At present it is unjustified to implicate a role for genetics in the success of East African runners when no genes have been identified as being important to their performance.

Favourable genetic endowment has been proposed as part of the explanation for the success of East African endurance athletes, but no evidence has yet been presented. The Y chromosome haplogroup distribution of elite Ethiopian athletes (n=62) was compared with that of the general Ethiopian population (n=95) and a control group from Arsi (a region producing a disproportionate number of athletes; n=85). Athletes belonged to three groups: marathon runners (M; n=23), 5-km to 10-km runners (5-10K; n=21) and other track and field athletes (TF; n=18). DNA was extracted from buccal swabs and haplogroups were assigned after the typing of binary markers in multiplexed minisequencing reactions. Frequency differences between groups were assessed by using contingency exact tests and showed that Y chromosome haplogroups are not distributed amongst elite Ethiopian endurance runners in the same proportions as in the general population, with statistically significant (P

Chromosome configurations and chiasma frequency during the metaphase I stage of spermatogenesis in the male horse are characterized in this work. The genome-wide frequency and distribution of chiasmata was detected as 49.45 ± 2.07 for 14 fertile stallions. All X and Y chromosomes shared a single chiasma at their pseudoautosomal region, while 1-4 chiasmata were observed in autosomal chromosomes. The chiasma frequency and distribution were further studied for 8 different bivalents identified by FISH in 5 fertile stallions. Genetic length was calculated from chiasmata data for the whole genome as well as for these 8 chromosomes. The findings complement the genetic linkage data and provide insight into the genetic basis of spermatogenesis in normal stallions.

In order to demonstrate the sequence diversity of mitochondrial D-loop DNA in the Taiwanese Han population, we established a database of 155 unrelated individuals. For each individual, the complete 980bp DNA region from the 5' end of HVI to 3' end of HVII segment was sequenced. In these 155 sequence data, 149 different haplotypes were observed, amongst these haplotypes, 144 were unique, 4 were found in 2 individuals and 1 was found in 3 individuals. When compare to the Anderson sequence, 144 transitions, 24 transversions, 5 insertions and 5 deletions were found. Eight positions exhibited more than one polymorphic sequence, six exhibited two variants while two exhibited three variants. Over the 1024bp that was analysed, pairwise differences between the sequences were 11.35+/-3.53bp. The sequence and nucleotide diversity were 0.9994 and 0.0116, respectively. The probability of two individuals randomly matching over the entire control region was 0.007. The diversity in the mitochondrial D-loop indicates the value of this locus for casework within Taiwan.

The expansion of premodern humans into western and eastern Europe approximately 40,000 years before the present led to the eventual replacement of the Neanderthals by modern humans approximately 28,000 years ago. Here we report the second mitochondrial DNA (mtDNA) analysis of a Neanderthal, and the first such analysis on clearly dated Neanderthal remains. The specimen is from one of the eastern-most Neanderthal populations, recovered from Mezmaiskaya Cave in the northern Caucasus. Radiocarbon dating estimated the specimen to be approximately 29,000 years old and therefore from one of the latest living Neanderthals. The sequence shows 3.48% divergence from the Feldhofer Neanderthal. Phylogenetic analysis places the two Neanderthals from the Caucasus and western Germany together in a clade that is distinct from modern humans, suggesting that their mtDNA types have not contributed to the modern human mtDNA pool. Comparison with modern populations provides no evidence for the multiregional hypothesis of modern human evolution.

An anthropological study of the remains has indicated uniformity of the ancient human Jety-Asar population (Central Asia) and suggests a mixed Euro-Mongoloid genesis. DNA was extracted from teeth from three Caucasoid skulls recovered from a burial site dated at approximately 2000 years ago. Ancient mitochondrial DNA (mtDNA) was analysed by restriction fragment length polymorphism (RFLP) analysis for the A, B, C and D haplogroups and the sequencing of hypervariable region I of the mtDNA control region. The full set of mtDNA control region variants determined for Jety-Asar specimens was only found among a modern Mongolian population (Mongoloid people), indicating some discordance of molecular and morphological data.

