Identification Of Human DNA Research Articles

Population genetics data for 22 autosomal STR loci in European, South Asian and African populations using SureID® 23comp Human DNA Identification Kit

Allele frequency data for 22 short tandem repeat loci; D18S1364, D1S1656, D13S325, D5S2800, D9S1122, D4S2366, D3S1744, D12S391, D11S2368, D21S2055, D20S482, D8S1132, D7S3048, D2S441, D19S253, D10S1248, D17S1301, D22-GATA198B05, D16S539, D6S474, D14S1434 and D15S659 from the SureID® 23comp Human DNA Identification Kit have been determined for unrelated individuals in European, South Asian and African populations. Deviations from Hardy–Weinberg equilibrium were observed in loci D1S1656 and D19S253 in European; D18S1364, D6S474 and D14S1434 in South Asian; and D9S1122 and D8S1132 in African populations (p-value <0.05). However, after Bonferroni correction no significant deviations were observed (p-value <0.002). The most discriminating loci were D1S1656 and D12S391 for European (PD=0.977), D21S2055 for South Asian (PD=0.980), and D21S2055 and D7S3048 for African (PD=0.972) populations. The match probabilities were 1 in 6.7×1025 for European, 1 in 1.4×1026 for South Asian and 1 in 1.6×1026 for African populations. These findings established the high discriminatory capacity and robustness of the tested STR loci for forensic identification and kinship testing.

Identification of Human DNA in Mixture of Human and Chicken Blood Using PCR with Specific Primer of Cytochrome B Gene

This study aimed to identify human DNA from mixing human and chicken blood samples by utilizing Polymerase Chain Reaction (PCR) and cytochrome b gene primer. The cytochrome b gene is a gene located in mitochondrial DNA and has high variation of sequence relation between one species and another. PCR analysis was performed using human cytochrome b gene primer in variation of DNA templates (0 ng, 0.01 ng, 0.1 ng, 1 ng, 10 ng and 100 ng), human blood percentages (100%, 50%, 40 %, 25%, 10%, 5%, 1%, 0%) and sample age before analysis (0 day, 3 days, 7 days, 10 days, and 15 days). The minimum DNA template obtained in this study was 0.01 ng and minimum percentage of human blood in the mixture was 1%. Blood spots on cloth isolated on days 0, 3, 7, 10 and 15 could still be analyzed and the resulting of DNA band (157 bp) had the same intensity/thickness. From the results of this study, it can be concluded that human blood in the mixture of human and chicken blood can be identified using PCR with specific primers of cytochrome b gene. PCR using specific primer of cytochrome b gene may help forensic practitioners to identify human sample in mixed biological samples.

A Systems Security Analysis of Issuance and Verification of Birth Documents Enhanced with DNA Profiles

The use of biometrics to enhance identification has been explored and utilized to various extents. DNA is the most reliable and stable biometric that remains unchanged throughout an individual’s lifetime. Advancements in DNA analysis, in terms of reduced cost and faster processing times, make the use of DNA as a biometric more feasible over time. Since DNA data is of a sensitive nature, privacy and ethical concerns would have to be carefully considered before large-scale adoption for use in identity documents. Birth certificates are a fundamental document used by a person for identification. However, it does not contain any means of authentication beyond possession of the document. This paper examines the security measures that would be required if birth certificates were embedded with DNA profile information. The U.S. FBI CODIS approach is referred to, being an established standard for human DNA profiling and identification. Effects on the issuance and verification network for birth certificate documents are explored, in addition to the security threats.

Loss of heterozygosity detected at three short tandem repeat locus commonly used for human DNA identification in a case of paternity testing.

Short tandem repeat (STR) is widely used for DNA profiling in forensic sciences for its stable inheritance. Genomic variations in STR loci may affect the results of the genotyping. In this study, using STR profiling and genome-wide chromosomal microarray assay, we detected the incidence of uniparental disomy or copy-neutral loss of heterozygosity (LOH) in a case of a parental testing, which altered the genotype of three commonly used STR markers including D2S1338, D2S441 and D2S1776. To the best of our knowledge, this is the first time found that LOH affect the genotyping of STR markers commonly used for paternity testing. Our findings demonstrated that the incidence of LOH in the genome may dramatically alter the results of DNA identification, and suggested that genomic structure variation need to be taking into consideration in the DNA identification using STR markers.

