DNA Typing Results Research Articles

PCR analysis from cigaret butts, postage stamps, envelope sealing flaps, and other saliva-stained material.

AbstractThe polymerase chain reaction (PCR) has offered the forensic scientist a new range of sensitivity in the examination of forensic samples. PCR has been successfully used to amplify specific DNA fragments from extremely small amounts of DNA present on cigaret butts (1,2), postage stamps (3), envelope sealing flaps, and other saliva-stained materials (4). In addition to DNA typing results, it is at times desirable to confirm the presence of saliva by the simultaneous employment of an amylase assay. This chapter describes methods that are routinely and successfully used on forensic casework samples.KeywordsSodium Dodecyl SulfateForensic SampleSterile ForcepsPostage StampPolymerase Chain Reaction ReagentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

DNA typing from stained sperm-positive vaginal smears: four rape cases

DNA typing from stained sperm-positive vaginal smears was performed in four cases of sexual assault. In Cases 1 and 2, a 6-month-old and a 6-week-old, Papanicolaou-stained vaginal smear with 99% propanol fixation underwent DNA analysis respectively. In Case 1, the HLA-DQα type of the sperm fraction of the smear was able to be determined following PCR, and the possibility of the suspect being the rapist could not be excluded. In Case 2, the possibility that the suspect was the rapist in the crime was excluded based on the results of DNA typing by means of two different single-locus probes. In the remaining two cases, H-E-stained smears after air drying for 4 or 5 months were the material for DNA analysis, and DNA typing was attempted by PCR-based STR system. In Case 3, there was no genetic information from sperm fraction of the smear due to the small number of spermatozoa on the glass slide. Therefore, it was impossible to determine whether the suspect was the rapist or not. In Case 4, sperm DNA from the smear was amplified and typed, and the suspect could be excluded. These four cases demonstrate that stained vaginal smears containing many spermatozoa can be used for DNA analysis in sexual assault cases.

CLINICAL RELEVANCE OF HLA-DPB LOCUS MATCHING FOR CADAVER KIDNEY RETRANSPLANTS

Matching for the HLA class I loci A and B and for the HLA class II locus DRB is known to influence the survival rate of kidney transplants. It is unknown whether matching for the HLA class II locus DPB also exerts an influence on graft outcome. The influence of matching for the HLA-DPB locus was analyzed based on DNA typing results obtained in more than 3600 first and 1300 repeat cadaver kidney transplants. HLA-DPB mismatches had no deleterious influence on the outcome of first cadaver transplants. However, the influence was statistically significant for retransplants. One-year graft survival rates were 83+/-2% with no mismatch (n=345), as compared with 76+/-2% with one mismatch (n=702, P=0.02), and 73+/-3% with two mismatches (n=258, P=0.003). The deleterious influence of HLA-DPB mismatches was particularly strong in retransplant recipients with >50% reactivity of preformed lymphocytotoxic antibodies, for which the 1-year graft survival rate was 70+/-4% with no mismatch, as compared with 69+/-3% with one mismatch (P=0.05) and 61+/-5% with two mismatches (P=0.003). These results indicate that HLA-DPB is a clinically relevant histocompatibility locus in cadaver kidney retransplantation. It is proposed that prospective typing and matching for HLA-DPB should be implemented for cadaver kidney retransplants.

A paternity case with apparently conflicting multilocus and single-locus DNA typing results.

A paternity case is presented in which DNA typing with thirteen variable number of tandem repeat (VNTR) systems was in apparent conflict with an initial multilocus DNA analysis using oligonucleotide probe (CAC)5. A dual approach was necessary, because the multilocus analysis yielded a proportion of aberrant nonmaternal offspring bands far too small and level of band-sharing too high for non-paternity. The case could finally be solved by reference to the joint likelihood ratio combining both approaches. Arguments are presented that summation of log likelihoods is indeed valid even if no information is available regarding linkage between the VNTR loci and the loci contributing to a multilocus DNA fingerprint.

Effect of environmental factors on PCR-DNA analysis from dental pulp.

This study was designed to observe the results of DNA typing on teeth subjected to aging, different temperatures and various environmental factors. A total of 570 teeth were studied. The study included the analysis of the PCR-based polymorphisms HLA DQA1, D1S80, HUMTH01, HUMFES/FPS and the XY homologous gene amelogenin. In general the best results were obtained with the XY homologous gene amelogenin, followed by the two STRs studied (HUMTH01 and HUMFES/FPS). The small fragment sizes and the method of detection used after PCR amplification are the main factors explaining this fact. In general, teeth submerged in water gave the poorest results. Teeth exposed to outdoor conditions provided better results than teeth buried in sand or soil, but even in these cases good results were obtained. Up to 4 degree C, temperature had only a slight influence on the results. Positive results were obtained in most cases at high temperatures (400 degrees C for 2 min) which are rarely reached in practical casework. Positive typing results for the XY homologous gene amelogenin and the STRs were obtained from teeth 10-30 years old. The usefulness of dental pulp for identification purposes is exemplified in some actual cases.

P579 - International cell exchange-comparison of class I serologic and DNA typing results

P579 - International cell exchange-comparison of class I serologic and DNA typing results

Prevalence of HPV DNA in cervical lesions in patients from Ecuador and Japan.

