Lack of association between urinary creatinine and ethanol concentrations and urine/blood ratio of ethanol in two successive voids from drinking drivers

Primer extension preamplification (PEP), which can amplify sequence-independently a limited quantity of DNA as a whole, allows multiple DNA analyses by polymerase chain reaction (PCR). This technique may be applicable to forensic cases, especially in cases where only small amounts of DNA are available. To define the accuracy and sensitivity of PEP, the DNA typing results of nine loci (TH01, MCT118, HLA-DQ alpha, amelogenin, LDLR, GYPA, HBGG, D7S8, GC) by PCR with PEP (PEP-PCR) were compared with those by PCR without PEP. Both results were identical to each other for each sample tested. Furthermore, amplification of an initial genomic DNA by PEP was found to range from 15 to 600 times of the initial quantity and the efficiency of PEP may depend on the sequences flanking the loci tested. These results indicate that PEP can increase typing potential of PCR from forensic samples.     

Ultrasound biomicroscopy of the anterior segment of the eyes of infants

Multiplex PCR amplification has been useful for gene mapping with polymorphic short tandem repeat (STR) loci. We have tested the four loci D20S470, D13S325, HumFOLP23 and D10S2325 for the simultaneous typing of more than 100 unrelated Koreans. This analysis allows a single base pair resolution and rapid typing with silver staining. The allele and genotype distributions are in accordance with Hardy - Weinberg expectations. These STR loci have proven useful for forensic analysis and paternity tests in which the variable number of tandem repeat (VNTR) loci have some limitations.     

Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence

The detection of restriction fragment length polymorphisms (RFLP) (1) in DNA extracted from forensic samples remains impossible in a significant number of cases due to deterioration and contamination of the biological material and the extremely low quantities of DNA isolated. The polymerase chain reaction (PCR) is a recent and particularly convenient method for analysing and typing very small amounts (10-20 ng) of degraded human DNA. DNA analysis at the level of a few cells present in forensic samples such as bloodstains, semen stains, vaginal swabs and head hair bulbs now appears possible using DNA amplification. A PCR protocol was adapted to simultaneously amplify a Y-specific DNA repeat sequence from the DYZ1 locus and an X-specific DNA repeat sequence from the DXS424 locus. The co-amplified Y-specific DNA fragment (102 bp) and X-specific DNA fragments (181-199 bp) were visualized on an ethidium bromide-stained 4% agarose gel. The male or female type of the amplified DNA extracted from blood samples, bloodstains, semen stains, vaginal swabs, brain tissue and 1, 2, 5, or 10 head hair bulbs was determined.     

Genetic typing of HLA class II genes in Swedish populations: application to forensic analysis

In an attempt to determine the value of DNA based typing of HLA class II loci to forensic analysis, allele and genotype frequencies at DQA1, DQB1, DPB1, and DRB1 were determined in samples from two Swedish populations using hybridization with sequence specific oligonucleotides to PCR amplified DNA. Significant allele frequency differences were observed at the DQB1 and DRB1 loci between the two populations, as well as between one of the Swedish and a Norwegian population. The average heterozygosity varies between 0.74 to 0.91 and the power of discrimination between 0.90 to 0.98, with the highest values obtained for the DRB1 locus. The probability of genotype identity by chance differs on average 2% between the populations. When applied to a paternity case with one parent deceased and a criminal case, typing of class II loci proved in both cases informative. Analyses of DR and DQ genes does not increase the power of discrimination, due to strong linkage, but offers through the reconstruction of putative haplotypes an internal control for the consistency of the typing results at several loci. Typing of the DRB1 and DPB1 loci was found to result in an approximate combined average probability of genotype identity by chance of one in a thousand.     