Review: domestic animal forensic genetics - biological evidence, genetic markers, analytical approaches and challenges.

This review highlights the importance of domestic animal genetic evidence sources, genetic testing, markers and analytical approaches as well as the challenges this field is facing in view of the de facto 'gold standard' human DNA identification. Because of the genetic similarity between humans and domestic animals, genetic analysis of domestic animal hair, saliva, urine, blood and other biological material has generated vital investigative leads that have been admitted into a variety of court proceedings, including criminal and civil litigation. Information on validated short tandem repeat, single nucleotide polymorphism and mitochondrial DNA markers and public access to genetic databases for forensic DNA analysis is becoming readily available. Although the fundamental aspects of animal forensic genetic testing may be reliable and acceptable, animal forensic testing still lacks the standardized testing protocols that human genetic profiling requires, probably because of the absence of monetary support from government agencies and the difficulty in promoting cooperation among competing laboratories. Moreover, there is a lack in consensus about how to best present the results and expert opinion to comply with court standards and bear judicial scrutiny. This has been the single most persistent challenge ever since the earliest use of domestic animal forensic genetic testing in a criminal case in the mid-1990s. Crime laboratory accreditation ensures that genetic test results have the courts' confidence. Because accreditation requires significant commitments of effort, time and resources, the vast majority of animal forensic genetic laboratories are not accredited nor are their analysts certified forensic examiners. The relevance of domestic animal forensic genetics in the criminal justice system is undeniable. However, further improvements are needed in a wide range of supporting resources, including standardized quality assurance and control protocols for sample handling, evidence testing, statistical analysis and reporting that meet the rules of scientific acceptance, reliability and human forensic identification standards.

E6/E7 mRNA testing for human papilloma virus-induced high-grade cervical intraepithelial disease (CIN2/CIN3): a promising perspective.

Since the introduction of biomolecular testing for the identification of high-risk human papillomavirus DNA (hrHPV-DNA) in cervical cancer preventive strategies, many interesting aspects have emerged in this field; firstly, HPV-DNA testing has been demonstrated to have better sensitivity than conventional cytology in several settings: screening, triage of ASC-US and in follow-up after treatment. Despite this, some limitations of these new technologies have also been underlined: the major issue is the low specificity of the tests, which cannot discriminate between regressive and progressive infections. Thus, recent research has moved the attention towards novel markers of progression that could more precisely detect cases at real risk of cancer development. In view of the fact that progression to cancer is dependable of the E6/E7 proteins integration and transforming action, the overexpression of E6/E7 transcripts has been seen as a valuable marker of this risk. This review aims to summarise the literature data on this topic and to provide a clear view of the emerging perspectives.

Genetic variability of X-linked STR markers in Siberian populations

X-chromosome microsatellite markers are a convenient tool for studying genetic variability in human populations and DNA identification, especially during the decreased informativeness of autosomal markers. The results of genetic analysis of Siberian populations by ten X-linked microsatellite markers (DXS8378, GATA172D05, DXS7132, DXS9898, DXS7423, DXS8377, DXS101, DXS6809, DXS6789, and HPRTB) are presented. The allele frequencies, criminalistics parameters, and genetic relations between populations were calculated. The average level of expected heterozygosity (He) was 0.73 in populations. The total level of genetic differentiation in ten populations was relatively low (Fst = 0.031) compared to the level determined by autosomal and Y chromosome markers. The high probability of the establishment of differences (PD) between two unrelated individuals using a ten X-STR marker system was demonstrated. The average PD value in the panel of ten X chromosome microsatellite markers was 0.9999999997 in women and 0.999998 in men. The total level of genetic differentiation in a pool of ten populations is 0.03186.

