Optimizing Storage and Handling of DNA Extracts.

Nucleic acid sample storage is of paramount importance in forensic science as well as in epidemiological, clinical, and genetic laboratories. Millions of biological samples, including cells, viruses, and DNA/RNA, are stored every year for diagnostics, research, and forensic science. PCR has permitted the analysis of minute sample quantities. Samples such as bone, teeth, touch samples, and some sexual assault evidence may yield only low-quality and low-quantity DNA/RNA. Efficient storage of the extracted DNA/RNA is needed to ensure the stability of the sample over time for retesting of the CODIS STRs, mtDNA, YSTRs, mRNA, and other future marker-typing systems. Amplification of some or all of these markers may fail because the biological material has been highly degraded, contains inhibitors, is too low in quantity, or is contaminated with contemporary DNA. Reduction in recovery has been observed with refrigerated liquid DNA extracts and also those exposed to multiple freeze-thaw cycles. Therefore, the development of optimal storage and amplification methods is critical for successful recovery of profiles from these types of samples since, in many cases, retesting is necessary. This review is divided into three sections. The Introduction and Background covers forensic DNA storage, factors that influence DNA stability, and a brief review of molecular strategies to type non-optimal DNA. Section I covers the importance of DNA extract storage in forensic and non-forensic DNA databanks and the mechanisms responsible for loss during storage. Finally, Section II covers strategies and technologies being utilized to store DNA.

The impact of additional Y-STR loci on resolving common haplotypes and closely related individuals.

Commercial Y-STR kits have permitted laboratories to go beyond the original nine minimal haplotype loci (MHL) and to discover the advantage of additional Y-STR loci in resolving common haplotypes. In an effort to examine the impact of Y-STR markers beyond the 17 loci now available in commercial kit form, new Y-STR loci are being investigated on a common set of samples representative of the major U.S. population groups. Additional Y-STRs can also increase the power of discrimination between closely related male individuals, which is important not only in forensics but also in the paternity and genetic genealogy communities.

Turkish population data with the CODIS multiplex short tandem repeat loci.

Allele frequencies for 13 tetrameric short tandem repeat (STR) loci, CSF1PO, D18S51, D3S1358, D21S11, D5S818, FGA, D7S820, HUMTH01, D8S1179, TPOX, D13S317, VWA, and D16S539 were determined on 198 Turkish blood samples.

NIST mixed stain studies #1 and #2: interlaboratory comparison of DNA quantification practice and short tandem repeat multiplex performance with multiple-source samples.

The Mixed Stain Study 1 (MSS1, Apr.-Nov. 1997) and Mixed Stain Study 2 (MSS2, Jan.-May 1999) evaluated multiplexed short-tandem repeat (STR) DNA typing systems with samples containing DNA from more than one source. These interlaboratory challenge studies evaluated forensic STR measurement, interpretation, and reporting practice using well-characterized samples of very different analytical difficulty. None of the relatively few errors reported in either exercise resulted in a false identification of a reference source; several errors in evaluating the unknown source in three-source samples would hinder matching the profile in any archival database. None of the measurement anomalies reported is associated with any particular STR multiplex; all DNA amplification anomalies are associated with inefficient DNA extraction, inaccurate DNA quantitation, and/or analytical threshold policies.

A review of the DNA standard reference materials developed by the National Institute of Standards and Technology.

The Standard Reference Materials Program at the US National Institute of Standards and Technology (NIST) has three human DNA standard reference materials (SRM 2390, SRM 2391a, and SRM 2392) currently available [1, 2]. Both the DNA profiling SRM 2390 and the polymerase chain reaction (PCR)-based DNA profiling SRM 2391a are intended for use in forensic and paternity identifications, for instructional law enforcement, or for non-clinical research purposes and are not intended for clinical diagnostics. The mitochondrial DNA (mtDNA) SRM 2392 is to provide standardization and quality control when performing PCR and sequencing any segment or the entire 16,569 base pairs that comprise human mitochondrial DNA. SRM 2392 is designed for use by the forensic, medical, and toxicological communities for human identification, disease diagnosis or mutation detection.

Preparation and value assignment of Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum.

Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for retinal, delta-, gamma-, and alpha-tocopherol, trans- and total beta-carotene, and cholesterol in human serum. Values are also reported for 16 additional compounds including lutein, zeaxanthin, alpha- and beta-cryptoxanthin, lycopene, alpha-carotene, retinyl palmitate, and 25-hydroxyvitamin D. The certified values for the fat-soluble vitamins and carotenoids in SRM 968c were based on the agreement of results from the means of at least two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the medians from an interlaboratory comparison study among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol in the SRM are the means of results obtained using the NIST definitive method, gas chromatography-isotope dilution mass spectrometry.