In the Ras Al Khaimah DNA Forensic Unit Chelex ® -100 is used when processing biological evidence. However, in many laboratories solid phase extraction techniques have gained popularity. Before changing methodology we wanted to compare the performance of two commonly used methods, the QIAamp ® DNA Investigator Kit (Qiagen, Germany) and the PrepFiler™ lysis buffer using the AutoMate Express™ System (Applied Biosystems). Fresh samples of blood, semen and saliva were placed onto cotton swabs and air-dried before extraction. Evaluation was based on the extracted DNA quantity and quality using real-time quantification (Quantifiler ® Human, Applied Biosystems). Additional criteria such as consistency, ease of use and cost efficiency were also considered. All extraction methods yielded good quality DNA with an IPC value between 27C t and 29C t with the exception of the Chelex ® -100 method when blood samples were processed. One-step analysis of variance showed significant differences in the quantity of DNA recovered between the methods tested ( p =0.0003 for blood, p =6.83e −5 for semen and p =0.002 for saliva). Chelex ® -100 gave the highest yield for semen and saliva and the AutoMate Express™ gave the highest yield when processing blood. Although QIAamp ® DNA Investigator gave lower yields it did produce the most consistent results, with little variation between extracts. Overall the AutoMate Express™ was the most favorable method tested; however Chelex ® -100 is a much cheaper alternative, which does not compromise on DNA yield and in practice rarely suffers from inhibition when processing routine casework.

Pakistan is a large country with a diverse population consisting of four significant population groups besides many smaller isolated populations. Male population samples were collected from indigenous populations and the extracted DNA was used to amplify seven Y STRs. Allele frequencies within each population were similar as were the haplotype frequencies. The data showed that individual populations were diverse in spite of a high rate of consanguineous marriages. This work indicates that, even with smaller number of loci, significant amount of genetic and phylogenetic information can be gained from the data.

Advances in molecular cytogenetics have provided the opportunity to study events during prophase I of meiosis. Immunofluorescent localization of different meiotic protein components were used to characterize the early stages of the first meiotic division in horse spermatocytes. The frequency and distribution of recombination events during prophase I were investigated using the mutL homolog 1 (MLH1) protein that is known to be associated with these events. The frequency and distribution of MLH1 foci were investigated in pachytene nuclei of 6 fertile stallions, and the average relative synaptonemal complex length was found to be highly correlated with the average number of MLH1 foci. The frequency and distribution of MLH1 foci were found to closely correspond to the frequency and distribution of chiasmata on metaphase I chromosomes, and genetic length, calculated from MLH1 foci data, for the whole genome was 2,505.5 cM.

Mitochondrial deoxyribonucleic acid (mtDNA) has been important in understanding the evolutionary histories of both modern humans and archaic hominins, especially the Neanderthals. mtDNA to date (2012) has been successfully extracted from 13 specimens recovered from the geographical range of the Neanderthals. Primer extension capture along with next generation sequencing has been used along with standard shotgun sequencing to determine the entire mtDNA genome of six specimens: two from Kleine Feldhofer Grotte in Germany, one from El Sidron cave in Spain, two from Vindija Cave in Croatia and one from Mezmaiskaya Cave in Russia. New mtDNA sequences have enhanced the data available from earlier studies that analysed the hypervariable regions of several specimens. The Neanderthal mtDNA pool is distinct from modern human mtDNA and forms a separate phylogenetic clade; genetic diversity of Neanderthals is estimated to be approximately one-third of extant modern human populations. In addition to analysis of the mtDNA, next generation sequencing has allowed the first insights into the Neanderthal nuclear genome. Key Concepts: The mtDNA genomes of six Neanderthals from four countries have been fully sequenced. The hypervariable regions of several other specimens have also been analysed. The Neanderthals form a distinct genetic clade from modern humans; the two lineages diverged approximately 500 000 years before the present. Genetic diversity in the Neanderthal population appears to be considerably reduced in comparison to the modern human populations. Based on the mtDNA analysis, the Eurasian Neanderthal population appeared to be fragmented into subpopulations with separate demographic histories. The likelihood of admixture between Neanderthals and modern humans is so low that either behavioural or biological barriers to interbreeding may have existed. Keywords: Neanderthal; ancient DNA; PCR; mitochondrial DNA