Generating Rapid DNA Profiles from Crime Scene Samples Commonly Encountered in the United Arab Emirates

Short tandem repeat (STR) DNA profiles are routinely generated from blood and saliva stains found on items of evidence collected at crime scenes. Current study utilized the fully integrated RapidHIT™ Human DNA Identification System (IntegenX, Pleasanton, CA, USA) to generate STR profiles from simulated crime scene evidence samples, similar to the ones commonly encountered at indoor crime scenes in the United Arab Emirates (UAE). The two body fluids were deposited on each of these items and processed. Data generated from 120 samples using this automated system and the “Run Other Samples” instrument protocol were in complete concordance with the data generated with traditional method used in forensic laboratories. Sensitivity study performed with this instrument indicated that complete profiles can be obtained from 0.25 μL of blood and 10.0 μL of saliva. Varying amounts of both of the body fluids were deposited on 25 different types of substrates containing potential inhibitors such as dyes and soil. Fifteen of these substrates containing body fluids were incubated at 48°C heat and 43% humidity for 24 hours. These conditions were used to mimic indoor crime scenes at the UAE during the Summer Season. Complete and concordant profiles were generated from most of these samples.

Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

RapidHIT™ 200, a promising system for rapid DNA analysis

DNA analysis systems suited for rapid, mobile, and non-expert operation are emerging in forensics. We show that the first version of the IntegenX RapidHIT™ 200 Human DNA Identification System enables reliable, correct and fast DNA profiling when buccal swabs are used, which is in agreement with the designed functionality. Genotyping information was also obtained for saliva, semen, skin and hair specimens, but not from blood. Standard DNA procedures were much more sensitive than this first RapidHIT release, but promise lies in the finding that mock casework samples show informative profiling results, also when the system is placed in a mobile vehicle.

DNA investigative lead development from blood and saliva samples in less than two hours using the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System produces full STR DNA profiles (sample in, results out) in 90min. Here we present results from a new protocol on the RapidHIT System, designed for crime scene samples. Data are presented for blood and saliva samples, with results for a range of samples and substrates. Success rates and sensitivity display a high level of performance for the sample types evaluated in the study and demonstrate the efficacy of the new protocol for crime scene samples.

Developmental validation studies on the RapidHIT™ Human DNA Identification System

The RapidHIT™ Human DNA Identification System is a fully integrated system (sample in, result out) capable of rapidly generating STR DNA profiles in around 90min. Here we present a portion of the results from the developmental validation studies performed on the RapidHIT System.

Identification of cytomegalovirus and human herpesvirus-6 DNA in a patient with corneal endotheliitis

To report the case of a patient with unilateral corneal endotheliitis in which both cytomegalovirus (CMV) and human herpesvirus-6 (HHV6) DNA was identified in the aqueous humor. A 67-year-old man with corneal endotheliitis OD was referred to us for decreased visual acuity. Local corneal stromal edema, pigmented keratic precipitates, a coin-shaped lesion and minimal anterior chamber reaction were observed by slit-lamp biomicroscopy. Cells with owl's eye appearance in the endothelial cell layer were observed by in vivo laser confocal microscopy. The patient had rheumatoid arthritis, which was treated by oral prednisolone and intravenous abatacept. Polymerase chain reaction analysis of aqueous humor samples detected both CMV and HHV6 DNA, but not other HHVs. Treatment with topical ganciclovir and systemic valganciclovir resulted in a clear cornea. A patient with corneal endotheliitis had both CMV and HHV6 DNA identified in the aqueous humor. Although both viruses were identified in this case, clinical manifestations resembled CMV corneal endotheliitis, and it was unclear whether HHV6 could affect the clinical course. Systemic abatacept and corticosteroid therapy might play a positive role in cases with both CMV and HHV6 DNA in this corneal endotheliitis.