A microsatellite-based multilocus phylogeny of the Drosophila melanogaster species complex

Uncovering the genealogy of closely related species remains a major challenge for phylogenetic reconstruction. It is unlikely that the phylogeny of a single gene will represent the phylogeny of a species as a whole [1], but DNA sequence data across a large number of loci can be combined in order to obtain a consensus tree [2]. Long sequences are needed, however, to minimize the effect of (infrequent) base substitutions, and sufficient individuals must be sequenced per species to account for intraspecific polymorphisms, an overwhelming task using current DNA sequencing technology. By contrast, microsatellites are easy to type [3], allowing the analysis of many loci in multiple individuals. Despite their successful use in mapping [4,5], behavioural ecology [6] and population genetics [7], their usefulness for the phylogenetic reconstruction of closely related taxa has never been demonstrated, even though microsatellites are often conserved across species [8-10]. One drawback to microsatellite use is their high mutation rate (10(-4)-10(-2)), combined with an incomplete understanding of their mutation patterns. Many microsatellites are available for Drosophila melanogaster, and they are distributed throughout the genome [11]. Most can be amplified in the D. melanogaster species complex [12,13] and have low mutation rates [14, 15]. We show that microsatellite-specific distance measurements [16] correlate with other multilocus distances, such as those obtained from DNA-DNA hybridization data. Thus microsatellites may provide an ideal tool for building multilocus phylogenies. Our phylogenetic reconstruction of the D. melanogaster complex provides strong evidence that D. sechellia arose first, followed by a split between D. simulans and D. mauritiana.     

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes

Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.     

Application of DNA fingerprinting to obstetrics and gynecology

Restriction fragment length polymorphisms (RFLP) are variations in the size of restriction fragments of genomic DNA that hybridize to specific probes. They are the consequence of changes in primary DNA sequences, most of which result from small-scale changes in DNA. Recently minisatellite DNA probes that detect many regions of great variability within the human genome have been described. Minisatellite probes consist of multiple repeated copies of a common 10-15 base pair core sequence. On hybridization to restriction enzyme digests of human DNA, they simultaneously detect many highly polymorphic minisatellites at different loci in the genome, and produce band patterns that are individual specific. The band patterns are called "DNA fingerprints" or "DNA barcode" which can be used for individual identification on forensic and legal medicine. In addition to forensic and legal medicine, DNA fingerprinting can be used in both basic research and clinical examination of obstetrics and gynecology. The RFLP bands in DNA fingerprinting are inherited as single Mendelian co-dominants and we can use such minisatellite DNA probe for the determination of zygosity in multiple pregnancy. This probe can be used for the determination of androgenesis as a cause of complete hydatidiform mole. Each polymorphic band in molar tissues could be identified as being of paternal but not maternal origin. Some polymorphic bands of paternal origin were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole (androgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complexes

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.     

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.     

Right laparoscopic lumbar sympathectomy]

In three separate shooting incidents involving multiple gunshots, two FMJ bullets and one bullet fragment found at the scene (one from each case) were investigated for the presence of biological material from the victim after perforation. The surface of the missiles, which did not show obvious tissue traces when examined under a macroscope, was swabbed. PCR typing of up to five STR loci was performed on the small amounts of DNA extracted, which were seen below the detection limit of the slot blot quantification in one case. Nevertheless, individualisation of cellular material from the perforating projectiles was successful in each of the three cases presented. Consequently, identification of the victim wounded by a perforating bullet can reliably be achieved if contamination or removal of evidentiary material by improper handling is prevented. This technique is especially useful in cases where more than one person has fired a gun because the bullet carrying DNA can be linked to the firearm by investigation with a comparison microscope. As a by-product of this investigation, a variant allele 14 (14+4) at the VWA locus was detected.     

