Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness.

Digital polymerase chain reaction (dPCR) methodology has been asserted to be a "potentially primary" analytical approach for assigning DNA concentration. The essence of dPCR measurements is the independent dispersal of fragments into multiple reaction partitions, amplifying fragments containing a target nucleotide sequence until the signal from all partitions containing at least one such fragment rises above threshold, and then determining the proportion of partitions with an above-threshold signal. Should originally double-stranded DNA (dsDNA) fragments be converted into two single strands (ssDNA) prior to dispersal, the dPCR measurements could be biased high by as much as a factor of two. Realizing dPCR's metrological potential therefore requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. To meet this need, we have investigated several approaches to this determination: A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR enzyme. In our hands, only the endonuclease-based approach provides adequately accurate estimates for relatively small ssDNA proportions. We present four (enzyme, assay) pairs that provide self-consistent results for human nuclear DNA extracts. However, the proportion of ssDNA differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the assay's target sequence. While materials extracted by us have no more than 6% ssDNA content even after long storage, a commercially obtained PCR assay calibrant contains ≈18% ssDNA. Graphical abstract.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Evaluating Droplet Digital Polymerase Chain Reaction for the Quantification of Human Genomic DNA: Lifting the Traceability Fog.

Digital polymerase chain reaction (dPCR) end point platforms directly estimate the number of DNA target copies per reaction partition, λ, where the partitions are fixed-location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both λ and the partition volume, V, must be metrologically traceable to some accessible reference system, ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the critical dPCR assumptions that are essential for establishing the SI traceability of λ. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable partition volume specifications. Our colleagues at the National Institute of Standards and Technology (NIST) have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus, neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split samples with both cdPCR and ddPCR platforms can transfer the λ traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR estimates of CRM target concentration.

Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles.

Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. We have successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. We are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials.

Match criteria for human cell line authentication: where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

A Brief Screening and Assessment Tool for Opioid Use in Adults: Results from a Validation Study of the Tobacco, Alcohol, Prescription Medication, and Other Substances Tool.

OBJECTIVES: This secondary analysis evaluated opioid-specific validation results of the Tobacco, Alcohol, Prescription Medication, and Other Substances (TAPS) tool for screening in primary care. METHODS: This study is a secondary data analysis of the TAPS validation study. Performance of the TAPS tool for screening for unhealthy opioid use (with a score of 1+ for heroin and/or prescription opioids representing a positive screen) was evaluated. Discriminative ability was examined in comparison with reference standard measures across the spectrum of unhealthy opioid use: timeline follow-back with and without oral fluid testing identifying past-month use and the modified Composite International Diagnostic Interview for past-year problem use, opioid use disorder (OUD), and moderate-severe OUD. RESULTS: In a sample of 2000 primary care patients, 114 screened positive for opioids on the TAPS tool. With a TAPS cutoff equal to 1+, the TAPS accurately identified past-month use, problem use, any OUD, and moderate-severe OUD (sensitivities = 68%-85%, specificities = 97%-98%, area under the curve = 0.80-0.91). When past-month use was expanded to include timeline follow-back with oral fluid testing, accuracy declined (52% sensitivity [95% confidence interval, 43%-60%], 98% specific [95% confidence interval, 97%-98%]). CONCLUSIONS: While further testing in a larger population sample may be warranted, given their brevity, simplicity, and accuracy when self-administered, the TAPS opioid items can be used in primary care settings for a spectrum of unhealthy opioid use; however, self-disclosure remains an issue in primary care settings.

Implementation of substance use screening in rural federally-qualified health center clinics identified high rates of unhealthy alcohol and cannabis use among adult primary care patients.

