D1S80 Typing Research Articles

Comparative study of D1S80 typing by capillary electrophoresis and sequencing

The D1S80 locus is very useful for personal identification in Japan. To obtain a correct allele over 45, we examined PCR amplification product of the allele over 45 both by direct sequencing and fragment analysis using capillary electrophoresis. Direct sequencing finally determined the allele as being 57. However, it was calculated to be an allele of 56 by comparison with size markers for capillary electrophoresis. The difference could be attributed to the electrophoretic size markers. This finding indicates that the direct sequencing may be useful to determine the allele over 45 in the D1S80 locus.

The risk of incorrect typing of D1S80 by unstable minisatellite

The D1S80 locus is very useful for personal identification in Japan. To analyze PCR amplification products at the D1S80 locus, DIG-labeled primer was used for PCR amplifications. After electrophoresis, the PCR products were transferred to a nylon membrane and detected with alkaline phosphatase-labeled anti-DIG antibody (AP-DIG Ab). Numerous extra bands were detected on the membranes, indicating that PCR amplification products at the D1S80 locus contain many extra products which cause the undesirable bands to appear during D1S80 typing. To obtain a correct genotype, it was necessary to perform Southern blotting using an oligonucleotide that includes an internal sequence of the amplification products as a probe.

Influence of post-mortem changes on DNA typing (D1S80, TH01, HLA DQA 1, and PM typing system): case studies for personal identification

Between 1996 and 2002, we tested a total of 20 unidentified bodies for DNA typing. We describe here the relationships among detection rates achieved by four DNA typing systems (D1S80 typing, TH01 typing, HLA DQA1 typing, and PM typings), the post-mortem interval, types of specimens (bone, nail, and blood), post-mortem changes, and the site at which the corpse was found (indoors, outdoor, or in the sea). Detection rates for PM typings, HLA DQA1 typing, TH01 typing, and D1S80 typing in all cases were 94.7, 90.0, 73.7, and 50.0%, respectively. The success of the typings was highly influenced by the post-mortem interval. Using blood, almost all DNA types were detected, while the nail showed comparatively higher detection rates than bone. The detection rate decreased in order with indoor, outdoor, sea, and soil as the site at which the corpse was found. It is important to consider the specimen, the site at which the corpse was found, and the post-mortem interval to successfully achieve DNA typing.

Influence of post-mortem changes on DNA typing (D1S80, TH01, HLA DQA 1, and PM typing system): case studies for personal identification

Between 1996 and 2002, we tested a total of 20 unidentified bodies for DNA typing. We describe here the relationships among detection rates achieved by four DNA typing systems (D1S80 typing, TH01 typing, HLA DQA1 typing, and PM typings), the post-mortem interval, types of specimens (bone, nail, and blood), post-mortem changes, and the site at which the corpse was found (indoors, outdoor, or in the sea). Detection rates for PM typings, HLA DQA1 typing, TH01 typing, and D1S80 typing in all cases were 94.7, 90.0, 73.7, and 50.0%, respectively. The success of the typings was highly influenced by the post-mortem interval. Using blood, almost all DNA types were detected, while the nail showed comparatively higher detection rates than bone. The detection rate decreased in order with indoor, outdoor, sea, and soil as the site at which the corpse was found. It is important to consider the specimen, the site at which the corpse was found, and the post-mortem interval to successfully achieve DNA typing.

A new sequenced allelic ladder marker for D1S80 typing.

A sequenced allelic ladder marker that contains 32 alleles consisting of alleles 13-44 was developed for D1S80 ( MCT118) typing. Each allele, after amplification with D1S80 primers, was cloned to the pT7Blue T-Vector. The plasmid DNAs of these clones were mixed and amplified with D1S80 primers to construct the allelic ladder marker. All the alleles prepared in this study were sequenced in both directions, using two types of forward primers and one reverse primer. The sequencing results revealed that all the alleles in our allelic ladder marker had the correct length corresponding to the allele number. This marker can be used for more effectively typing D1S80.

Quality and quantity of DNA in cadavers' serum

We investigated the quality and quantity of DNA in sera obtained from 16 cadavers who were left at room temperature or placed in a refrigerator for cadavers within 3 days of their death. The causes of death were as follows: asphyxia, 4; hemorrhage, 4; fire, 3; cold, 1; subdural bleeding, 1; sepsis, 1; cerebral contusion, 1; and pulmonary thromboembolism, 1. When examined using agarose gel electrophoresis, the samples from the victims who died from sepsis and pulmonary thromboembolism exhibited smear patterns, while the other samples exhibited ladder patterns. The size of each band was an integer multiple of approximately 180 bp, considered to be characteristic of apoptosis. The DNA concentration in the cadavers' sera ranged from 3.0 to 258.9 ng/ml. DNA types (D1S80, HLADQA1, TH01, and PM) were determined for all the samples except for the sample from the victim who died from sepsis. The detected types were the same as those obtained from paired bloodstain samples, except for in the asphyxia victim. D1S80 typing of the serum sample from the asphyxia victim showed the loss of the longer allelic hetero-band. These results suggest that the quantity of DNA in serum from cadavers is sufficient for DNA typing, but that the degradation of DNA may proceed quickly, depending on the temperature of the area in which the cadaver is left as well as the pathological conditions that the victim had experienced.