Analysis of allele distribution for six short tandem repeat loci in the French Canadian population of Québec

Short tandem repeat (STR) loci represent a rich source of highly polymorphic markers in the human genome which are useful for the purposes of forensic identification and determination of biological relatedness of individuals. Here, as a part of an ongoing extensive study, we report the analysis of a multilocus genotype survey of 642 to 870 chromosomes in the French Canadian Caucasian population of Québec at six STR loci. The loci HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMFESFPS, and HUMvWA were typed using two multiplex polymerase chain reactions (PCR). Amplified DNA samples were subsequently analyzed by polyacrylamide gel electrophoresis followed by silver staining. The heterozygote frequencies of the loci range from 0.614 to 0.820 (0.661 to 0.818 expected) and the number of alleles from 7 to 12 per locus. Although statistically significant deviation from Hardy-Weinberg expectations of genotype frequencies was noted at some loci by one or more tests, in general, the genotype frequencies are well estimated from the product of allele frequencies at all loci. The most frequent six-locus genotype is expected to occur in the French Canadian population with a frequency of 3.50 by 10(-5) and together, these six loci have an average probability of discrimination of 0.9999985. The study presented here indicates that these six STR loci are informative genetic markers for identity testing purposes in the French Canadian Caucasian population of Québec.     

Maternal identification from skeletal remains of an infant kept by the alleged mother for 16 years with DNA typing

This is a case study concerning maternal identification by DNA typing at various loci. An infant skeleton was found in the alleged mother's apartment after it was kept for 16 years. We obtained the skeletal remains as well as saliva stains from the alleged mother. DNA typing was conducted for three loci in the HLA class II region (HLA-DQA1, -DPB1, and DRB1), five loci with the AmpliType PM kit (LDLR, GYPA, HBGG, D7S8, and GC), five STR loci (LPL, vWA, F13B, TH01, and TPOX) and D-loop region in mtDNA for maternal identification. Sex determination was accomplished using fluorescent DNA capillary electrophoresis typing. Approximately 5 ng of human DNA was recovered from 1 g of femur bone retrieved from the infant skeletal remains. The probability of two unrelated Japanese sharing the same genotypes was estimated as 7.2 x 10(-11). The combined probability of exclusion that an individual is not the mother was also calculated at 0.998. We therefore conclude that the skeleton is from a female infant, and that there is no inconsistency in the claim that the infant was a daughter of the alleged mother.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

Additional approaches to DNA typing of skeletal remains: the search for "missing" persons killed during the last dictatorship in Argentina

DNA typing techniques are among the most advanced tools for human identification and can contribute to the identification of poorly preserved skeletal remains. Ten thousand people are thought to have been killed during the last dictatorship in Argentina (1976-1983) and there are few official records on the identity of the victims or the location of burials. A mass grave containing 340 skeletons was excavated using archeological methods. A small number of individuals was identified by traditional forensic methods and one family group by mitochondrial DNA (mtDNA) analysis. Due to the lack of antemortem physical information on many of the victims, the application of molecular methods is imperative to speed up the identification process. We have tested two molecular screening methods, Y chromosome-specific short tandem repeats (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) and amplification of autosomal microsatellites using nested primers. These methods can complement solely matrilineal mtDNA sequence data in the identification of "missing" persons.     

Allele frequency distributions at several variable number of tandem repeat (VNTR) and short tandem repeat (STR) loci in a restricted Caucasian population from south Italy and their evaluation for paternity and forensic use

Allele frequencies at six VNTR loci, 11 STR loci, and at the HLA-DQA1 locus were evaluated in a well-defined population from Campania (South Italy). The allele frequencies of three VNTR loci, 11 STR loci, and the HLA-DQA1 locus were compared with data obtained from a general Caucasian reference population in the USA. The aim of this study was to determine the power of each single locus and group of loci for forensic and paternity testing purposes. Significant differences between the allele frequencies of the two populations were found in two VNTR loci, four STR loci and in the HLA-DQA1 locus. The two populations were in Hardy-Weinberg equilibrium for the STR loci, but as expected, not for some VNTR loci. It was also found that: (i) the discriminatory power of two STR systems (nine and 11 loci, respectively) is similar in the two populations analysed; and (ii) that the allele frequencies for the STR systems of a large reference population can always be applied to subjects of a small subpopulation. In conclusion, for forensic purposes and for paternity testing, most of the 11 STR loci examined can be analysed using allele frequencies from a general Caucasian reference population without typing subpopulations, whereas the VNTR loci must be subtyped.     