BACKGROUND: Screening for substance use in rural primary care clinics faces unique challenges due to limited resources, high patient volumes, and multiple demands on providers. To explore the potential for electronic health record (EHR)-integrated screening in this context, we conducted an implementation feasibility study with a rural federally-qualified health center (FQHC) in Maine. This was an ancillary study to a NIDA Clinical Trials Network study of screening in urban primary care clinics (CTN-0062). METHODS: Researchers worked with stakeholders from three FQHC clinics to define and implement their optimal screening approach. Clinics used the Tobacco, Alcohol, Prescription Medication, and Other Substance (TAPS) Tool, completed on tablet computers in the waiting room, and results were immediately recorded in the EHR. Adult patients presenting for annual preventive care visits, but not those with other visit types, were eligible for screening. Data were analyzed for the first 12 months following implementation at each clinic to assess screening rates and prevalence of reported unhealthy substance use, and documentation of counseling using an EHR-integrated clinical decision support tool, for patients screening positive for moderate-high risk alcohol or drug use. RESULTS: Screening was completed by 3749 patients, representing 93.4% of those with screening-eligible annual preventive care visits, and 18.5% of adult patients presenting for any type of primary care visit. Screening was self-administered in 92.9% of cases. The prevalence of moderate-high risk substance use detected on screening was 14.6% for tobacco, 30.4% for alcohol, 10.8% for cannabis, 0.3% for illicit drugs, and 0.6% for non-medical use of prescription drugs. Brief substance use counseling was documented for 17.4% of patients with any moderate-high risk alcohol or drug use. CONCLUSIONS: Self-administered EHR-integrated screening was feasible to implement, and detected substantial alcohol, cannabis, and tobacco use in rural FQHC clinics. Counseling was documented for a minority of patients with moderate-high risk use, possibly indicating a need for better support of primary care providers in addressing substance use. There is potential to broaden the reach of screening by offering it at routine medical visits rather than restricting to annual preventive care visits, within these and other rural primary care clinics.

Assessment of Screening Tools to Identify Substance Use Disorders Among Adolescents.

Importance: Efficient screening tools that effectively identify substance use disorders (SUDs) among youths are needed. Objective: To evaluate the psychometric properties of 3 brief substance use screening tools (Screening to Brief Intervention [S2BI]; Brief Screener for Tobacco, Alcohol, and Drugs [BSTAD]; and Tobacco, Alcohol, Prescription Medication, and Other Substances [TAPS]) with adolescents aged 12 to 17 years. Design, Setting, and Participants: This cross-sectional validation study was conducted from July 1, 2020, to February 28, 2022. Participants aged 12 to 17 years were recruited virtually and in person from 3 health care settings in Massachusetts: (1) an outpatient adolescent SUD treatment program at a pediatric hospital, (2) an adolescent medicine program at a community pediatric practice affiliated with an academic institution, and (3) 1 of 28 participating pediatric primary care practices. Participants were randomly assigned to complete 1 of the 3 electronic screening tools via self-administration, followed by a brief electronic assessment battery and a research assistant-administered diagnostic interview as the criterion standard measure for Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) diagnoses of SUDs. Data were analyzed from May 31 to September 13, 2022. Main Outcomes and Measures: The main outcome was a DSM-5 diagnosis of tobacco/nicotine, alcohol, or cannabis use disorder as determined by the criterion standard World Mental Health Composite International Diagnostic Interview Substance Abuse Module. Classification accuracy of the 3 substance use screening tools was assessed by examining the agreement between the criterion, using sensitivity and specificity, based on cut points for each tool for use disorder, chosen a priori from previous studies. Results: This study included 798 adolescents, with a mean (SD) age of 14.6 (1.6) years. The majority of participants identified as female (415 [52.0%]) and were White (524 [65.7%]). High agreement between screening results and the criterion standard measure was observed, with area under the curve values ranging from 0.89 to 1 for nicotine, alcohol, and cannabis use disorders for each of the 3 screening tools. Conclusions and Relevance: These findings suggest that screening tools that use questions on past-year frequency of use are effective for identifying adolescents with SUDs. Future work could examine whether these tools have differing properties when used with different groups of adolescents in different settings.

Subthreshold opioid use disorder prevention (STOP) trial: a cluster randomized clinical trial: study design and methods.

