Validation studies for the genetic typing of the D1S80 locus for implementation into forensic casework

A series of validation experiments were designed to evaluate, according to the Technical Working Group on DNA Analysis Methods (TWGDAM) guidelines, the analysis of the D1S80 locus for casework implementation. Approximately 400 samples from three different populations (Minnesota Caucasian, Minnesota African Americans, and Minnesota Native Americans) were typed to determine allele frequencies. Simulated forensic type specimens (blood, saliva, hair and semen, or vaginal secretions) were typed to demonstrate that deoxyribonucleic acid (DNA) extracted from various tissues of an individual yield the same D1S80 type. Dilution studies were performed and it was determined that a wide range of input DNA (0.5 ng to 40.0 ng) will consistently yield typeable results. The evaluation of DNA from various animals showed that the D1S80 locus is specific to human DNA within the limits of the parameters tested. The reproducibility of the system was tested by duplicate analysis of approximately 200 population samples. Duplicate samples were analyzed on both horizontal and vertical gel systems. In addition, simulated forensic specimens were analyzed by two independent laboratories: the Minnesota Forensic Science Laboratory (MFSL) and the Roche Biomedical Laboratories (RBL). All analyses, including extraction, quantitation, amplification and typing, were performed independently. All typing results for both laboratories were in agreement. By the analysis of mixtures from various simulated casework type mixtures, it was demonstrated that the D1S80 typing system is suitable for analyzing mixtures. In addition to the simulated casework, evidentiary samples from several adjudicated cases previously analyzed by restriction fragment length polymorphism (RFLP) analysis and/or DQA1 were typed at the D1S80 locus. The D1S80 results were consistent with previous RFLP and/or DQA1 results regarding inclusions/exclusions.     

Spectral measurements of intercalated PCR-amplified short tandem repeat alleles

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.     

Hypervariable digital DNA codes for human paternal lineages: MVR-PCR at the Y-specific minisatellite, MSY1 (DYF155S1)

We describe the first haploid minisatellite, the human Y chromosome-specific locus, MSY1. It consists of an array of 48-114 AT-rich 25 bp repeats of at least five different variant types. A minisatellite variant repeat PCR (MVR-PCR) system gives Y-specific DNA codes, with a virtual heterozygosity of 99.9%, making MSY1 by far the single most variable locus on the Y. African populations contain the most diverged MSY1 structures. MSY1 is the only Y-chromosomal system where the characteristics of large numbers of mutations can be studied in detail: it provides a uniquely powerful tool both for the investigation of mutation in a haploid system, and for the dating of paternal lineages.     

Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.     

TWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis

Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.     

Arteriolar remodeling in skeletal muscle of rats exposed to chronic hypoxia

Simple and rapid detection of HLA-DRB polymorphism has been performed using AMPLICOR HLA-DRB Typing Kit. We tried to apply this kit to various forensic samples. When DNA was extracted from the forensic samples using conventional phenol-chloroform method, addition of 7.5 mM MgCl2 was required to PCR amplification. HLA-DRB types were detected from DNA more than 0.1 ng by PCR amplification. Typing of unrelated 50 Japanese showed 38 different patterns, of which 30 patterns occurred once in the group. A total of 16 serotypes were deduced from the HLA-DRB DNA types. Out of them, high frequency serotypes were DR4 (24%), DR9 (18%) and DR15/16 (14%). This kit was very useful in forensic cases such as rape and in paternity cases. When we tried to detect HLA-DRB types from a single hair shaft of 3 cm in length, we were successful in detection from only one of five persons.     

Chronic renal insufficiency and secondary hyperparathyroidism in rats. Biochemical and histological evaluation

Rearrangement of the BCL-2 gene is the molecular consequence of the t(14;18) chromosomal translocation, which is found in approximately 60-90% of follicular lymphomas. To investigate the ability of the polymerase chain reaction (PCR) to detect this rearrangement in fixed-tissue samples, we studied 48 cases of follicular lymphoma using DNA extracted from paired samples of fresh-frozen tissue and formalin-fixed, paraffin-embedded tissue. A standard phenol-chloroform DNA extraction method was used for both types of tissue. Rearrangements of the major breakpoint region (MBR) and minor cluster sequence (MCS) were examined. Three segments of the human beta-globin gene were also amplified to estimate the degree of DNA degradation in the fixed-tissue samples. PCR of fresh-tissue (intact) DNA revealed amplifiable products in 29 of the 48 follicular lymphomas (60%), whereas the fixed-tissue (degraded) DNA studies were positive in 24 (50%). MBR products were detected in 24 fresh-tissue samples, and varied from 80 bp to > 1.5 kb. Twenty of these cases yielded MBR products in the corresponding fixed-tissue DNA, ranging from 80 to 276 bp. Five fresh-tissue and four fixed-tissue samples produced MCS segments that ranged from 340 bp to 1.2 kb. Four of the five samples with no detectable MBR or MCS translocations using degraded DNA had products greater than 1.0 kb in the fresh-tissue studies. A 175-bp segment of the beta-globin gene was amplified in all 29 fixed-tissue samples; a 324 bp fragment was produced in 20 samples (69%), and a 676 bp segment was detected in 13 (45%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Titration of varicella-zoster virus DNA in throat swabs from varicella patients by combined use of PCR and microplate hybridization