Comparison of bacterial DNA profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners

It is crucial to identify the owner of unattended footwear left at a crime scene. However, retrieving enough DNA for DNA profiling from the owner's foot skin (plantar skin) cells from inside the footwear is often unsuccessful. This is sometimes because footwear that is used on a daily basis contains an abundance of bacteria that degrade DNA. Further, numerous other factors related to the inside of the shoe, such as high humidity and temperature, can encourage bacterial growth inside the footwear and enhance DNA degradation. This project sought to determine if bacteria from inside footwear could be used for footwear trace evidence. The plantar skins and insoles of shoes of volunteers were swabbed for bacteria, and their bacterial community profiles were compared using bacterial 16S rRNA terminal restriction fragment length polymorphism analysis. Sufficient bacteria were recovered from both footwear insoles and the plantar skins of the volunteers. The profiling identified that each volunteer's plantar skins harbored unique bacterial communities, as did the individuals' footwear insoles. In most cases, a significant similarity in the bacterial community was identified for the matched foot/insole swabs from each volunteer, as compared with those profiles from different volunteers. These observations indicate the probability to discriminate the owner of footwear by comparing the microbial DNA fingerprint from inside footwear with that of the skin from the soles of the feet of the suspected owner. This novel strategy will offer auxiliary forensic footwear evidence for human DNA identification, although further investigations into this technique are required.

Recovery of human DNA profiles from poached deer remains part 2: Improved recovery protocol without the need for LCN analysis

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total - one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5pg±43.2pg, 31× greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFℓSTR® SGM Plus® kit at 30cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690×10(-05) to 2.2706×10(-14). The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.

Optimization and validation of a fast amplification protocol for AmpF lSTR ® Profiler Plus ® for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpF lSTR ® Profiler Plus ® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpF lSTR ® Profiler Plus ® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpF lSTR ® Profiler Plus ® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.

Assessment of DNA degradation and the genotyping success of highly degraded samples

DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples. We report a comparative study of genotyping using STRs, mini-STRs and single nucleotide polymorphisms (SNPs) with template at different levels of degradation in varying amounts. Two methods of assessing quantity and quality of a DNA sample prior to genotyping were investigated. The QIAxcel capillary gel electrophoresis system provided a rapid, cost effective screening method for assessing sample quality. A real-time quantitative PCR (qPCR) assay was able to simultaneously quantify total human DNA, male DNA, DNA degradation and PCR inhibition. The extent of DNA degradation could be assessed with reasonable accuracy to 62.5pg and genomic targets could be quantified to a lower limit of 15.6pg. The qPCR assay was able to detect male DNA to a lower limit of 20pg in a 1:1,000 background of female DNA. By considering the amount of DNA and the degradation ratio of a sample, a general prediction of genotyping success using AmpFlSTR® Profiler Plus®, MiniFiler™ kits and SNP analysis can be made. The results indicate mini-STRs and SNP markers are usually more successful in typing degraded samples and in cases of extreme DNA degradation (≤200bp) and template amounts below 250pg, mini-STR and SNP analysis yielded significantly more complete profiles and lower match probabilities than corresponding STR profiles.

Characterization of various promoter regions of the human DNA helicase-encoding genes and identification of duplicated ets (GGAA) motifs as an essential transcription regulatory element

DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells.

Genotyping mitochondrial DNA single nucleotide polymorphisms by PCR ligase detection reactions

The identification of human mitochondrial DNA (mtDNA) sequence variations, especially single nucleotide polymorphisms (SNPs), is important for many applications. The PCR-ligase detection reaction (LDR) method can reduce false-positives and eliminate the need for both post-PCR and post-ligation purifications in SNP analyses. In addition, it has been successfully employed to detect point mutations in various nuclear genes. In this study, we used the PCR-LDR platform to characterize mtDNA SNPs. Multiplex PCR-LDRs were used to genotype 19 mtDNA single nucleotide polymorphic sites from 812 samples. Performance of the method was assessed by direct sequencing of 44 samples. We established an overall 97.4% success rate with 99.2% accuracy using the multiplex PCR-LDR methodology. The PCR-LDR mtDNA genotyping technique is simple, highly accurate, has high-throughput, and is cost-effective. Therefore, this method is applicable to mtDNA haplotyping in various applications.