A selective difference between human Y-chromosomal DNA haplotypes

DNA analysis is making a valuable contribution to the understanding of human evolution [1]. Much attention has focused on mitochondrial DNA (mtDNA) [2] and the Y chromosome [3] [4], both of which escape recombination and so provide information on maternal and paternal lineages, respectively. It is often assumed that the polymorphisms observed at loci on mtDNA and the Y chromosome are selectively neutral and, therefore, that existing patterns of molecular variation can be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. The coalescence of the molecular phylogenies of mtDNA and the Y chromosome to recent common ancestors in Africa [5] [6], for example, has been taken to reflect a recent origin of modern human populations in Africa. An alternative explanation, though, could be the recent selective spread of mtDNA and Y chromosome haplotypes from Africa in a population with a more complex history [7]. It is therefore important to establish whether there are selective differences between classes (haplotypes) of mtDNA and Y chromosomes and, if so, whether these differences could have been sufficient to influence the distributions of haplotypes in existing populations. A precedent for this hypothesis has been established for mtDNA in that one mtDNA background increases susceptibility to Leber hereditary optic neuropathy [8]. Although studies of nucleotide diversity in global samples of Y chromosomes have suggested an absence of recent selective sweeps or bottlenecks [9], selection may, in principle, be very important for the Y chromosome because it carries several loci affecting male fertility [10] [11] and as many as 5% of males are infertile [11] [12]. Here, we show that one class of infertile males, PRKX/PRKY translocation XX males, arises predominantly on a particular Y haplotypic background. Selection is, therefore, acting on Y haplotype distributions in the population.     

Validation of highly polymorphic fluorescent multiplex short tandem repeat systems using two generations of DNA sequencers

Validation studies are a crucial requirement before implementation of new genetic typing systems for clinical diagnostics or forensic identity. Two different fluorescence-based multiplex DNA profiling systems composed of amelogenin, HumD21S11 and HumFGA (referred to as multiplex 1A), and HumD3S1358, HumD21S11 and HumFGA (multiplex 1B) have been evaluated for use in forensic identification using the Applied Biosystems Model 373A and Prism 377 DNA Sequencers, respectively. Experiments were aimed at defining the limit of target DNA required for reliable profiling, the level of degradation that would still permit amplification of the short tandem repeat (STR) loci examined, and the robustness of each locus in the multiplexes after samples were exposed to environmental insults. In addition, the specificity of the multiplexes was demonstrated using nonhuman DNAs. Forensically relevant samples such as cigarette butts, chewing gum, fingernails and envelope flaps were processed using both an organic extraction procedure and a QIAamp protocol. DNAs and resultant multiplex STR profiles were compared. The validation of the triplex STR systems was extended to include over 140 nonprobative casework specimens and was followed with a close monitoring of initial casework (over 300 exhibits). Our results document the robustness of these multiplex STR profiling systems which, when combined with other multiplex systems, could provide a power of discrimination of approximately 0.9999.     

The participation of the genetic apparatus in the mechanisms of memory trace formation: the role of the calcium-regulatory system of the neurons in rats

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.     

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].     

Co-amplification of the amelogenin and HLA DQ alpha genes: optimization and validation

An optimized PCR-based system allowing the co-amplification of the HLA DQ alpha and the amelogenin genes has been developed and validated, enabling simultaneous identity testing and sex determination. Sensitivities below 100 pg of human DNA were obtained, using a convenient, high resolution agarose gel system and ethidium bromide staining. Comparison of several co-amplification methods revealed that the best sensitivities and most consistent results were obtained using a hotstart technique employing an inactivating antibody to Taq polymerase. HLA DQ alpha typing results were reliably obtained using the co-amplification process. The sensitivity and ease of this system rendered it directly applicable to forensic analyses. The optimized techniques described here have been validated and successfully applied to forensic cases including People vs. Trujillo, in which the California Superior Court accepted these techniques as scientifically reliable and admissible. Work currently in progress has demonstrated that the described protocol may also be used to co-amplify the amelogenin gene with the AmpliType PM (polymarker) system, allowing identity testing at six loci in addition to sex determination.