Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

Direct PCR Improves the Recovery of DNA from Various Substrates

This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs^™. Swabs (n = 90) were either processed using the DNA IQ^™ kit or, for direct PCR, swab fibers (~2 mm²) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.     

The Use of Adhesive Tape for Recovery of DNA from Crime Scene Items

Abstract: The selection of the appropriate method of collection of biological material from crime scene items can be crucial to obtaining a DNA profile. The three techniques commonly used for sampling items are: cutting, swabbing, and taping. The tape sampling technique offers an advantage, in that it enables the collection of a potentially highly informative source of DNA, shed epithelial cells, from selected areas on crime scene items (the inside fingers of a glove, for instance). Furthermore, surface collection of biological material by taping reduces co‐sampling of known PCR inhibitors such as clothing dyes. The correct choice of tape for crime scene item sampling is important. Not all tapes are suitable for biological trace evidence collection as well as DNA extraction. We report on one tape that met both these criteria. Three different cases are presented which demonstrate the usefulness of adhesive tape sampling of crime items. Finally, the advantages of the tape collection technique are discussed and guidelines for preferred areas of tape sampling on various casework items are presented.     

Allele frequency profile of three STR loci in nine North Indian populations

POPULATION: Bhargavas (n=120), Chaturvedis (n=120), Brahmins (n=120), Muslim Sunni (n=120), Muslim Shiya (n=120), Kayastha (n=120), Mathurs (n=120), Rastogies (n=120), and Vaish (n=120).     

Trace DNA Sampling Success from Evidence Items Commonly Encountered in Forensic Casework

Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from “handler” and “wearer” areas of 764 items commonly submitted for examination. One hundred and fifty‐five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T‐shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison.     

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

Tyrosine Hydroxylase TH01 9.3 Allele in the Occurrence of Sudden Infant Death Syndrome in Swiss Caucasians

Catecholamines, especially noradrenalin, are essential in the control of respiration and arousal. Thus, an impaired production of these neurotransmitters may contribute to the occurrence of sudden infant death syndrome (SIDS). The first step of the noradrenergic synthesis pathway is catalyzed by the enzyme tyrosine hydroxylase (TH). The TH‐encoding gene contains a tetrameric short tandem repeat in intron 1 (TH01), with allele 9.3 reported to be associated with SIDS in German infants. We investigated the allelic frequency of the TH01 marker in 171 Swiss SIDS infants and 500 healthy and gender‐matched Caucasian adults. In our study population, the allelic frequency of the 9.3 allele is similarly distributed in SIDS cases and controls (27.2% vs. 25.6%; p‐value = 0.562). Nevertheless, the TH‐encoding gene is only one of several genes involved in the noradrenergic biosynthesis pathway. Therefore, further genetic investigations are required with focus on the whole noradrenergic signaling system.     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.     

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a “complete” Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.     

Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases

In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.     

Higher Capillary Electrophoresis Injection Settings as an Efficient Approach to Increase the Sensitivity of STR Typing

Abstract:  Evidentiary traces may contain low quantities of DNA, and regularly incomplete short tandem repeat (STR) profiles are obtained. In this study, higher capillary electrophoresis injection settings were used to efficiently improve incomplete STR profiles generated from low-level DNA samples under standard polymerase chain reaction (PCR) conditions. The method involves capillary electrophoresis with higher injection voltage and extended injection time. STR peak heights increased six-fold. Inherent to the analysis of low-level DNA samples, we observed stochastic amplification artifacts, mainly in the form of allele dropout and heterozygous peak imbalance. Increased stutter ratios and allele drop-in were rarely seen. Upon STR typing of 10:1 admixed samples, the profile of the major component did not become overloaded when using higher injection settings as was observed upon elevated cycling. Thereby an improved profile of the minor component was obtained. For low-level DNA casework samples, we adhere to independent replication of the PCR amplification and boosted capillary electrophoresis.