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.

The National Institute of Standards and Technology (NIST), formerly the National Bureau of Standards, was established by the U.S. Congress in 1901 and charged with establishing a measurement foundation to facilitate U.S. and international commerce. NIST provides reference materials and reference instruments that are used to ensure the accuracy, metrological traceability, and comparability of measurement results in many diverse fields of science, industry, and technology. This document provides and explains the vocabulary used at NIST to describe the reference materials and reference instruments available to NIST s customers. This document also describes the methods used in developing NIST reference materials. The definitions and descriptions provided supersede those used in the original versions of this publication, NIST SP260-136 and SP260-136-2020.

Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately.Such identification is not intended to imply recommendation

From 1984 to 2017, the National Institute of Standards and Technology (NIST) Micronutrients Measurement Quality Assurance Program (MMQAP) coordinated 79 Round Robin (RR) interlaboratory studies designed to improve measurement comparability among laboratories measuring fat soluble vitamins and carotenoids in human serum and plasma. There were 22 participants in the initial study; participation increased to 58 in 1997 and then declined in stages to 29 by 2017. A total of 138 academic, commercial, governmental, or non-governmental organizations actively participated in at least one RR; two laboratories participated in 77 of the 79 RRs. A total of 350 human serum or plasma samples were distributed in the RRs, typically three to five per RR. One hundred thirty one (131) of these samples were unique materials. While the initial RRs focused on just retinol (vitamin A), ? tocopherol (vitamin E), and ? carotene (provitamin A) over the life of the program 57 vitamin related measurands were reported at least once. Fifteen (15) measurands were reported sufficiently often to enable analysis of measurement performance over time: total retinol, retinyl palmitate, ? tocopherol, ? plus ? tocopherol, total ? carotene, trans ? carotene, total cis ? carotene, total ? carotene, total lycopene, trans lycopene, total ? cryptoxanthin, total lutein, total zeaxanthin, total lutein plus zeaxanthin, and coenzyme Q10. In addition to documenting the number and nature of the MMQAP's participants, measurands, materials, and reported measurements, this report explores the evolution of among-participant concordance and within-participant apparent precision as functions of calendar date documents and the relationships among results reported for materials delivered to participants both as liquid-frozen and lyophilized samples.

Certain commercial entities, equipment, or materials may be identified in this document to describe an experimental procedure or concept adequately.Such identification is not intended to imply recommendation

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coordinated the Micronutrients Measurement Quality Assurance Program (MMQAP) for laboratories that measure fat-soluble vitamins and carotenoids in human serum and plasma.This report describes the design of and results for the Spring and Fall 1987 MMQAP measurement comparability improvement studies: 1) Round Robin IX Fat-Soluble Vitamins and Carotenoids in Human Serum and 2) Round Robin XI Fat-Soluble Vitamins and Carotenoids in Human Serum.The first participant results for Round Robin IX were received April 22, 1987; the last results were received June 23, 1987.The first participant results for Round Robin XI were received July 8, 1987; the last results were received September 4, 1987.The analytes in Round Robin X were selenium and zinc and are not discussed in this report.

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.

Standard Reference Material (SRM) 2372a is intended for use in the value assignment of human genomic deoxyribonucleic acid (DNA) forensic quantitation materials.A unit of SRM 2372a consists of three well-characterized human genomic DNA materials solubilized in 10 mmol/L 2-amino-2-(hydroxymethyl)-1,3 propanediol hydrochloride (Tris HCl) and 0.1 mmol/L ethylenediaminetetraacetic acid disodium salt (disodium EDTA) pH 8.0 buffer (TE -4 ).The three component genomic DNA materials, labeled A, B, and C, are respectively derived from a single male donor, a single female donor, and 1:3 mixture of a male and a female donor.A unit of the SRM consists of one 0.5 mL tube of each component, each tube containing approximately 55 µL of DNA solution.Each of these tubes is labeled and is sealed with a color-coded screw cap.This publication documents the production, analytical methods, and statistical evaluations involved in production of this SRM.

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.

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.

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.

• We report Swedish population data on PowerPlex ESI 17 markers and compare to other population data reported. • We report probability and power of exclusion. • Expected and observed heterozygosity values, p-values from test of Hardy-Weinberg equilibrium are described. • Sequence data on the new allele 16.1 in D1S16156 is described.

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.

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.