NIST micronutrients measurement quality assurance program: characterizing the measurement community's performance over time.

The Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology was created in 1984 with the goal of improving among-participant measurement comparability for fat-soluble vitamin-related compounds in human serum. We recently described improved tools for evaluating comparison exercise data; we here extend and apply these tools to the evaluation of the measurement community's performance over the entire 15-year history of the M2QAP. We here display measurement performance characteristics for the 14 measurands most commonly reported by the M2QAP community. We confirm that among-participant comparability for total beta-carotene cannot be much improved without improving average long-term within-participant measurement stability. We demonstrate that improved measurand definition and/or identification of interferences may help participants improve comparability for many of the M2QAP's other commonly reported measurands. The reported measurement performance characteristics may be of interest to clinical, nutritional, and epidemiological studies involving any of these measurands. The data analysis techniques utilized may be applicable to other programs.

NIST micronutrients measurement quality assurance program: characterizing individual participant measurement performance over time.

The mission of the Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology is enhanced interlaboratory measurement comparability for fat-soluble vitamin-related measurands in human serum. We recently described improved tools for evaluating individual participant measurement performance in single interlaboratory comparison exercises; we here apply and extend these tools to the evaluation of participant performance over the entire 15-year history of the M2QAP. We describe and illustrate a set of interconnected graphical reporting tools for identifying long-term trends and single-exercise events. We document and discuss recurrent patterns we observe in the measurement performance characteristics for M2QAP participants. The graphical analysis techniques utilized may be applicable to other interlaboratory comparison programs.

Micronutrients Measurement Quality Assurance Program: helping participants use interlaboratory comparison exercise results to improve their long-term measurement performance.

Over the past decade, the Micronutrients Measurement Quality Assurance Program (M2QAP) at the National Institute of Standards and Technology (NIST) has administered nearly 40 interlaboratory comparison exercises devoted to fat-soluble vitamin-related analytes in human serum. While M2QAP studies have been used to help certify reference materials and to document the performance of analytical systems, the primary focus of the M2QAP has been, and remains, the improvement of among-participant measurement comparability for target analytes. Recent analysis of historical measurement performance indicated the most efficient mechanism for further improving measurement comparability among participants is the improvement of long-term (months to years) comparability within each laboratory. The summary reports for the M2QAP studies are being redesigned to provide more chemist-friendly analyses of participant performance, dissecting systematic and random components of measurement incomparability as functions of analyte level and time. This report documents the semantic and graphical tools developed to help interlaboratory-comparison-exercise participants interpret their own measurement performance.

The stability of retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in liquid-frozen and lyophilized serum.

The concentrations of retinol, alpha-tocopherol, and trans-beta-carotene in lyophilized serum stored at -25 degrees C and -80 degrees C have been monitored for 10 years. There was no evidence of degradation of any of these compounds over the 10-year period. Retinol, alpha-tocopherol, and trans-beta-carotene were less stable at -25 degrees C in liquid-frozen serum than they were in lyophilized serum. At -80 degrees C, trans-beta-carotene levels were stable for up to 3 years of storage in liquid-frozen serum. Both retinol and alpha-tocopherol appeared stable in liquid-frozen serum for at least 5 years at -80 degrees C. The effect of repeated freeze/thaw cycles on retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in liquid-frozen and reconstituted lyophilized serum both stored at -20 degrees C was also studied. Retinol, alpha-tocopherol, trans-lycopene, and trans-beta-carotene in reconstituted lyophilized serum stored at -20 degrees C were stable for at least 3 days with minimal (< 5) freeze/thaw cycles.

Interlaboratory evaluation of short tandem repeat triplex CTT.

An interlaboratory comparison of typing results for Short Tandem Repeats (STRs) at the GenBank loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMVWFA31 using the "CTT triplex" and "CTTv quadruplex" has been evaluated. These STRs all have a nominal four basepair (bp) repeat. Seven different samples were distributed to 41 laboratories. The 34 laboratories that returned results used a wide variety of analytical systems. Comparable results were obtained for all samples at all loci when results were reported as an allelic name. Raw sizing results obtained from internal-lane sizing standards differed by nearly five bp at some loci. Many different factors contribute to this observed sizing variability, including choice of sizing standards and matrix composition. Although sizing results can be made more comparable by locus-specific offsets or calibration to a comprehensive set of alleles at each locus, samples typed to the allelic name can now be validly compared regardless of analytical method. Interlaboratory comparison of raw allelic size remains problematic.