A seemingly routine paternity case which involved the testing of a mother, child and man led to inconclusive results as an exclusion involving only a single repeat was found at one of the 14 loci tested. This led to the testing of further loci. Only the single exclusion was found after profiling a total of 21 loci, with the addition of a further single locus providing a second exclusion. Even with both mutation events incorporated into the calculation a paternity index (PI) of 4957 was calculated, still a significant figure. However, when a second likelihood ratio was calculated assessing the likelihood of the results if the biological father of the child was the tested man or the tested man's brother, then the results were not significant, only 0.15. This analysis led to the profiling of the tested man's brother who matched at all 19 loci that were profiled and was concluded to be the biological father.

A study was undertaken to assess the utility of mtDNA as a forensic tool in the Malay Peninsular. Two populations, modern Malay and Orang Asli, were sampled and their mitochondrial DNA (mtDNA) analysed. Comparing the different haplogroups that were found, there are clear differences between the two populations. As a forensic tool, the use of mtDNA in the Orang Asli is limited as many individuals share the same haplotype, reducing the power of discrimination. In the Malay population, the power of discrimination is sufficiently high to make it a valuable tool.

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit, developed and supplied by Applied Biosystems, complements the AmpFlSTR Identifiler polymerase chain reaction amplification kit (Applied Biosystems, Warrington, U.K.) by improving the success rate when profiling DNA that is degraded or contains inhibitors. Before applying the MiniFiler kit to casework, the profiles from 200 unrelated Kuwaitis were compared to Identifiler profiles. Concordance was observed for 99.875% (1598 of 1600) of the compared STR loci. The two discordant profiles displayed allelic dropout: one at the D13S317 locus due to nonamplification of allele 10 in the MiniFiler profile, and one at the D18S51 locus due to nonamplification of allele 18 in the Identifiler profile.

The maternal inheritance of mitochondrial DNA (mtDNA) has enabled construction of detailed phylogenies. Analysis of key polymorphisms from these phylogenies allows mtDNA to be assigned to haplogroups, which have been associated with elite endurance performance. Purpose: To compare the frequencies of mtDNA haplogroups found in elite Kenyan athletes with those in the general Kenyan population. Methods: DNA samples were obtained from 221 national level Kenyan athletes (N), 70 international Kenyan athletes (I), and 85 members of the general Kenyan population (C). mtDNA haplogroups were classified by sequencing 340 bases of hypervariable section (HVS I) and by genotyping known restriction sites. Frequency differences between groups were assessed using exact tests of population differentiation. Results: The haplogroup distribution of national (P = 0.023) and international athletes (P < 0.001) differed significantly from controls, with international athletes showing a greater proportion of L0 haplogroups (C = 15%, N = 18%, I = 30%) and lower proportion of L3* haplogroups (C = 48%, N = 36%, I = 26%). Although a high number of international athletes originated from the Rift Valley province relative to controls (C = 20%, N = 65%, I = 81%), subjects from this province did not differ in haplogroup distribution from other regions (P = 0.23). Nor did Bantu subjects differ from Nilotic (P = 0.12) despite an overrepresentation of Nilotic languages among the athletes. Conclusions: International athletes differed in their mtDNA haplogroup distribution relative to the general Kenyan population. They displayed an excess of L0 haplogroups and a dearth of L3* haplogroups. These findings suggest that mtDNA haplogroups are influential in elite Kenyan distance running, although population stratification cannot be ruled out.

