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.

STRBase: a short tandem repeat DNA database for the human identity testing community.

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.

Prokaryotic expression, purification, and reconstitution of biological activities (Antiprotease, antitumor, and heparin-binding) for tissue factor pathway inhibitor-2.

We report the expression of tissue factor pathway inhibitor-2 (TFPI-2) (also known as PP-5, placental protein-5; MSPI, matrix-associated serine protease inhibitor) in E. coli as a 25-kDa nonglycosylated protein with a glycine substituted for aspartic acid at the amino terminus. High-level expression of TFPI-2 was obtained with pRE1 expression vector under the transcriptional and translational controls of the lambdaP(L) promoter and lambdacII ribosome-binding site, respectively, with ATG initiation codon. TFPI-2 was produced as inclusion bodies and accounted for 25-30% of the total E. coli proteins. The inclusion bodies containing TFPI-2 were solubilized with urea, sulfitolyzed, purified, and refolded through a disulfide interchange reaction. The refolded E. coli TFPI-2 inhibited plasmin with an inhibition constant (K(i)) of 5 nM that is similar with the TFPI-2 expressed in a mammalian system. The refolded E. coli TFPI-2 bound heparin and also inhibited plasmin, regardless of whether the enzyme was in the fluid phase or was bound to the membranes of HT-1080 fibrosarcoma cells. In addition, refolded E. coli TFPI-2 inhibited radiolabeled matrix degradation and Matrigel matrix invasion by HT-1080 fibrosarcoma cells and B16-F10 melanoma cells. Together, our results suggest that glycosylation is not essential for antiprotease, antitumor, and matrix-binding activities of TFPI-2. Based on these collective data, we conclude that a biologically active nonglycosylated TFPI-2 can be produced in E. coli and that the protein can be produced in high-enough quantities to conduct in vivo studies for determination of the role of this inhibitor in tumor invasion and metastasis.

RFLP band size standards: NIST standard reference material 2390.

The procedural standard for DNA profiling developed by the U.S. advisory board on DNA quality assurance methods mandates annual confirmation of forensic DNA measurement systems against an appropriate reference material supplied by or traceable to the National Institute of Standards and Technology (NIST). NIST Standard Reference Material (SRM) 2390 is a suitable and appropriate standard for HaeIII restriction enzyme-based restriction fragment length polymorphism (RFLP) profiling systems. Originally issued in 1992, an among-laboratory SRM 2390 recertification study was initiated in 1997. Using data provided by the 20 state, local, or commercial forensic laboratory participants, quantitative band sizes values (expected mean values and associated bivariate tolerance intervals) are established for two different-source DNAs (female cell line K562 and healthy male "TAW") for genetic loci D1S7, D2S44, D4S139, D5S110, D1OS28, and D17S79. Methods for validating an RFLP measurement system, validating a control material or other secondary standard, and for tracing a particular set of RFLP measurements to NIST SRM 2390 are described in detail.

RFLP band size standards: cell line K562 values from 1991 to 1997 proficiency studies.

Cell line K562 is the defacto forensic control material for forensic restriction fragment length polymorphism (RFLP) DNA profiling in the U.S. Fifty-one proficiency tests conducted from 1991 through 1997 enable a detailed description of RFLP measurement performance during this period. Sufficient data are available to define reference distributions for all commonly utilized and many less commonly reported genetic loci, for both HaeIII- and HinfI-based RFLP systems. The average measured size of HaeIII locus D1S7 and D5S110 bands has varied slightly over time; while relatively small, these temporal changes add to the overall interlaboratory measurement uncertainty. The characteristic standard deviation for HinfI RFLP system measurements has a nearly identical dependence on expected band size as does the standard deviation for HaeIII measurements. The ellipsoidal distance, K, is suggested for use as an RFLP data quality metric; the critical threshold value that on average excludes 1% of plausibly valid proficiency data for a given polymorphic locus is K1% = 14.2.

Long PCR for VNTR analysis.

The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.

Impact of DNA typing on standards and practice in the forensic community.

