Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl2, and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

Forensic animal DNA analysis using economical two-step direct PCR.

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/µL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire(®) Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

Ethical issues in DNA identification of human biological material from mass disasters.

Each mass disaster has its own characteristics and will involve a different approach, so the safeguarding and collection of forensic evidence have to be considered as part of the field response procedure. DNA typing has played a more prominent role in the identification of human remains, and particularly so for highly decomposed and fragmented remains. Although the ultimate goal is to obtain the identification, the specific context of each application of human identity testing has its specific problems, ranging from technical approach, through statistical interpretation, to ethical issues. The preparedness plan of the forensic genetics laboratory needs to include policies for family notification, long-term sample storage, and data archiving. For this reason, DNA sample collection and a strategy for DNA-based victim identification needs to be part of the preparedness plan. In this paper, the authors seek to define three of these ethical aspects: (1) the humanitarian importance of identification; (2) resource allocation in the victims' DNA identification; and (3) the secondary use for research of the samples initially collected for identification purposes. DNA analysis for the purpose of identifying victims of mass disasters has complex implications that demand much more rigorous examination than they have received until now.

Testing the effectiveness of an international conservation agreement: marketplace forensics and CITES caviar trade regulation.

BACKGROUND: The international wildlife trade is a key threat to biodiversity. Temporal genetic marketplace monitoring can determine if wildlife trade regulation efforts such as the Convention on International Trade in Endangered Species (CITES) are succeeding. Protected under CITES effective 1997, sturgeons and paddlefishes, the producers of black caviar, are flagship CITES species. METHODOLOGY/PRINCIPAL FINDINGS: We test whether CITES has limited the amount of fraudulent black caviar reaching the marketplace. Using mitochondrial DNA-based methods, we compare mislabeling in caviar and meat purchased in the New York City area pre and post CITES listing. Our recent sampling of this market reveals a decrease in mislabeled caviar (2006-2008; 10%; n = 90) compared to pre-CITES implementation (1995-1996; 19%; n = 95). Mislabeled caviar was found only in online purchase (n = 49 online/41 retail). CONCLUSIONS/SIGNIFICANCE: Stricter controls on importing and exporting as per CITES policies may be having a positive conservation effect by limiting the amount of fraudulent caviar reaching the marketplace. Sturgeons and paddlefishes remain a conservation priority, however, due to continued overfishing and habitat degradation. Other marine and aquatic species stand to benefit from the international trade regulation that can result from CITES listing.

Bodies of science and law: forensic DNA profiling, biological bodies, and biopower.

How is jurisdiction transferred from an individual's biological body to agents of power such as the police, public prosecutors, and the judiciary, and what happens to these biological bodies when transformed from private into public objects? These questions are examined by analysing bodies situated at the intersection of science and law. More specifically, the transformation of 'private bodies' into 'public bodies' is analysed by going into the details of forensic DNA profiling in the Dutch jurisdiction. It will be argued that various 'forensic genetic practices' enact different forensic genetic bodies'. These enacted forensic genetic bodies are connected with various infringements of civil rights, which become articulated in exploring these forensic genetic bodies''normative registers'.

Sudden death: ethical and legal problems of post-mortem forensic genetic testing for hereditary cardiac diseases.

Hereditary non-structural diseases such as catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT, and the Brugada syndrome as well as structural disease such as hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) cause a significant percentage of sudden cardiac deaths in the young. In these cases, genetic testing can be useful and does not require proxy consent if it is carried out at the request of judicial authorities as part of a forensic death investigation. Mutations in several genes are implicated in arrhythmic syndromes, including SCN5A, KCNQ1, KCNH2, RyR2, and genes causing HCM. If the victim's test is positive, this information is important for relatives who might be themselves at risk of carrying the disease-causing mutation. There is no consensus about how professionals should proceed in this context. This article discusses the ethical and legal arguments in favour of and against three options: genetic testing of the deceased victim only; counselling of relatives before testing the victim; counselling restricted to relatives of victims who tested positive for mutations of serious and preventable diseases. Legal cases are mentioned that pertain to the duty of geneticists and other physicians to warn relatives. Although the claim for a legal duty is tenuous, recent publications and guidelines suggest that geneticists and others involved in the multidisciplinary approach of sudden death (SD) cases may, nevertheless, have an ethical duty to inform relatives of SD victims. Several practical problems remain pertaining to the costs of testing, the counselling and to the need to obtain permission of judicial authorities.

A low-cost, high-throughput, automated single nucleotide polymorphism assay for forensic human DNA applications.

Single nucleotide polymorphism (SNP) analysis of human DNA for the purpose of identification has some promising attributes. The question of approach is critical to the eventual adoption of this technology. The use of a low-volume open array platform was tested with a small selected set of eight SNP primers that have a low F(ST) (the proportion of the total genetic variance contained in a subpopulation [S subscript] relative to the total genetic variance [T subscript]) in human populations. Because multiple SNPs must be interrogated, issues concerning DNA concentration, total DNA, and whole genome amplification were investigated. Excellent correlations were obtained for seven of the eight SNP assays on a set of DNA samples of known configuration over a broad concentration range spanning 25-150ng/microl in blind studies. These seven SNP assays were then applied to 39 DNA samples in a population from southern India. These SNPs were sufficient to individualize each member of this sample population. In a paternity study, these same SNPs showed clear parental relationships. For low amounts of genomic DNA, the use of a commercially available whole genome amplification kit showed promise for genotyping sub-nanogram samples. Discrimination against nonhuman DNA was also demonstrated successfully. Because of the very low quantities of reagents used in the assay, the cost per test becomes reasonably inexpensive. Overall, using commercially available SNP assays, the OpenArray platform showed excellent promise as a highly automated, low-volume, high-throughput system for SNP analysis with potential applications to relevant forensic analyses such as identification and paternity.