Fragments of the non-coding portion of mtDNA of various lengths have been successfully isolated from a total of eight Neanderthal specimens. This has provided an insight into the mtDNA gene pool and has enabled some aspects of the diversity and age of the Neanderthal lineage to be assessed. No admixture between modern humans and Neanderthals has been detected, but the limited number of samples available for molecular analysis limit the conclusions that can be made with respect to potential admixture. Other explanations for the lack of Neanderthal lineages in the modern mtDNA gene pool, in particular genetic drift, can also explain the results, especially as the conclusions are based on the analysis of one haploid locus, the mtDNA. Further analysis will provide a better view of the Neanderthal gene pool, but the number of potential samples is limited: in total 70 sites have yielded Neanderthal bones (Klein 2003). Many of the sites, particularly those from southern Europe, do not show good molecular preservation (Cooper et al. 1997; Smith et al. 2003).

A recent dispersal of modern humans out of Africa is now widely accepted, but the routes taken across Eurasia are still disputed. We show that mitochondrial DNA variation in isolated “relict” populations in southeast Asia supports the view that there was only a single dispersal from Africa, most likely via a southern coastal route, through India and onward into southeast Asia and Australasia. There was an early offshoot, leading ultimately to the settlement of the Near East and Europe, but the main dispersal from India to Australia ∼65,000 years ago was rapid, most likely taking only a few thousand years.

Development of a new multiplex comprising of 13 Rapidly Mutating Y STRs (RM Y-STR) is presented. The multiplex will aid investigating the human genetic structure of United Arab Emirates (UAE) populations and would also be used to investigate unresolved forensic cases in Dubai Forensic Laboratory. Thirteen markers have been included in the multiplex. A mini validation of the new multiplex has been carried out including specificity, sensitivity and mixture studies. These markers have been analysed in 600 male samples from UAE population. Allelic frequencies, haplotype diversity and discrimination capacity were determined for the 13 RM Y-STRs. Mutations pattern analysis of the RM Y-STR loci in a typical UAE family has also been carried out.

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59bp to 115bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM ® 310 and 3500. Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex ® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp ® DNA mini kit (Qiagen) and Differex™ with the DNA IQ ® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH 2 O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler ® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex ® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp ® DNA mini kit and involved fewer liquid transfers.

Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch ® gDNA Plant Kit (Life Technologies), DNA IQ TM System Kit (Promega), DNeasy ® Blood & Tissue Kit (Qiagen) and PrepFiler ® BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq ® qPCR Master Mix (Promega) using an Applied Biosystems ® 7500 Real-Time PCR System and the extracts were amplified using an in-house multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch ® gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition.

East African runners are continually successful in international distance running. The extent to which genetic factors influence this phenomenon is unknown. The insertion (I) rather than deletion (D) of a 287 bp fragment in the human angiotensin converting enzyme (ACE) gene is associated with lower circulating and tissue ACE activity and with endurance performance amongst Caucasians. To assess the association between ACE gene variation and elite endurance athlete status in an African population successful in distance running, DNA samples were obtained from 221 national Kenyan athletes (N), 70 international Kenyan athletes (I), and 85 members of the general Kenyan population (C). Blood samples were obtained from C and assayed for circulating ACE activity. ACE I/D (rs????—from NCBI SNPdb first time poly mentioned) genotype was determined, as was genotype at A22982GD (rs????—from NCBI SNPdb first time poly mentioned) which has been shown to associate more closely with ACE levels in African subjects than the I/D polymorphism. ACE I/D and A22982G genotypes explained 13 and 24% of variation in circulating ACE activity levels (P = 0.034 andless than0.001 respectively). I/D genotype was not associated with elite endurance athlete status (df = 4, χ2 = 4.1, P = 0.39). In addition, genotype at 22982 was not associated with elite endurance athlete status (df = 4, χ2 = 5.7, P = 0.23). Nor was the A allele at 22982, which is associated with lower ACE activity, more prevalent in N (0.52) or I (0.41) relative to C (0.53). We conclude that ACE I/D and A22982G polymorphisms are not strongly associated with elite endurance athlete status amongst Kenyans.