This article reviews the history of DNA-based human identification from its inception in 1985. Since the development of the technology, experts called for setting of standards and use of proficiency tests for quality assurance measures. The response of the National Institute of Standards and Technology to DNA forensic standards needs was catalyzed by the Technical Working Group on DNA Analysis Methods, sponsored by the Federal Bureau of Investigation with funding provided by the National Institute of Justice. Standard reference materials were developed for the original technologies used in DNA identification and for the newer polymerase chain reaction-based technologies. Adoption of recommended standards developed through the Federal Bureau of Investigation-commissioned DNA Advisory Board show the acceptance of National Institute of Standards and Technology standards for calibration of laboratory protocols. New technologies will require a process of validation and continued testing through the use of proficiency tests, such as those provided through the College of American Pathologists. Robotics and parallel processing of samples will lead to increased efficiency in DNA testing. The use of DNA data banks of convicted felons will increase dramatically with the the Federal Bureau of Investigation's national implementation of a computerized identification system known as the Combined DNA Index System. This system that will make major use of short, tandem, repeat genetic systems and will be the major driver of technology for the next 5 to 10 years. Finally, sample collection and training are of major concern for those who look at the long-term impact of DNA testing in forensic laboratories.

Graphical tools for RFLP measurement quality assurance: laboratory performance charts.

Forensic restriction fragment length polymorphism analyses typically provide two band size results at each genetic locus for each sample. In collaboration with the member laboratories of the Technical Working Group for DNA Analysis Methods, we have developed graphical techniques that compactly summarize even large numbers of such paired measurements. This paper provides a detailed description of the basic tool, a modified bivariate control chart for data from multiple samples and/or multiple loci. We demonstrate how various modifications and combinations of these "laboratory performance charts" can be used for quality control, quality assurance, and quality demonstration.

A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection.

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM 2392 includes DNA from two lymphoblast cell cultures (CHR and 9947A) and cloned DNA from the CHR HV1 region, which contains a C stretch and is difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area or the entire mtDNA (16,569 bp) to be amplified and sequenced. While none of the differences in these three templates correspond to published mutations associated with specific diseases, some of these differences did result in animo acid changes compared with that published by S. Anderson et al. (1981, Nature 290: 457-465). An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified and sequenced correctly.

Interlaboratory comparison of autoradiographic DNA profiling measurements: precision and concordance.

Knowledge of the expected uncertainty in restriction fragment length polymorphism (RFLP) measurements is required for confident exchange of such data among different laboratories. The total measurement uncertainty among all Technical Working Group for DNA Analysis Methods laboratories has previously been characterized and found to be acceptably small. Casework cell line control measurements provided by six Royal Canadian Mounted Police (RCMP) and 30 U.S. commercial, local, state, and Federal forensic laboratories enable quantitative determination of the within-laboratory precision and among-laboratory concordance components of measurement uncertainty typical of both sets of laboratories. Measurement precision is the same in the two countries for DNA fragments of size 1000 base pairs (bp) to 10,000 bp. However, the measurement concordance among the RCMP laboratories is clearly superior to that within the U.S. forensic community. This result is attributable to the use of a single analytical protocol in all RCMP laboratories. Concordance among U.S. laboratories cannot be improved through simple mathematical adjustments. Community-wide efforts focused on improved concordance may be the most efficient mechanism for further reduction of among-laboratory RFLP measurement uncertainty, should the resources required to fully evaluate potential cross-jurisdictional matches become burdensome as the number of RFLP profiles on record increases.

Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence.

Increased sensitivity and improved quantitation of analytical tests used in biotechnology and clinical chemistry are goals of many laboratories. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell line in a background of mRNA from breast cancer cells. An electrochemiluminescence detection procedure was used as a readout system for this study. Biotinylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine)ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hybrid was subsequently captured by streptavidin-coated Dynabeads. When either the QPCR System 5000 or the Origen 1 Analyzer System were used, the luminescence emitted by the TBR-chelate of the captured specific post-PCR product was assessed. Tyrosinase-specific mRNA isolated from approximately 1-10 melanoma cells in a background of 10(7) cells could be detected. We improved the sensitivity and logistics of the assay through the use of rTth for reverse transcription and amplification. Tyrosinase mRNA was detected in blood from 7 of 16 melanoma patients, whereas none of the 5 healthy donor bloods were positive (P = 0.01; Wilcoxon test).

Rapid mitochondrial DNA typing using restriction enzyme digestion of polymerase chain reaction amplicons followed by capillary electrophoresis separation with laser-induced fluorescence detection.

