DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

Reliable and Robust Molecular Sexing of the Hen Harrier (Circus cyaneus) Using PCR‐RFLP Analysis of the CHD 1 Gene

The hen harrier (Circus cyaneus) is a bird of prey that is persecuted in the United Kingdom, and there is a need for a DNA‐based individual identification and sexing system for the use in forensic investigations. This study reports a new set of PCR primers for the chromo‐helicase‐DNA‐binding protein 1 gene, which allows sexing using PCR‐RFLP. Instead of exonic primers that amplify across a large intron, this set consists of a primer within the intron, enabling reduction in amplicon sizes from 356 to 212 bp and 565 to 219 bp in W and Z chromosomes. DNA degradation and dilution experiments demonstrate that this set is significantly more robust than one that amplifies across the intron, and sequencing of the intronic primer‐binding region across several individuals shows that it is highly conserved. While our objective is to incorporate this primer set into an STR‐based individualization kit, it may in the meantime prove useful in forensic or conservation studies.     

Mitochondrial DNA Validation in a State Laboratory*,†

Abstract: Because of the inception of the FBI Regional mitochondrial DNA (mtDNA) laboratories, many do not see establishing state/local mtDNA processing laboratories as a priority. Yet there is a long‐term need for mtDNA processing that will exceed the capabilities of the FBI Regional mtDNA laboratories and the few other laboratories that are currently processing mtDNA, and that need can be fulfilled by state/local laboratories. Thus, the DNA Unit of the Delaware Office of the Chief Medical Examiner (OCME‐DNA Unit) completed validation of in‐house mtDNA testing in January 2007. The validation plan for mtDNA processing included the following sections: preliminary research, sensitivity and contamination studies, ExoSAP‐IT^® optimization, BigDye^® optimization, sequencing and 310 optimization, sample preparation and extraction optimization, heteroplasmy, mixtures, and reproducibility. All sections of the validation were successfully completed, and mtDNA processing of skeletal remains, teeth, and hairs, as well as blood and buccal reference samples was adopted by the OCME‐DNA Unit.     

A Y‐Chromosomal Haplotype with Two Short Tandem Repeat Mutations*

Abstract:  The male‐specific Y‐chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR^® Yfiler^TM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y‐STR profile with a null allele at the DYS448 locus and an off‐ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N₄₂ region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92‐bp fragment that was off‐ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)₁₀. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.     

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance

Sequence analysis of the mitochondrial DNA (mtDNA) control region is of central importance for forensic identity testing as well as for studies of human evolution. Here we report the sequencing data of the hypervariable regions I and II from 50 unrelated individuals from a western German population (Rhine area). In regions I and II, 52 and 26 sites of sequence polymorphism, respectively, were noted. Nucleotide substitution rather than insertion/deletion was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Furthermore we investigated uniparental inheritance in seven CEPH families each family with 7–9 maternal descendants. Most maternal relatives shared identical mtDNA sequences. Additionally sequences were compared for father:child pairs and as expected no evidence for paternal transmission of mtDNA was observed. The high variability of mtDNA control region sequences permits utility in forensic identity investigations. The data also indicate that the neomutation rate seems to be very low from one generation to the other.     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

Development of a Protein‐based Human Identification Capability from a Single Hair

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10^?2 and 6.0 × 10^?9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.     

A new technology in mtDNA sequencing: Success rates vs time

Study of mitochondrial DNA (mtDNA) control region is a current practice in forensic genetics. In our service, mtDNA analysis is performed in many evidentiary specimens. Evaluation of this methodology is important to improve quality, increase efficiency and decrease artefacts, in order to reduce costs and time consuming.A case with 12 reference samples (bucal swabs) and 190 telogenic hair specimens extracted with DNA IQ™ System Tissue and Hair Extraction Kit (Promega) is reported. HVS-1 and HVS-2 control regions were sequenced with BigDye^® Terminator v1.1 Kit (Applied Biosystems), using BetterBuffer (Microzone Limited), followed by a simple bead purification method (XTerminator) to remove unincorporated terminators. Application of this procedure had success in 180 hair samples within a very short time comparing to dRhodamine/ethanolic precipitation sequencing strategy and also demonstrated that better results are achieved with clean sequence data closer to the primer.The quality of data produced by the BigDye/BetterBuffer/XTerminator (BDX) procedure has been demonstrated to be very high. Besides that the BDX procedure can significantly reduce overall processing time and cost per reaction. This new methodology has additional advantages like fewer reagent transfers and smaller amounts of DNA.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real‐time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re‐purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.     

Forensic casework analysis using the HVI/HVII mtDNA linear array assay

The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.     

Sequence polymorphism in the coding region of mitochondrial genome encompassing position 8389–8865

Analysis of the polymorphic sequences in mitochondrial DNA (mtDNA) has been widely applied to forensic tests and anthropology studies. However, these polymorphic data in human have thus far been derived from the displacement-loop and intergenic regions only. Here, we report the identification of clustered polymorphic sites in the mitochondria coding region encompassing position 8389–8865. The DNA sequences of 119 unrelated Chinese were determined by PCR amplification and direct sequencing. The results showed that heteroplasmy was found in five individuals, 39 sites were noted in this 477 bp region, and 41 haplotypes were identified. The probability of identity and allelic diversity were estimated as 0.1265 and 0.8809, respectively. The results suggest that sequence polymorphism from position 8389–8865 in human mtDNA can be used as a marker for identity investigation.     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

Genome‐wide Screen for Individual Identification SNPs (IISNPs) and the Confirmation of Six of Them in Han Chinese with Pyrosequencing Technology*

Abstract: Single nucleotide polymorphisms (SNPs) offer promise to forensic DNA analysts, but it remains uncertain whether a panel of individual identification SNPs can be as informative as the Combined DNA Index System short tandem repeats. Based on the highly accurate and publicly available HapMap SNP database (r21a) and a minor allele frequency cutoff of ≥0.45, we completed a genome‐wide screen through 3,905,819 SNPs with internally modified computer programs and identified 1439 SNPs with high heterozygosity and low F_st values among four populations (Utah Caucasian, Han Chinese, Tokyo Japanese, and Nigerian Yoruba). Using pyrosequencing technology, we studied six loci in a relatively large group of samples to determine whether these loci were as informative as the HapMap data suggest. These SNPs performed as expected in the Han Chinese in terms of heterozygosity and F_st. The 1439 identified SNPs should provide a comprehensive and reliable set of loci for identity and relationship testing.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Mitochondrial DNA cytochrome oxidase I gene: potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia

Forensic entomology requires the fast and accurate identification of insects collected from a corpse for estimation of the postmortem interval (PMI). Identification of specimens is traditionally performed using morphological features of the insect. Morphological identification may be complicated however by the numerical diversity of species and physical similarity between different species, particularly in immature stages. In this study, sequencing was performed to study the mitochondrial DNA (mtDNA) as the prospective basis of a diagnostic technique. The sequencing focused on a section of the cytochrome oxidase I encoding region of mtDNA. Three species of calliphorid (blow flies) commonly associated with corpses in western Australia, Calliphora dubia, Chrysomya rufifacies and Lucilia sericata, in addition to specimens of Calliphora augur and Chrysomya megacephala were studied. Phylogenetic analysis of data revealed grouping of species according to genus. The DNA region sequenced allowed identification of all species, providing high support for separation on congeneric species. Low levels of variation between some species of the same genus however indicate that further sequencing is required to locate a region for development of a molecular-based technique for identification.     

High‐Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high‐resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual‐dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.