Previous studies have hypothesised that mitochondrial DNA (mtDNA) polymorphisms may influence aerobic performance. The matrilineal inheritance and accumulation of polymorphisms in mtDNA means that mtDNA haplogroups, characterised by key polymorphisms, are often represented at different frequencies in different populations. The present study aimed to compare the mtDNA haplogroup distribution of elite Ethiopian athletes relative to the general Ethiopian population. The haplogroup distribution of 76 endurance athletes (E), members of the Ethiopian national athletics team, was compared to 108 members of the general Ethiopian population (C). DNA was extracted from buccal swabs and haplogroups assigned by sequencing part of the hypervariable sequence (HVS-I), followed by analysis of key coding-region polymorphisms. A high proportion of African 'L' haplogroups was found in athletes and controls (C=53%; E=55%). Haplogroup distribution of endurance runners did not differ from that of C (P=0.63). Elite Ethiopian athletes are not a mitochondrially distinct group relative to the Ethiopian population. It appears that environment and, perhaps, polymorphisms in the nuclear genome are more important determinants of Ethiopian running success than mtDNA polymorphisms.

INTRODUCTION - The dominance of East-African athletes in distance running remains largely unexplained; proposed reasons include favorable genetic endowment and optimal environmental conditions. PURPOSE - To compare the demographics of elite Ethiopian athletes with the general Ethiopian population and assess the validity of reports linking running long distances to school with endurance success. METHODS - Questionnaires, administered to 114 members (male and female) of the Ethiopian national athletics team and 111 Ethiopian control subjects (C) obtained information on place of birth, language, distance and method of travel to school. Athletes were separated into three groups according to athletic discipline: marathon (M; N = 34); 5,000-10,000 m (5-10 km; N = 42); and other track and field athletes (TF; N = 38). Frequency differences between groups were assessed using contingency chi-square tests. RESULTS Regional distributions of marathon athletes differed from controls (P < 0.001) and track and field athletes (P = 0.013), but not the 5- to 10-km athletes (P = 0.21). The 5- to 10-km athletes also differed from controls (P < 0.001). Marathon athletes exhibited excess from the regions of Arsi and Shewa (M: 73%; 5-10 km: 43%; TF: 29%; C: 15%). The language distribution of marathon athletes differed from all groups (P < 0.001), with a predominance of languages of Cushitic origin (M: 75%, 5-10 km: 52%, TF: 46%, C: 30%). A higher proportion of marathon athletes ran to school (M: 68%; 5-10 km: 31%; TF: 16%; C: 24%) and traveled greater distances. CONCLUSION - Elite endurance athletes are of a distinct environmental background in terms of geographical distribution, ethnicity, and also having generally traveled farther to school, often by running. These findings may reflect both environmental and genetic influences on athletic success in Ethiopian endurance athletes.

In order to assess DNA degradation in the model organisms chosen (pig and rabbit), two nuclear genes, Connexin 43 and RAG-1, were aligned to identify conserved regions. Primers were designed to amplify 70 bp, 194 bp, 305 bp and 384 bp amplicons. The primers were also designed to amplify human DNA, which allowed the use of commercially purchased DNA standards to be used as controls. Following DNA extraction PCR analysis was performed using the four primers sets in a multiplex (4-plex): the PCR was optimised so that it worked over a wide range of template amounts (0.1–75.83 ng). The multiplex (4-plex) PCR was found to work efficiently in triplicate samples with all three species down to 0.3 ng of DNA template. This multiplex has been used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. Full 4-plex profiles were generated until day 7 (112 ADD) from whole carcasses and body fragments. Future work will include; development of real-time PCR quantification assays, DNA fragment analysis and DNA preservation.

Allele frequencies for 10 STRs including the GenePrint PowerPlex 1.2 loci and also D3S1358, HumvWA and HumFGA were obtained from a sample of unrelated individuals from El-Minia City.