BACKGROUND: Preventing progression to moderate or severe opioid use disorder (OUD) among people who exhibit risky opioid use behavior that does not meet criteria for treatment with opioid agonists or antagonists (subthreshold OUD) is poorly understood. The Subthreshold Opioid Use Disorder Prevention (STOP) Trial is designed to study the efficacy of a collaborative care intervention to reduce risky opioid use and to prevent progression to moderate or severe OUD in adult primary care patients with subthreshold OUD. METHODS: The STOP trial is a cluster randomized controlled trial, randomized at the PCP level, conducted in 5 distinct geographic sites. STOP tests the efficacy of the STOP intervention in comparison to enhanced usual care (EUC) in adult primary care patients with risky opioid use that does not meet criteria for moderate-severe OUD. The STOP intervention consists of (1) a practice-embedded nurse care manager (NCM) who provides patient participant education and supports primary care providers (PCPs) in engaging and monitoring patient-participants; (2) brief advice, delivered to patient participants by their PCP and/or prerecorded video message, about health risks of opioid misuse; and (3) up to 6 sessions of telephone health coaching to motivate and support behavior change. EUC consists of primary care treatment as usual, plus printed overdose prevention educational materials and an educational video on cancer screening. The primary outcome measure is self-reported number of days of risky (illicit or nonmedical) opioid use over 180 days, assessed monthly via text message using items from the Addiction Severity Index and the Current Opioid Misuse Measure. Secondary outcomes assess other substance use, mental health, quality of life, and healthcare utilization as well as PCP prescribing and monitoring behaviors. A mixed effects negative binomial model with a log link will be fit to estimate the difference in means between treatment and control groups using an intent-to-treat population. DISCUSSION: Given a growing interest in interventions for the management of patients with risky opioid use, and the need for primary care-based interventions, this study potentially offers a blueprint for a feasible and effective approach to improving outcomes in this population. TRIAL REGISTRATION: Clinicaltrials.gov, identifier NCT04218201, January 6, 2020.

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

Dissemination of the CAPABLE Model of Care in a Medicaid Waiver Program to Improve Physical Function.

BACKGROUND/OBJECTIVES: Of older adults, 42% report problems with daily function, and physical function is the most important consideration for aging individuals. Thus, we implemented a model of care focused on improving physical function and examined health and use outcomes and satisfaction. DESIGN: A 3-year participatory, single-group pretrial/posttrial benchmarked to a usual care cohort that was evaluated prior to the study. SETTING: Four Medicaid home and community-based waiver sites in Michigan. PARTICIPANTS: The participants included 34 clinicians and 270 Medicaid beneficiaries 50 years and older. INTERVENTION: Community Aging in Place, Advancing Better Living for Elders (CAPABLE), an evidence-based model of care that improved physical function in older adults, was implemented using evidence-based strategies. MEASUREMENT: Characteristics (age, race, and sex), health outcomes (comorbidities, instrumental/activities of daily living [I/ADLs], pain, depression, and falls), and emergency department and hospitalization visits preintervention/postintervention and in the usual care cohort were examined. We also measured Medicaid beneficiary's satisfaction with care for those who received CAPABLE. RESULTS: Improved mean ± SD ADLs (preintervention, 8.51 ± 3.08; postintervention, 7.80 ± 2.86; P = .01) and IADLs (preintervention, 6.43 ± 1.31; postintervention, 5.62 ± 1.09; P < .01), a decrease in falls by 14% (from 34.8% preintervention to 20.8% postintervention; P < .01), and fewer hospitalizations (from 0.43 ± 1.51 preintervention to 0.23 ± 0.60 postintervention; P = .03) were found. Post-CAPABLE means were significantly better compared with a usual care cohort for IADLs (6.73 ± 1.27; P < .01) and hospitalizations (0.47 ± 2.66; P < .01). Satisfaction with care was high, and 98.1% recommended CAPABLE as a way to help remain living in the community. CONCLUSION: Improved ADLs and IADLs, a reduction in fall rates, fewer hospitalizations, and high satisfaction with care occurred in this population as a result of the use of CAPABLE. CAPABLE may be one solution to helping vulnerable, low-income older adults with poor physical function to remain living in the community. J Am Geriatr Soc 67:363-370, 2019.