We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.     

Evaluation of the ALF DNA sequencer for high-speed sizing of short tandem repeat alleles

DNA typing of short tandem repeat (STR) loci with automated real-time analysis of the fluorescent polymerase chain reaction (PCR) products has given forensic DNA analysis a new dimension. In the present work, the ALF DNA sequencer was evaluated for automated size determination of tetra-nucleotide STRs at high speed. Short gel plates, with a well-to-laser distance of 10 cm, allowed for the analysis of four STR loci (HUMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size determination was done with two external allelic ladders for each locus. Lane-to-lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR loci. The accuracy of sizing and allele detection within and between different gels was high (99.89%) for all four STR systems investigated and the gels could be reloaded without a decrease in accuracy of the allele size estimation. This way, the throughput of the system was increased, which is of interest for linkage studies, gene mapping, and population diversity studies.     

Optimization of a hexaplex DNA amplification from short tandem repeat and amelogenin loci

An automated DNA profiling system based on the multiplex amplification of highly polymorphic short tandem repeat (STR) markers and the amelogenin locus was developed. Five STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplifications, including HUMD1S103, HUMTH01, HUMD21S11, HUMD18S51, and HUMFIBRA. One primer for each locus was labeled with a fluorescent dye (fluorescein) which allows detection on the single wavelength ALF DNA Sequencer (Pharmacia Biotech). As part of the detailed evaluation of the suitability of the hexaplex system for routine forensic use, the effect of variation in amplification parameters on the efficiency of the system was examined. Polymerase chain reaction amplification conditions were optimized to provide specific, robust amplification of forensic samples.     

DYS19 and amelogenin in artificial blood stains with defined amounts of male and female cells

The sensitivity of the DYS19 and the amelogenin STR systems for amplifying Y-specific fragments was assayed using artificial bloodstains with varying amounts of male and female (non-template) DNA in different ratios. The study confirmed the high sensitivity of both systems in detecting male-specific PCR fragments in stains containing 10-25 template molecules even in the presence of large amounts of female DNA in the mixture by silver-stain detection. However, blood mixtures which contain less than 10% male cells could be reliably typed only when at least 100 template molecules were present in the artificial bloodstain, due to increasing amounts of hemoglobin from the female blood which is a PCR inhibitor.     

Amplification of a Y-chromosomal DNA sequence by the polymerase chain reaction for documentation of residual recipient cells in small samples from bone marrow, peripheral blood and cerebrospinal fluid after bone marrow transplantation

The long arm of the human Y-chromosome contains about 800 to 5000 copies of the tandemly repeated DNA sequence DYZ1. A major part of the repeating unit (pHY10) has been cloned and sequenced. Primers were designed to match a part of this repeat sequence for the amplification of a 154 bp fragment spanning the EcoRI restriction site of the unit. Typical dilution experiments showed that this polymerase chain reaction (PCR) method allows the detection of 5 to 10 male cells among 100,000 female cells, or in 500 microL of cerebrospinal fluid containing only one cell per microL. In addition, the quality of the DNA used for the amplification reaction is less critical, thus allowing analysis of long-term stored samples such as bone marrow smears or dried blood stains spotted onto filter paper, which might contain partially degraded DNA. We applied this technique to detect residual host cells in the clinical setting of human sex-mismatched bone marrow transplantation (BMT). Fourteen patients, receiving transplantations because of leukemias could be supervised so far. Throughout the whole period of monitoring (days +14 until +911 post BMT; median: 160 days), residual host cells were detected in all but three patients. Persistence of host cells in the early phase post-BMT was mostly transient and probably due to long-term surviving host T-lymphocytes. Reappearance of host cells several months after BMT is highly suspicious of relapse from the underlying malignancy. Due to its high sensitivity, PCR is a valuable tool in monitoring the switch from recipient to donor cell population.(ABSTRACT TRUNCATED AT 250 WORDS)     

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.     

Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts

Techniques have been developed for extracting, amplifying and directly sequencing mitochondrial DNA (mtDNA) from human hair shafts. The hair shaft is ground in a glass micro-tissue grinder, and the DNA is extracted with organic solvent and purified by filtration. The filtrate subsequently provides the mtDNA template for the PCR. The two hypervariable segments of the mtDNA control region are amplified in four separate reactions. After a purification step to remove unincorporated PCR primers, amplified products are quantitated by capillary electrophoresis and subjected to cycle sequencing. The products are separated and analyzed on an automated DNA sequencer. The mtDNA sequences from the hair shaft match the mtDNA sequences from blood samples taken from the same donor.     

Sequence variation of a variable short tandem repeat at the D18S535 locus

A short tandem repeat in the D18S535 locus was sequenced in 25 selected alleles. A total of 8 different alleles were found which can be designated according to the total number of repeats. This STR is a simple hypervariable STR consisting of blocks of (GATA) repeats with a basic sequence structure (GATA)1(GACA)1(GATA)1 (GAT)1(GATA)9-16. Population data showed that this is a highly polymorphic STR with a heterozygosity of more than 0.80, a simple structure and small size (130-158 bp) which makes this an interesting DNA polymorphism for forensic and genetic purposes.     

Virulent and avirulent Rhodococcus equi infection in T-cell deficient athymic nude mice: pathologic, bacteriologic and immunologic responses

We report a study of polymorphism for seven short tandem repeat (STR) loci in Japanese and Chinese populations. Among 104 to 134 individuals in the both population samples, eight alleles were revealed for locus PLA2, thirteen for D3S1359, eleven for FGA, eight for D8S315 (kw38), ten for D8S1132, five for CYP19, and seven for D3S2459. They correspondingly constituted 10 to 39 genotypes therein. For most of the STRs, there was only a single allele active as the most frequent one among the others, except locus D3S1359 in Chinese samples (two alleles, 206 bp and 210 bp, frequency = 0.273 each). Also, the population genotype configurations were locus specific, varying in the patterns of commonest genotypes on each locus, e.g., one pattern for loci CYP19, D3S1359, and D8S315, one and two for loci PLA2 and D3S2459, two for locus D8S1132, and one and four for locus FGA. The distributions of observed genotypes were in Hardy-Weinberg Equilibrium. Furthermore, the seven STRs were exhibited highly polymorphic and informative for the both populations, and the alleles could be easily separated in electrophoresis and correctly interpreted with side-to-side allelic ladders. Together, the results suggest that the tri- and tetra-meric STRs are useful genetic markers for forensic practice.     

Analysis of nine short tandem repeat (STR) loci in the Slovenian population

A polymerase chain reaction (PCR)-based short tandem repeat (STR) system consisting of nine loci has recently been introduced in Slovenia for use in routine forensic identity testing. Fluorescently labelled PCR products were analysed using an ABI PRISM 310 Genetic Analyzer. The STR loci analysed exhibit between 6 and 14 observed alleles per locus and have a combined matching probability of 2.3 x 10(-10).     

Use of polymerase chain reaction DNA studies in Hungarian legal practice

The authors survey the application of polymerase chain reaction (PCR)-based DNA polymorphisms in the Hungarian forensic practice. The combined application of the presented 17 PCR-based sequence- or length-polymorphic DNA systems to criminal cases gives the power of individualization to the hand of the forensic scientist. The joint application of these genetic markers to disputed paternity cases enables the verification of paternity for an unexcluded man with the highest legal category, namely "paternity practically proved". The investigation of sex-chromosome linked STRs and/or the analysis of mitochondrial DNA (mtDNA) D-loop region is useful in solving most of the problematic cases. The highlighted advantages of PCR over restriction fragment length polymorphism (RFLP)-based forensic DNA analyses clearly explain the overwhelming spread of PCR-based methods in the Hungarian forensic practice.     

Detection of pathogenic fungi in human blood by the polymerase chain reaction

The ability of the polymerase chain reaction (PCR) to detect pathogenic fungi in human blood was investigated. A DNA fragment of about 300 bp from the 18S rDNA, highly conserved in all fungi, was amplified with target DNA from 18 different species of fungi commonly isolated from clinical samples. The presence of PCR products was confirmed by hybridization with a fluorescein-labelled internal probe (21-mer). The PCR assay described is sensitive enough to detect 125 fg of purified Candida albicans DNA and 10 to 100 yeast cells per millilitre of blood.     

Detection of vancomycin-resistant enterococci in fecal samples by PCR

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.