Automation of the standard DNA differential extraction on the Hamilton AutoLys STAR system: A proof-of-concept study.

For several decades, a common approach for processing sexual assault evidence has been to use the "standard" differential extraction to separate the evidence into a non-sperm-cell fraction and a sperm-cell fraction for further analysis. In this standard approach (P. Gill et al., Nature 318 (1985) 577-579), an initial mild chemical lysis step preferentially digests the mainly epithelial cells, which allows for removing this lysate as a non-sperm-cell fraction. The undigested sperm cells in the remaining fraction may then be purified by a series of wash and centrifugation steps, after which more robust lysis conditions are used to digest this sperm-cell fraction. Although this standard approach has been generally effective, it has been difficult to fully automate, due to the variety of different types of manipulations required for sample processing (e.g., incubation, shaking, substrate separation by centrifugation, and multiple liquid transfers for sperm-pellet centrifugation and washing steps). We describe here a fully automated standard differential extraction procedure that uses the Hamilton AutoLys STAR liquid handling assay-ready workstation, which is configured with on-deck components for sample incubation, shaking and centrifugation steps, and works with unique AutoLys-A® sample tubes for front-end sample processing. In this proof-of-concept procedure, up to 24 samples may be processed, "hands-free," in a single automated workflow. The automated procedure was tested by performing differential extractions on mock sexual assault swabs. For comparison, manual differential extractions were performed on identically prepared swabs in a side-by-side manner. DNA quantification and STR typing results showed that similar levels of separation efficiency were achieved for the sperm-cell fractions using both automated and manual procedures, although the results suggest that somewhat higher male DNA yields may be achieved for samples with extremely low semen levels (<∼0.1 µL) using the manual processing procedure. In addition to these mock samples, automated differential extractions were also performed on a set of authentic post-coital swabs (24, 48, 72, and 96 hours, post-coitus); primarily male STR profiles for the sperm-cell fractions were obtained for each sample in this set.

Improving the efficacy of the standard DNA differential extraction method for sexual assault evidence.

The efficacy of a DNA differential extraction procedure relies on reducing the amount of non-sperm female DNA carryover into the sperm fraction, while providing a sufficient recovery of male DNA from the sperm cell component. A standard approach to this extraction is to use a mild initial lysis step to digest the female (epithelial cell) component in the mixture, followed by a series of centrifugation and wash steps to further purify the resulting sperm-pellet fraction. This sperm fraction is then digested in the presence of a chemical reducing agent in preparation for DNA extraction. This method has been employed with relatively few changes since its introduction in the mid-1980s, despite numerous attempts to develop new or improved procedures. In this report, we demonstrate that it is possible to improve the efficacy of the standard differential extraction by applying simple modifications that can reduce the amount of female DNA carryover into the sperm fraction, with no adverse effects on the recovery of male DNA. In one modification, the addition of a second mild lysis step at the beginning of the differential extraction procedure improved the average male-to-female DNA ratio in the sperm fraction by 3- to 6-fold. In another modification, a "tube transfer" step was added to move the re-suspended sperm pellet to a new tube for the second mild lysis and subsequent wash steps. With this modification, the average male-to-female DNA ratio in the sperm fraction was improved by 4- to 90-fold, relative to results obtained for the non-modified differential extraction method. These modifications may be accomplished using tools and reagents that are already present in most forensic DNA laboratories, so that implementation should be relatively low-cost and practical.

Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC©) procedures.

Evaluation of the RapidHIT™ 200 and RapidHIT GlobalFiler(®) Express kit for fully automated STR genotyping.

The RapidHIT™ 200 Human Identification System and RapidHIT GlobalFiler(®) Express kit were evaluated and validated for use with single-source reference samples. It was of primary interest to evaluate the system for its efficacy as an expert system and to estimate a first pass success rate, as well as to identify the technical variables impacting that result. While results indicated that this instrument/kit combination can be used to accurately type single-source buccal samples, substantial variability in sensitivity and intra-color balance were observed, as were multiple artifacts, requiring extensive manual editing of the profiles. Artifacts included dye "blobs" and spectral overlap (pull-up) peaks that often originated from relatively low intensity allele peaks. Reduced intra-color balance, in combination with low sensitivity, occasionally resulted in instances of allelic dropout. Overall, 50% of the buccal samples analyzed in this study would have been successfully typed to give full GlobalFiler(®) profiles without the need for manual review and editing.

Stochastic sampling effects in STR typing: Implications for analysis and interpretation.

