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Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Analysis of mitochondrial DNA in forensic samples is routinely carried out by direct sequencing of hypervariable regions within the non-coding displacement loop. Although the accuracy and sensitivity of this method cannot be questioned, it is both time-consuming and labor intensive. Finding a way to rapidly pre-screen forensic samples-prior to sequencing, to reduce the number of samples that need to be sequenced-would greatly benefit forensic laboratories. Herein, we describe an assay for discrimination of DNA from different individuals based on high-resolution melting analysis of the two hypervariable regions HVI and HVII of the mitochondrial genome. By clearly distinguishing the DNA melting curves of six different individuals, we show that this assay has the potential to function as a rapid and inexpensive pre-screening method for forensic samples prior to DNA sequencing.

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

More than 200 latent fingerprints were deposited on various surfaces under controlled conditions and then developed using nine different visualization techniques. DNA was extracted from the fingerprints and the samples were subsequently quantified for nuclear and mitochondrial DNA content, using real-time PCR. The results show that several of the evaluated visualization techniques (e.g., Wet Powder and black fingerprint powder) do not damage DNA and allow DNA analysis to a large extent. However, some of the visualization techniques (e.g., physical developer and silver nitrate) seem to eliminate DNA completely, highly degrade DNA, or introduce inhibitors, preventing subsequent analysis. Furthermore, the results demonstrate great variation in DNA amounts detected in samples developed with the same method. [ABSTRACT FROM AUTHOR]

Abstract: Analysis of Y chromosome STR markers has proven to be useful in forensic cases where the samples contain a mixture of DNA from several individuals. STR markers are commonly genotyped based on length separation of PCR products. In this study we evaluated if Pyrosequencing can be used as an alternative method for determining Y-STR variants. In total 70 unrelated Swedish males were typed for the Y chromosomal markers (DYS19, DYS389 I-II, DYS390, DYS391, DYS392, DYS393 and DYS438) using Pyrosequencing. Using the 8 markers, 57 unique haplotypes were observed with a discrimination capacity of 0.81. At four loci, the Pyrosequencing analysis revealed sequence variants. The sequence variants were found in the DYS389 II, DYS390, DYS391, and DYS393 loci in frequencies between 1.43% and 14.3%. Pyrosequencing has here been shown to be a useful tool for typing Y chromosomal STRs and the method can provide a complement to conventional forensic Y STR analyses. Moreover, the Pyrosequencing method can be used to rapidly evaluate novel markers. [Copyright &y& Elsevier]

Abstract: Single nucleotide polymorphism (SNP) typing will be useful in forensic analysis but is limited by the multiplexing capacity of multiple targets in the PCR. A strategy to circumvent this problem can be to use molecular inversion probes (MIPs). MIPs are linear oligonucleotides 70–100 nt in length, with end-segments that are complementary to a target sequence. In this study, 10 SNPs were selected for SNP detection performed by pyrosequencing. The high-multiplexing capacity of MIPs makes this technology very useful for large scale SNP genotyping, but may also be useful for DNA typing in forensic investigations. [Copyright &y& Elsevier]

Diversity of the hypervariable regions (HV) I and II of the mitochondrial genome was studied in maternally unrelated Sri Lankans ( N = 202) from six ethnic groups (i.e.: Sinhalese, Sri Lankan Tamil, Muslim, Malay, Indian Tamil and Vedda). DNA was extracted from blood and buccal swabs and HVI and HVII regions were PCR amplified and sequenced. Resulting sequences were aligned and edited between 16024–16365 and 73–340 regions and compared with revised Cambridge reference sequences (rCRS). One hundred and thirty-five unique haplotypes and 22 shared haplotypes were observed. A total of 145 polymorphic sites and 158 polymorphisms were observed. Hypervariable region I showed a higher polymorphic variation than hypervariable region II. Nucleotide diversities were quite low and similar for all ethnicities apart from a slightly higher value for Indian Tamils and a much lower value for the Vedda population compared to the other groups. When the total population was considered South Asian (Indian) haplogroups were predominant, but there were differences in the distribution of phylo-geographical haplogroups between ethnic groups. Sinhalese, Sri Lankan Tamil and Vedda populations had a considerable presence of West Eurasian haplogroups. About 2/3rd of the Vedda population comprised of macro-haplogroup N or its subclades R and U, whereas macro-haplogroup M was predominant in all other populations. The Vedda population clustered separately from other groups and Sri Lankan Tamils showed a closer genetic affiliation to Sinhalese than to Indian Tamils. Thus this study provides useful information for forensic analysis and anthropological studies of Sri Lankans. [ABSTRACT FROM AUTHOR]