DNA sequence analysis of long PCR amplified products at the D1S80 locus

Several PCR amplified products of larger alleles than 41 were observed frequently in D1S80 typing. These long alleles have rarely been analyzed. A total of seven amplified products of larger alleles than 41 at D1S80 were selected for sequence analysis. From the results, all of the samples have a repeat of only 16 base pairs in size, without the first repeat unit. In this study, the largest allele was determined to be the allele 72.

QIAamp Spin Columns as a Method of DNA Isolation for Forensic Casework

The Detroit Police Crime Lab has historically used Chelex as a method to isolate DNA for amplification and typing of bloodstains at the HLADQA1, PM and D1S80 loci. However, preliminary validation of several STR systems for casework has demonstrated that the Chelex procedure is not the best method of DNA isolation for STR amplifications for our purposes. Long term storage at -20 degrees C in the presence of unbuffered Chelex beads (approximately 1 year), combined with multiple freeze thaws, resulted in signal loss at a locus for many database samples. Therefore, we have employed the QIAamp spin column as an alternative method of DNA isolation for amplification and typing of STR loci currently being validated for use in the laboratory. Moreover, we determined that QIAamp isolated DNA is also suitable for HLADQA1, PM and D1S80 typing. A matrix study was performed to determine if the QIAamp DNA procedure would give better results on bloodstains deposited on "problem surfaces" such as leather, dirt and various dyed fabrics. Again, QIAamp isolated DNA was more readily typeable than Chelex isolated DNA. We successfully replaced the phenol/chloroform extraction steps utilized in our laboratory for differential extractions, a commonly used method for separating sperm and non-sperm fractions of sexual assault evidence, with the QIAamp spin columns. The QIAamp extracted DNA performed as well in all PCR amplification and typing procedures tested (PM, HLADQA1, D1S80, and STR (PowerPlex)) as the phenol/chloroform Centricon isolated or EtOH precipitated DNAs. Thus we concluded that QIAamp spin columns are a superior method for isolating DNA to be typed for a variety of loci.

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.

DNA typing of the D1S80 locus using capillary zone electrophoresis

DNA typing of the D1S80 locus was carried out using capillary polyacrylamide gel zone electrophoresis (CZE). The D1S80 allelic ladder was isolated within 40 min in polyacrylamide gel columns of 50 μm inner diameter and 30 cm length. The PCR products and ladder marker in the injection port were electrophoresed and detected by UV absorption (254 nm). The typing was determined according to migration of DNA fragments on the electropherograms, which permits simultaneous analysis of both the test sample polymerase chain reaction products and the ladder marker. The two-step sample injection method applied in this study allows us to use minute amounts of the ladder maker. This procedure provides a reliable, economical and rapid method for D1S80 typing.

D1S80 Typing of DNA from Simulated Forensic Specimens

The reliability of a D1S80 typing procedure has been evaluated using simulated forensic specimens. D1S80 alleles were detectable in DNA recovered from bloodstains exposed to sunlight for up to 20 weeks. However, D1S80 alleles were undetectable in semen stains after six weeks sunlight exposure. Analysis of blood and semen that had been deposited on a variety of substrates and examined over a twenty-week period, revealed no systematic influence of substrate on the ability to type D1S80. A study in which body fluids were exposed to household chemical substances, such as bleach, acids, oil, and gasoline, indicated that only HCl and bleach had a deleterious effect on the ability to type D1S80. In addition, personal care chemical products were without effect on D1S80 allele patterns derived from semen. Exposure of blood and semen to four different species of microorganisms resulted in no alteration of D1S80 genotype patterns in these body fluids. D1S80 genotypes could be reliably determined even when body fluids from different individuals were mixed. DNA from no animals other than humans and higher primates could be amplified at locus D1S80 when the DNA had been isolated through an organic procedure. These studies, in concert with the reports of others, indicate that the procedures for the amplification and detection of genetic variation at locus D1S80 are suitable for use on forensic evidentiary materials.

D1S80 typing in forensic practice

D1S80 typing in forensic practice

Analysis of the D1S80 locus by amplified fragment length polymorphism technique in the Chinese, Malays and Indians in Singapore

The distributions of the D1S80 alleles and genotypes in the Chinese, Malays and Indians in Singapore were determined by amplified fragment length polymorphism (AMP-FLP) analysis. The distributions of the observed genotypes for the three races conformed to Hardy-Weinberg expectations. The system was applied to 19 families whose paternity had been established by restriction fragment length polymorphism (RFLP) analysis. In all cases, Mendelian inheritance of the alleles at the D1S80 locus could be demonstrated. D1S80 typing on DNA recovered by differential extraction of forensic specimens which included vaginal swabs, urethral swabs and seminal stains yielded consistent results.

Analysis of the locus D1S80 by amplified fragment length polymorphism technique (AMP-FLP). Frequency distribution in Danes. Intra and inter laboratory reproducibility of the technique

DNA from the locus D1S80 was amplified by polymerase chain reaction (PCR) and analyzed by electrophoresis in vertical polyacrylamide gels followed by silverstaining. DNA samples from 119 unrelated Danes and 97 mother/child pairs were examined. The amplified fragment length polymorphism (AMP-FLP) analysis of the D1S80 locus demonstrated 21 alleles and a heterozygosity of 77%. Of the 231 possible phenotypes, 57 were observed. All mother/child pairs shared at least one D1S80 allele. The D1S80 typing results in 70 Danes were compared to the results obtained on the same samples in another laboratory and the results were concordant in all cases.