Cervical cancer is about 6 times more frequent in Ecuador than in Japan. We investigated the association between infection by the human papillomavirus (HPV) and the genesis of cervical cancer in specimens of lesions of the cervical epithelium obtained from patients in Ecuador and Japan. We also examined the results of HPV DNA detection and typing by the polymerase chain reaction (PCR) performed under the same technical conditions in areas with differing rates of cervical cancer. Purified tissue DNA from paraffin-embedded samples was amplified by PCR with universal and type-specific primers. HPV DNA was detected in 8 (20%) of 40 normal cervical epithelial samples from Ecuadorian patients, 19 (45%) of 42 low-grade squamous intraepithelial lesions (LSIL), 16 (50%) of 32 high-grade squamous intraepithelial lesions (HSIL), and 38 (81%) of 47 invasive squamous cell carcinomas (SCC) compared with 3 (10%) of 30 normal cervical specimens from Japanese patients, 107 (51%) of 210 HSILs, and 45 (71%) of 63 SCCs. The prevalence of HPV types 16 and 18 rose significantly with increasing histological grade (p < 0.05). The prevalence of HPV DNA decreased with increasing age in both Ecuadorian and Japanese patients. The detection rate and type-specific distribution of HPV DNA were not correlated with geographic location. Findings suggest that risk factors associated with poverty and underdevelopment may influence the prevalence of HPV infection and the sequence of events after HPV infection culminating in cervical cancer. These factors may help to explain the differing geographic distribution of this disease.

Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR

Randomly amplified polymorphic DNA PCR was used to generate unique and identifying DNA profiles for members of the cyanobacterial genera Anabaena and Microcystis, which are responsible for much of the production of nuisance blooms in various freshwater systems, including recreational and drinking water supplies. A method based on the combination of two 10-mer oligonucleotides in a single PCR was developed to provide specific and repeatable DNA fingerprints for cyanobacterial isolates. The strain-specific randomly amplified polymorphic DNA profiles made it possible to discriminate among all toxigenic cyanobacteria studied to the three taxonomic levels of genus, species, and strain. Analysis of DNA typing results obtained by the described method clearly distinguishes between the genera Anabaena and Microcystis. The markers produced for each strain were also applied to a phylogenetic analysis to infer genetic relatedness in this group of prokaryotes.

Molekularbiologische Methoden zur Bestimmung der NA-Antigene

Of the granulocyte-specific antigens, the NA1 and NA2 antigens are of most clinical importance. Since isolation of a sufficient number of granulocytes for serotyping is often not possible from the patient’s blood samples, we adapted the techniques of polymerase chain reaction (PCR) with sequence-specific primers (PCR-SSP) and PCR with subsequent hybridization of sequence-specific oligonucleotides (PCR-SSO) for DNA-based NA typing. The DNA typing results of altogether 114 individuals were compared with the phenotypes determined by the antigen capture assay MAIGA and the granulocyte immunofluorescence test. The DNA typing and the MAIGA results correlated perfectly, whereas by immunofluorescence mistyping rates of 15 and 17%, respectively, were observed. Our results indicate that for reliable NA typing a DNA-based technique or the MAIGA assay should be used.

Analysis of the HLA-DR gene locus by temperature gradient gel electrophoresis and its application for the rapid selection of unrelated bone marrow donors.

The human leucocyte antigen (HLA) class II compatibility of bone marrow donor and recipient is an essential prerequisite for the prevention of severe graft versus host disease and therefore for the successful outcome of bone marrow transplantation. In this study an efficient protocol was developed for the rapid analysis of the polymorphic HLA-DR gene locus, based on DNA-polymerase chain reaction (PCR) amplification of the variable exon II of the HLA class II DR genes and subsequent temperature gradient gel electrophoresis (TGGE). Computer-assisted melting map calculations were carried out to determine the melting behavior of the different HLA-DR fragments. Despite the high variability of the DR alleles on the nucleotide sequence level the calculations revealed a common melting domain structure of the different HLA-DR fragments, which was experimentally confirmed by perpendicular TGGE. On parallel TGGE, all samples were separated under the same electrophoretical conditions using a single PCR fragment without GC-clamp. TGGE was applied for the analysis of the DR alleles of numerous bone marrow receipt pairs and compared to the corresponding serological and DNA typing results. The TGGE patterns were found to be different for all samples with different HLA-DR typing results. Identical homoduplex and heteroduplex patterns occurred only in the case of complete genotypic HLA-DR identity as determined by direct sequencing of PCR products.

Report of a european collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.

Use of a ribosomal RNA gene probe for the epidemiological study of methicillin and ciprofloxacin resistantStaphylococcus aureus

Conventional bacteriophage typing was combined with ribotyping in the analysis of methicillin and ciprofloxacin resistant Staphylococcus aureus strains isolated in increasing frequency since the introduction of the new 4-quinolones as therapeutic agents in the Tel-Aviv Medical Center. Whole-cell DNA was digested with EcoRI and HindIII restriction endonucleases. Agarose gel electrophoresis, Southern blotting, and hybridization by biotinylated probe DNA coding for ribosomal RNA revealed 7 to 14 bands. Analysis of the patterns established a single DNA type in EcoRI as well as in HindIII digests for all strains except one. Control strains from other sources differed in their band patterns. Bacteriophage typing confirmed the results of DNA typing. Thus, the frequent occurrence of staphylococcal isolates resistant to 4-quinolones in the hospital was not due to mutational development of resistance in many strains, but to the spread of a resistant strain.

Benign muscular dystrophy: risk calculation in families with consanguinity.

This report concerns two families in which the index patients are sporadic cases of a benign form of muscular dystrophy. In both families the sisters of the patients have married a close relative. The respective risks for a child of these consanguineous marriages being affected with either X linked Becker muscular dystrophy or autosomal recessive limb girdle muscular dystrophy is calculated using pedigree information, results of serum creatine kinase determinations, and also, in one family, results of DNA typing using RFLPs from the short arm of the X chromosome.