The analysis and interpretation of forensic STR typing results can become more complicated when reduced template amounts are used for PCR amplification due to increased stochastic effects. These effects are typically observed as reduced heterozygous peak-height balance and increased frequency of undetected alleles (allelic "dropout"). To investigate the origins of these effects, a study was performed using the AmpFlSTR(®) Identifiler Plus(®) and MiniFiler(®) kits to amplify replicates from a dilution series of NIST Human DNA Quantitation Standard (SRM(®) 2372A). The resulting amplicons were resolved and detected on two different genetic analyzer platforms, the Applied Biosystems 3130xL and 3500 analyzers. Results from our study show that the four different STR/genetic analyzer combinations exhibited very similar peak-height ratio statistics when normalized for the amount of template DNA in the PCR. Peak-height ratio statistics were successfully modeled using the Poisson distribution to simulate pre-PCR stochastic sampling of the alleles, confirming earlier explanations that sampling is the primary source for peak-height imbalance in reduced template dilutions. In addition, template-based pre-PCR sampling simulations also successfully predicted allelic dropout frequencies, as modeled by logistic regression methods, for the low-template DNA dilutions. We discuss the possibility that an accurately quantified DNA template might be used to characterize the linear signal response for data collected using different STR kits or genetic analyzer platforms, so as to provide a standardized approach for comparing results obtained from different STR/CE combinations and to aid in validation studies.

An examination of the utility of a nuclear DNA/mitochondrial DNA duplex qPCR assay to assess surface decontamination of hair.

The goal of this study was to compare two commonly used methods for the surface decontamination of human hair shafts, and to evaluate the use of a duplex real-time qPCR assay to assess decontamination effectiveness for the purpose of mitochondrial DNA typing. Hair shafts of known mitochondrial DNA haplotype were coated with undiluted saliva, semen or blood, each of known mitochondrial haplotype distinct from the test hair. Surface decontamination was conducted by enzymatic treatment with Terg-a-zyme™ and by chemical treatment with dilutions of sodium hypochlorite (NaClO, bleach). Following DNA extraction, a duplex (nuclear and mitochondrial DNA) real-time qPCR assay was used to quantify mitochondrial DNA and to test for surface contamination by quantifying the exogenous nuclear DNA not removed from the hair shaft. The NaClO treatment was found to be more effective for removing surface contamination than the Terg-a-zyme™ treatment, and it was procedurally simpler to implement, resulting in a significant savings of sample processing time. Exposure to 3% NaClO for up to two minutes had no detrimental effect on quantity or typing of the mitochondrial DNA belonging to the hair. In addition, we demonstrated that the duplex real-time PCR assay is a convenient early-warning diagnostic method for the detection of the presence of external DNA contamination, providing an assessment of the purity of the sample prior to embarking on further analysis by more laborious mitochondrial DNA typing methods.

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence.

We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (≈ 2-6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.

The NucleoSpin® DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: an alternative to microdialysis filtration.

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon(®) and Microcon(®) centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin(®) XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin(®) XS column is approximately 30 min vs. approximately 1.5h with the Centricon(®) YM-100 filter device.

Searching for first-degree familial relationships in California's offender DNA database: validation of a likelihood ratio-based approach.

A validation study was performed to measure the effectiveness of using a likelihood ratio-based approach to search for possible first-degree familial relationships (full-sibling and parent-child) by comparing an evidence autosomal short tandem repeat (STR) profile to California's ∼1,000,000-profile State DNA Index System (SDIS) database. Test searches used autosomal STR and Y-STR profiles generated for 100 artificial test families. When the test sample and the first-degree relative in the database were characterized at the 15 Identifiler(®) (Applied Biosystems(®), Foster City, CA) STR loci, the search procedure included 96% of the fathers and 72% of the full-siblings. When the relative profile was limited to the 13 Combined DNA Index System (CODIS) core loci, the search procedure included 93% of the fathers and 61% of the full-siblings. These results, combined with those of functional tests using three real families, support the effectiveness of this tool. Based upon these results, the validated approach was implemented as a key, pragmatic and demonstrably practical component of the California Department of Justice's Familial Search Program. An investigative lead created through this process recently led to an arrest in the Los Angeles Grim Sleeper serial murders.

A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing.

A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a approximately 170-190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137 bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67 bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77 bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan and TaqManMGB detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01-nuSRY-nuCSF-IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing ("MiniSTRs") where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C(T) value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male-female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines.

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.

Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48.

Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots EZ1 or BioRobots M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1.

A quantitative PCR assay for the assessment of DNA degradation in forensic samples.

A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest. The targets were chosen to provide quantification and fragment length information relevant to the STR amplification targets commonly used for forensic genotyping. The longer target (nuTH01, 170-190 bp) spans the TH01 STR locus. Although not one of the longest loci used for STR genotyping, it was chosen as a good compromise given the target length limitations on qPCR efficiency with TaqMan detection. The shorter target (nuCSF, 67 bp) was designed in the upstream flanking region of the CSF1PO STR locus. In addition to these human nuclear targets, the assay includes an internal PCR control target sequence to allow for an assessment of PCR inhibition. The assay was rigorously tested on samples with varying amounts of degradation, and the ratio of nuCSF:nuTH01 quantifications was shown to provide a good estimation of the degree of degradation present in a sample. This estimate, along with the internal control for PCR inhibition, provides a valuable tool for post-extraction sample assessment.

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples.

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.