DNA Typing Methods Research Articles

LMD as a forensic tool in a sexual assault casework: LCN DNA typing to identify the responsible

We have previously studied the sensitivity of laser microdissection (LMD) techniques and have tested our capability to yield a complete genotype from 30 haploid sperm cells. Partial but significant genotype information have been obtained also from 5 to 10 haploid sperm cells. This experimental procedure has important applications both in pathological and in forensic fields. A real casework of a teenager sexually assaulted in a car in a small town in Sicily has induced us to evaluate the possibility to transfer the biological residuals present on a cut off from the car seat to a laser microdissection-prepared microscope slide. Biological traces have been at first examined with Crimescope CS-16 in order to evaluate the kind of traces we were to process; to avoid the loss of the forensic traces, we have reproduced the same conditions in several experimental procedures. We have settled several samples distinct for the amount of biological mixed traces, age of the traces and their exposure to different atmospheric agents. DNA typing was performed both with Identifiler STR loci kit and with another forensic kit based on shorter STR amplicons (MiniSTR). Laser microdissection techniques, coupled to high-sensitivity DNA typing methods as short STRs, allow forensic operators to isolate the different cell residuals whenever in front of mixed traces.

Parentage testing of Thoroughbred horse in Korea using microsatellite DNA typing

The present study was to construct a parentage testing system for Thoroughbred (TB) horse. A total number of 1,285 TB horse samples including 962 foals for parentage testing, 9 sires and 314 dams for individual identification were genotyped. Genomic DNA was extracted from 5 hair roots and genotyped by using 14 microsatellite markers (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS3, HMS6, HMS7, HTG4, HTG10, LEX3 and VHL20). This method consisted of multiplexing PCR procedure and showed reasonable amplification of all PCR products. Genotypes were determined by genetic analyzer. The number of alleles per locus varied from 3 to 9 with a mean value of 6.36 in TB horse. The expected heterozygosity was ranged from 0.548 to 0.831 (mean 0.699), and the total exclusion probability of 14 microstellite loci was 0.9998. Of the 14 markers, ASB2, ASB17, ASB23, HMS7 and HTG10 loci have relatively high PIC value (> 0.7). Of the 962 foals, 960 foals were qualified by compatibility according to the Mendelism. These results suggest that the DNA typing method has high potential for parentage verification and individual identification of TB horses.

A New DNA Typing Method of D1S80 Marker by Capillary Electrophoresis of ABI 310 Genetic Analyzer

A New DNA Typing Method of D1S80 Marker by Capillary Electrophoresis of ABI 310 Genetic Analyzer

Isolation of Nontuberculous Mycobacteria in Zambia: Eight Case Reports

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

DNA Identifications After the 9/11 World Trade Center Attack

The attack on the World Trade Center on 9/11/2001 challenged current approaches to forensic DNA typing methods. The large number of victims and the extreme thermal and physical conditions of the site necessitated special approaches to the DNA-based identification. Because of these and many additional challenges, new procedures were created or modified from routine forensic protocols. This effort facilitated the identification of 1594 of the 2749 victims. In this Policy Forum, the authors, who were were members of the World Trade Center Kinship and Data Analysis Panel, review the lessons of the attack response from the perspective of DNA forensic identification and suggest policies and procedures for future mass disasters or large-scale terrorist attacks.

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA) 4, (GTG) 5 and (GAC) 5 enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA) 4 enabled a higher level of discrimination than primers (GAC) 5 and (GTG) 5. The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA) 4 and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.

Identification of Exhumed Remains of Fire Tragedy Victims Using Conventional Methods and Autosomal/Y-Chromosomal Short Tandem Repeat DNA Profiling

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.

The null allele HLA-B*5111N accounted for a discrepancy between serological and low resolution DNA typing methods

The null allele HLA-B*5111N accounted for a discrepancy between serological and low resolution DNA typing methods

Analysis and Frequency of Bovine Lymphocyte Antigen (BoLA-DRB3) Alleles in Iranian Holstein Cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.