The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expense of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR-amplified mtDNA using RsaI and MnlI and then capillary electrophoresis (CE) to separate and size the PCR-RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T-->C transition at position 16189, which gives rise to the so-called "C-stretch" in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.

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.

Interlaboratory comparison of autoradiographic DNA profiling measurements. 4. Protocol effects.

The observed total interlaboratory uncertainty in restriction fragment length polymorphism (RFLP) measurements is sufficiently small to be of little significance given current forensic needs. However, as the number of RFLP data increase, further reduction in the total uncertainty could help minimize the resources required to evaluate potential profile matches. The large number of data available enable quantitative estimation of the within-laboratory imprecision and among-laboratory bias contributions to the total uncertainty. Some small but consistent among-laboratory measurement biases can be attributed to specific procedural or materials differences. The bias direction is often fragment-specific and thus unpredictable for unknown samples. Actions that would minimize currently recognized sources of interlaboratory bias include the following: (1) all laboratories should use the same algorithm for data interpolation, (2) all laboratories should use the same sizing ladders, (3) each laboratory should prepare control DNA and sample DNA in the same manner and with the identical reagents, (4) all laboratories should adopt a uniform policy on ethidium bromide use, and (5) all laboratories should adopt the same control DNA sizing acceptability criteria.

Interlaboratory comparison of autoradiographic DNA profiling measurements. 3. Repeatability and reproducibility of restriction fragment length polymorphism band sizing, particularly bands of molecular size > 10K base pairs.

The observed interlaboratory standard deviation (SD) associated with the restriction fragment length polymorphism (RFLP) measurement of DNA fragment size is a predictable function of the observed mean band size (MBS). For DNA fragments of size 1,000 base pairs (bp) to the largest resolved component of commonly used "sizing ladder" calibration materials (about 20,000 bp), the variation in the sizing data from the Technical Working Group on DNA Analysis Methods (TWGDAM)-sponsored "Large Fragment Study" is well-described by SD = 7.5 (1 + MBS/19 500)7.1. This sizing variability arises from a 0.1-0.4% SD in the relative positions of sample and calibration bands among electrophoretic gels. Statistically significant sizing differences do exist for bands above 10,000 bp among laboratories that use different calibration materials. The Large Fragment Study was efficiently accomplished through the use of a designed set of DNA samples, requiring but one gel in each of 20 participating laboratories.

The use of a new gel matrix for the separation of DNA fragments: a comparison study between slab gel electrophoresis and capillary electrophoresis.

TreviGel-500, a new polysaccharide matrix containing AgaCryl, commercially available as a powder for slab gel electrophoresis, is now being applied to the separation of DNA fragments in capillary electrophoresis. The capillary mode allows the use of one to two orders of magnitude lower mass fractions of matrix and approximately five to six orders of magnitude lower sample quantities than the slab gel electrophoresis counterpart for optimal separation of DNA fragments in the 100 to 2,000 base pair size range. In the capillary mode, this new separation matrix forms a semi-rigid gel that demonstrates enhanced selectivity for DNA fragments in the 1,000 to 7,000 base pair size range relative to alternative size-sieving polymer solutions. In addition, this matrix offers the advantages of lower toxicity than acrylamide. Comparisons are drawn between the use of this matrix in both slab gel electrophoresis and capillary electrophoresis for the separation of DNA fragments with respect to the mass fraction of the matrix in buffer, the buffer composition and sample loading or injection parameters.

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.

Local synthesis of apolipoprotein J in the eye.

Apolipoprotein J (apoJ), a secretory glycoprotein known to transport lipids and to regulate terminal complement function, is present in the human eye in both aqueous and vitreous, as well as in the retina. Ocular apoJ is the product of local synthesis, rather than plasma contamination, as demonstrated by its distinct structural properties and the presence of abundant apoJ mRNA in retina and retina pigment epithelium. ApoJ mRNA is also present in mouse eye, with a developmentally regulated pattern of expression. In fetal mouse, apoJ mRNA is present in retina, lens and cornea. In contrast, adult eye apoJ mRNA is present in retina and ciliary body. We propose that apoJ is important in tissue remodeling and in stabilizing hydrophobic molecules which are required for vision and/or which would otherwise be deleterious and membrane-active.