NIST/NCI Micronutrients Measurement Quality Assurance Program: measurement repeatabilities and reproducibilities for fat-soluble vitamin-related compounds in human sera.

The NIST/NCI Micronutrient Measurement Quality Assurance Program has conducted 33 interlaboratory comparison exercises for fat-soluble vitamin-related compounds in human sera over the past 12 years. Periodic reanalysis of lyophilized serum samples prepared from more than 70 different sera has enabled estimation of the short- and long-term measurement characteristics. Median- and interquartile-range-based statistics adequately estimate the distribution of results from laboratories that are in analytical control from total distributions that include a significant minority of outlier data. Short-term interlaboratory reproducibility standard deviations (SDs) are predictable functions of analyte concentration, with an asymptotic limit at low analyte concentration and a linear relationship at high concentrations. Long-term trends in the interlaboratory reproducibility can be estimated by standardizing the short-term SD at the observed analyte concentration to an expected SD at a given physiologically significant analyte concentration. The "average" laboratory's same-day analytical repeatability SD is about one-third of the estimated interlaboratory reproducibility; repeatability for longer periods between analyses is, on average, on better than the reproducibility. While a few exceptional laboratories have maintained excellent repeatability over the entire decade, long-term study measurements generated within a single laboratory are not generally more internally consistent than results from multiple laboratories. Enhanced and more consistently implemented intralaboratory quality control and quality assurance methods are required to further improve and maintain interlaboratory measurement comparability.

Certification of fat-soluble vitamins, carotenoids, and cholesterol in human serum: Standard Reference Material 968b.

In Standard Reference Material 968b, fat-soluble vitamins and cholesterol in human serum, certified values are provided for cholesterol, retinol, retinyl palmitate, alpha-tocopherol, trans-beta-carotene, total beta-carotene ( trans plus cis isomers), total alpha-carotene, and lutein. Non-certified values are also reported for gamma-tocopherol (includes beta-tocopherol), delta-tocopherol, zeaxanthin, beta-cryptoxanthin, trans-lycopene, trans-lycopene, trans-alpha-carotene, total lycopene, 9- cis-betacarotene, 13- plus 15- cis-beta-carotene, and 15- cis-beta-carotene. Both certified and non-certified values are based on the agreement among results from three different liquid chromatographic analytical procedures developed at NIST and from an interlaboratory comparison exercise among institutions that participate in a NIST-managed Micronutrients Measurement Quality Assurance Program. Cholesterol is certified in this material using the NIST isotope dilution/mass spectrometric definitive method.

Intercomparison of DNA sizing ladders in electrophoretic separation matrices and their potential for accurate typing of the D1S8O locus.

The 100-1,000 basepair size typical of PCR-amplified DNA fragments demands high resolution electrophoretic gels for the adequate characterization of small differences among samples. We have studied the behavior of a number of commercial sizing ladders in three classes of separation systems: polyacrylamides with discontinuous buffer, proprietary acrylamides with continuous buffer, and agarose-like materials with continuous buffer. None of the ladders examined perform adequately in any of these systems using vendor-supplied nominal ladder component basepair sizes. All ladders successfully typed D1S8O alleles after calibration with the allelic ladder (replacing the nominal size values with the least squares estimate of allele/matrix-specific apparent sizes). Some ladders and matrices are qualitatively better than others. No one ladder proved consistently better than others; a polyacrylamide gel with ribose modifier provided the most precise results in this study. Appropriately calibrated electrophoretic apparent sizes must be used for results to be validly exchanged among laboratories. Appropriate allelic ladders or a well defined subset of known alleles can serve as the calibration system.

Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence--capillary electrophoresis.

Recent developments in the chemical synthesis of DNA-binding dyes have enhanced detection of polymerase chain reaction (PCR) products by capillary electrophoresis. These dyes are dimers of thiazole orange (TOTO) or oxazole orange (YOYO) and have a very high binding affinity for DNA (Haugland, 1992). These dyes show enhanced fluorescence signals when they bind to double-stranded DNA and their fluorescence in the unbound state is almost zero, making them extremely useful in detecting minute (fg) quantities of DNA. We report here the utility of these dyes in DNA typing applications using a laser-induced fluorescence detector in conjunction with a capillary electrophoresis system.