Association of clinical features with HLA in chronic pulmonary thromboembolism

The aetiology of chronic thromboembolic pulmonary hypertension (CTEPH) is largely unknown and may be heterogeneous, because there are several ethnic differences in the clinical characteristics of CTEPH. Female predominance and a higher ratio of chronic to acute pulmonary thromboembolism have been reported in Japan as compared with the USA. Because such ethnic differences may be controlled by genetic factors, the current study investigated HLA polymorphisms in Japanese patients with CTEPH. HLA typing by serological and/or DNA typing methods was performed (for HLA-A, B, DPB1, DRB1) in 80 patients and 678 controls, and the association of clinical characteristics with HLA alleles was studied. The frequencies of HLA-B*5201 (40 versus 24%) and DPB1*0202 (19 versus 6%) were significantly higher in the patients. HLA-B*5201 positive patients showed a significant female predominance. Total pulmonary vascular resistance and mixed venous oxygen tension were better in the HLA-B*5201 positive patients. In contrast, cardiac index and gas exchange parameters were worse in the HLA-DPB1*0202 positive patients. In the patients carrying HLA-B*5201 and/or -DPB1*0202, the frequency of deep vein thrombosis was significantly lower than the other patients. These observations suggested that both the susceptibility and clinical characteristics of chronic thromboembolic pulmonary hypertension were controlled in part by the HLA-B and -DPB1 loci.

Analysis of HLA Class I and Class II Gene Polymorphisms in Japanese Patients with Human T-Cell Lymphotropic Virus Type 1-Associated Uveitis

Purpose: To investigate the immunogenetic background of human T-cell lymphotropic virus type 1 (HTLV-1)-associated uveitis (HAU) that presents immune-mediated reactive changes in the uvea. Methods: HLA class I and class II genes were studied in 51 patients with HAU, 192 asymptomatic HTLV-1 carriers, and 266 HTLV-1-seronegative controls using a high-resolution method of HLA DNA typing. The HLA alleles of HAU were compared with those of HTLV-1 carriers and healthy controls. Results: We identified 62 distinct alleles of HLA-A, HLA-Cw, and HLA-B and 49 distinct alleles of HLA-DRB1 and HLA-DQB1 in patients with HAU, asymptomatic HTLV-1 carriers, and healthy controls. The relative frequencies of these HLA alleles did not differ among the three groups. Conclusion: The results suggest that HLA class I and class II genes do not contribute to susceptibility to HAU.

Evaluation of genetic determinants involved in β-lactam- and multiresistance in a surgical ICU

Antibiotic resistance is a major and well-known problem in intensive care units (ICUs) world-wide and previously susceptible isolates become resistant through the acquisition of resistance determinants from other bacteria or the development of mutations, as is the case in β-lactam resistance. We evaluated the presence of resistance determinants involved in β-lactam resistance and multi-resistance in order to establish the contribution of horizontal gene transfer to the spread of resistance in a surgical ICU during an antibiotic rotation study. Pseudomonas aeruginosa and Enterobacteriaceae isolates were selected and iso-electric focusing (IEF), DNA-typing methods such as specific β-lactamase and specific integron PCRs were performed to determine the presence of β-lactamases. The PCRs specific for IMP-1, OXA-1, and VIM-type β-lactamases performed on the selected P. aeruginosa and Enterobacteriaceae isolates with MICs for cephalosporins >1 mg/l did not demonstrate any of these β-lactamases. IEF for 14 pseudomonads, representing 7 genotypes from 9 patients, showed a β-lactamase with a p I larger than 8.5 in 13 of the isolates. The integrase PCR was positive for only five isolates from three patients and conserved segment PCR showed integrons of variable sizes (700, 900, 1400 and 1500 bp). Each patient had its own integron types. It can be concluded that integrons and associated resistance determinants played only a minor role in the surgical ICU and β-lactam resistance among P. aeruginosa isolates was most likely due to the derepression of its AmpC gene.

Bacterial Population Association with Phytoplankton Cultured in a Bivalve Hatchery

Bacterial populations association with phytoplankton cultures used as food for bivalve larvae were enumerated and identified from their partial 16S rDNA gene sequences. Microalgae were provided from different European hatcheries during the larval production season. Average concentration (direct counts) of bacteria ranged from 1.3 x 10(5) to 5.3 x 10(8) mL(-1) while culturable bacteria represented from 10% to >60% of total bacteria. In most cases, three to six representatives of each type of colony were collected on solid medium. The identity of isolates from the same colony type was checked by two different randomly amplified polymorphic DNA (RAPD) typing methods, after which the 16S rDNA gene of one to three isolates by colony type were partially sequenced. Algae harbored a large spectrum of bacteria belonging to the alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, Cytophaga- Flavobacterium- Bacteroides (CFB) group, Actinobacteria, and Bacillus. Members of the Roseobacter clade and CFB group were the most abundant. In the majority of cases one strain constituted 50% or more of the culturable bacterial flora. About half of the isolates were common to two hatcheries or at least two microalgal cultures. Several isolates were closely related to bacteria associated with harmful dinoflagellates in culture. Thus, the algal cultures seemed to favor certain bacterial species which belonged to distantly separated groups. As some of them could disturb the development of bivalve larvae, the control of bacterial populations would undoubtedly make it possible to reduce larval losses in bivalve rearing.