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples.

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.

NIST interlaboratory studies involving DNA mixtures (MIX05 and MIX13): Variation observed and lessons learned.

Interlaboratory studies are a type of collaborative exercise in which many laboratories are presented with the same set of data to interpret, and the results they produce are examined to get a "big picture" view of the effectiveness and accuracy of analytical protocols used across participating laboratories. In 2005 and again in 2013, the Applied Genetics Group of the National Institute of Standards and Technology (NIST) conducted interlaboratory studies involving DNA mixture interpretation. In the 2005 NIST MIX05 study, 69 laboratories interpreted data in the form of electropherograms of two-person DNA mixtures representing four different mock sexual assault cases with different contributor ratios. In the 2013 NIST MIX13 study,108 laboratories interpreted electropherogram data for five different case scenarios involving two, three, or four contributors, with some of the contributors potentially related. This paper describes the design of these studies, the variations observed among laboratory results, and lessons learned.

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits.

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.

Allele frequencies for 27 Y-STR loci with U.S. Caucasian, African American, and Hispanic samples.

A total of 263 U.S. Caucasians, 260 African Americans and 140 U.S. Hispanics or a subset of 31 Caucasians, 32 African Americans, and 32 Hispanics were typed for 27 Y-chromosome short tandem repeat (Y-STR) markers: DYS444, DYS446, DYS449, DYS463, DYS485, DYS490, DYS495, DYS504, DYS505, DYS508, DYS520, DYS522, DYS525, DYS532, DYS533, DYS534, DYS540, DYS556, DYS557, DYS570, DYS575, DYS576, DYS594, DYS632, DYS635, DYS641, and DYS643. Allele frequencies for each locus are reported along with nomenclature based on sequence analysis.

Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation.

Y-chromosome short tandem repeat (Y-STR) markers are being used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA as is present in many sexual assault samples. Usually single copy Y-STR loci produce a single amplicon in single source samples, and thus the observation of multiple peaks at such a locus could suggest to an analyst that a mixture of more than one male contributor is present in the tested sample. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation. Reasons for the presence of duplications at multiple loci within a single sample are explored in the context of Y-STR marker location along the chromosome. True male-male mixtures commonly exhibit more than one locus-specific PCR product across multiple Y-STR loci that are not adjacent to one another on the Y-chromosome. In addition, duplicated loci typically possess alleles that differ by only a single repeat unit and possess similar peak heights.

Results from the NIST 2004 DNA Quantitation Study.

For optimal DNA short tandem repeat (STR) typing results, the DNA concentration ([DNA]) of the sample must be accurately determined prior to the polymerase chain reaction (PCR) amplification step in the typing process. In early 2004, the National Institute of Standards and Technology (NIST) conducted an interlaboratory study to help assess the accuracy of DNA quantitation in forensic DNA laboratories. This study was designed with four primary purposes: (1) to examine concentration effects and to probe performance at the lower DNA concentration levels that are frequently seen in forensic casework; (2) to examine consistency with various methodologies across multiple laboratories; (3) to examine single versus multiple source samples; and (4) to study DNA stability over time and through shipping in two types of storage tubes. Eight DNA samples of [DNA] from 0.05 ng/microL to 1.5 ng/microL were distributed. A total of 287 independent data sets were returned from 80 participants. Results were reported for 19 different DNA quantitation methodologies. Approximately 65% of the data were obtained using traditional slot blot hybridization methods; 21% were obtained using newly available quantitative real-time PCR (Q-PCR) techniques. Information from this interlaboratory study is guiding development of a future NIST Standard Reference Material for Human DNA Quantitation, SRM 2372.

Mitochondrial DNA typing screens with control region and coding region SNPs.

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.