A SURVEY OF HOSPITAL ACQUIRED INFECTIONS IN OBAFEMI AWOLOWO UNIVERSITY TEACHING HOSPITAL, ILE-IFE

A well-structured infection control programme plays a vital role in reducing mortality, morbidity and cost resulting from nosocomial infections in hospitalized patients. However infection-control activities in developing countries is severely constrained by lack of infection control infrastructure and lack of strong commitment by hospital clinicians and administrators as well as the level of socio-political and economic development prevalent in the developing world. The Infection Control Program (ICP) of Obafemi Awolowo University Teaching Hospital Complex (OAUTHC) was established in 1995. An analysis of data of a hospital wide surveillance obtained between January 1995 and December 1999 is hereby presented, highlighting our experience with the pattern of nosocomial infection seen in this hospital. From January 1995 to December 1999, a total of 19,471 patients were discharged during this period. Of this, 515 cases of infection were recorded while on admission, giving an annual prevalence rate of 2.7% for nosocomial infection per year. (X2 = 47.34, df = 4, p = 0.000). The highest infection rate was recorded from the orthopaedic ward (12.8%), followed by the intensive care unit (ICU)(8.4%) while the neonatal ward (NNW) and the paediatric ward (PW) recorded relatively low figures (0.5%) and (0.4%) respectively. Gram-negative rods (GNR) 491(78.8%) were commonly encountered as pathogens implicated in hospital-acquired infection (HAI) followed by Staphylococcus aureus and “other” organisms 17(2.7%). Current methods are inadequate because phenotypic typing alone has limited discriminatory power. DNA typing method is now the ‘gold' standard for epidemiological and routine investigation of HAI. African Journal Of Clinical And Experimental Microbiology Jan 2004 Vol.5 No.1 108-118

Two subtypes of hepatitis B virus-associated glomerulonephritis are associated with different HLA-DR2 alleles in Koreans.

Hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) is occurring at high prevalence in most Asian endemic areas. There have been some reports on human leucocyte antigen (HLA) associations with HBV infections; however, HLA association with HBV-GN has been rarely reported. Forty-six adult Korean patients with HBV-GN (42 male and four female patients, age 20-66), 100 HBsAg (-) healthy controls, and 89 individuals with chronic HBV infection were studied for HLA-DRB1 and DQB1 gene polymorphisms using high-resolution DNA typing methods. In HBV-GN patients, a strong association with HLA-DR2 was observed compared with HBsAg (-) controls (OR = 4.0). Different HLA-DR2 alleles were associated with different pathologic subtypes of HBV-GN: DRB1*1502 with membranoproliferative glomerulonephritis (MPGN, n = 35) (OR = 14.5) and DRB1*1501 with membranous nephropathy (MN, n = 11) (OR = 3.8). HLA-DQB1*0601, strongly linked to DRB1*1502, was also associated with MPGN subtype of HBV-GN (OR = 4.3). All these associations were also significant compared with chronic HBV infection group. For chronic HBV infection per se, DRB1*1302, DQB1*0402, and DQB1*0604 had some protective effect (OR = 0.4, OR = 0.3, and OR = 0.1, respectively), and DRB1*1101 was weakly associated (OR = 4.6) in Koreans. These results suggest that HLA-DR or related genetic factor is associated with disease susceptibility to HBV-GN in Koreans, and different pathologic subtypes of HBV-GN are influenced by the genetic factors of the patients.

Genotyping ofEntamoebaSpecies in South Africa: Diversity, Stability, and Transmission Patterns within Families

Using a recently described polymerase chain reaction-based DNA typing method for Entamoeba histolytica and E. dispar, we investigated the genetic diversity of these species in a geographically restricted region of South Africa. Patterns were stable over time in the same infection, and, with few exceptions, infected family members carried the same strain. However, both species exhibited remarkable variation, with no 2 family groups being infected with the same strain of E. histolytica. Mixed infections were rare. The results indicate that this typing method will be useful in identifying epidemiological linkage between infections.

Complement C4 protein and DNA typing methods.

Complement C4 protein and DNA typing methods.

Pyrosequencing™ : A one-step method for high resolution HLA typing

While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.

Discrimination of monozygotic twins (and clones) on the DNA level

Recent DNA typing methods (RFLP, STR, RAPD; e.g., [ Naturwissenschaften 84 (1997) 181] [3]) do not allow discrimination of monozygotic twins. To overcome this restriction, we suggest the use of variable DNA sequences of bone-marrow-derived memory B lymphocytes that are likely to be different even in monozygotic twins. Since memory B cells are transported in the blood stream, they can be found in blood stains on crime scenes and checked for a match to the cells of a living twin pair of twins.

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays.

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.