AmpliType PM Research Articles

Brazilian population data on the polymerase chain reaction-based loci LDLR, GYPA, HBGG, D7S8, and Gc.

Gene and genotype frequencies in relation to the low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group specific component (Gc) loci were determined in a sample of 344 unrelated individuals (250 whites and 94 mulattoes) living in the city of São Paulo, Brazil. DNA was extracted from 5 mL of peripheral blood obtained from each of the 344 volunteers by the salting-out procedure. Polymerase chain reaction and reverse dot-blot analysis were performed with the Amplitype PM PCR Amplification and Typing Kit (Polymarker Multiplex; Applied Biosystems, Foster City, CA) under conditions recommended by the manufacturer. Estimated allele frequencies in the white sample were in the usual range of that of other United States and European population groups. In any case, genotype distributions for these loci did not deviate significantly from Hardy-Weinberg equilibrium proportions. Only 1 marginally significant (0.01 < P < 0.05) association, between loci HBGG and Gc, was detected in our mulatto sample out of a total of 20 possible pairwise comparisons of the 5 loci for both data sets. Allele frequencies were significantly different (P < 0.001) at the HBGG and Gc loci when the white and mulatto samples were compared. Biologic relationship exclusion probabilities (test powers) were calculated for the data. A Brazilian database has thus been established for the loci LDLR, GYPA, HBGG, D7S8, and Gc, 5 polymerase chain reaction-based loci systems that have been shown to be a useful tool for biologic relationship identification and exclusion.

HLA-DQA1 and Polymarker Locus Allele Frequencies in the Northeast Region of Colombia (Departments of Santander, Norte de Santander, Boyaca and Casanare)

Abstract Whole blood samples were obtained in the genetics laboratory of the UIS (Universidad Industrial of Santander) in collaboration with ICBF (Instituto Colombiano de Bienestar Familiar) from unrelated Colombian donors. Genomic DNA was extracted using the salting out procedure. The samples were typed for HLA-DQA1/PM loci by using the Amplitype PM and DQA1 kit following manufacturer's instructions (PE-Biosystems, Foster City, CA). Statistical analysis was performed as previously reported (1). The complete data set is available to any interested researcher upon request from Oscar Garcia, Area de Laboratorio Ertzaintza, Av. Montevideo 3, 48002-Bilbao, Spain.

HLA-DQA1 and Polymarker Allele Frequencies in Peru

Abstract Whole blood samples were obtained after informed consent from 100 unrelated Peruvian donors. Genomic DNA was extracted by a standard phenol/chloroform extraction procedure. The extracted DNA was quantitated by slot-blot hybridization using the QuantiblotR Human DNA Quantitation kit (PE-Biosystems, Foster City, CA). The samples were typed for HLA-DQA1/PM loci by using the Amplitype PM and DQA1 kit following manufacturer's instructions (PE-Biosystems, Foster City, CA). Exact tests were performed by using the computer program GDA for checking the Hardy-Weinberg expectations (1). Data were analyzed by Power-Stats (2). The complete data set is available to any interested researcher upon request from Oscar Garcia, Ph.D., Area de Laboratorio Ertzaintza, Av. Montevideo 3, 48002-Bilbao, Spain.

Allele Frequencies of HLADQA1 and PM in Istanbul (Turkey) Population

Abstract Blood samples were obtained from randomly selected, healthy, unrelated individuals from both sexes and were examined in order to estimate the allele and genotype frequencies of HLADQA1, LDLR, GYPA, HBGG, D7S8, GC loci. DNA was extracted using Chelex 100 extraction method (1). The amplification was carried out according to the instructions of Amplitype PMDQA1 PCR amplification kit. The samples were typed for HLADQA1/PM loci using the Amplitype PM + DQA1 kit (Perkin Elmer, Foster City, CA) as per the manufacturer's instructions. The statistical analysis was done by the gene count method and it revealed no significant deviation from Hardy-Weinberg expectations. The complete data including the comparison with various Turkish and different populations (2) and the statistical parameters of medico/legal interest are available at http://istanbul.edu.tr/enstituler/forensic/popgen-04.htm.

Distribution of HLA-DQA1 and Amplitype PM Locus Alleles in Aegean Region of Turkey

Abstract The samples were collected from unrelated individuals randomly selected from criminal cases. The DNA was extracted from fresh blood leucocytes, bloodstains, and single hairs by Chelex 100 method (1).

Distribution of HLA-DQA1 and Amplitype PM Locus Alleles in a Turkish Population Sample

Abstract The samples were collected from unrelated individuals randomly selected from criminal cases. The DNA was extracted from fresh blood leucocytes, bloodstains, and single hairs by Chelex 100 method (1).

A Standard of AmpliType PM Typing from Aged Evidentiary Samples

In analyzing aged samples by the AmpliType PM PCR amplification and Typing kit, it was occasionally observed that color developed typing strips had dark allele dots on PM loci but no visible S dot. Since the S dot acts as a minimum dot intensity control to determine positive alleles on the PM loci, it is necessary to apply another control system. To achieve positive PM typing from a degraded DNA sample that is inferred to be derived from a single donor, a standard has been adopted wherein loci from which sufficient PCR products are observed on agarose gel can be typed. The objective determination of sufficient PCR was done by comparison between band peak height of each locus generated from a sample and that of the corresponding locus generated from two nanograms (recommended minimum quantity as template DNA) of the control DNA provided in the kit.

The Application of Minisatellite Variant Repeat Mapping by PCR (MVR-PCR) in a Paternity Case Showing False Exclusion Due to STR Mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.

Association between LDLR polymorphism and diseases in the Japanese population: aging and distribution of the polymorphism

A number of DNA polymorphisms have been found to be associated with the pathophysiology of some common disease. If the LDLR polymorphism is directly or indirectly related to some fatal disease, the distribution of the polymorphism may vary with age. We therefore investigated the aging-associated distribution of the LDLR polymorphism. Blood samples were collected from Japanese cadavers (aged 0–91) at autopsy. The LDLR polymorphism was detected using a AmpliType PM PCR Typing kit. When the LDLR genotype was examined in cadavers divided according to age into 0–29 year group, 30–59 year group, and 60–91 year group, there were significant differences in genotype among the three age groups and between the 0–29 year group and 60–91 year group. The LDLR-A genotype tended to be lower in the older cadavers. The present study revealed that there were aging-dependent differences in the distribution of the LDLR polymorphism in autopsy samples, suggesting that a common mutation involved in the occurrence of fatal diseases may be present near the LDLR-A polymorphism locus.

NaOH Treatment to Neutralize Inhibitors of Taq Polymerase

The introduction of polymerase chain reaction (PCR) into the forensic field has greatly extended the ability to analyze DNA from small or degraded samples. However, one significant problem with PCR analysis is the sensitivity of Taq Polymerase to inhibitors found in many substrates commonly encountered with evidentiary materials. We hypothesize that the most problematic of these compounds intercalate into double stranded DNA (dsDNA) and have significantly less affinity for single stranded DNA (ssDNA). This study presents a comprehensive analysis of a novel method for the neutralization of Taq inhibitors by denaturation and washing with NaOH in Microcon-100 filtration units. The data show that DNA recovered following NaOH repurification routinely amplifies when other inhibitor neutralization techniques are unsuccessful. Genetic profiles have been obtained with both AmpliType PM + DQA1 and D1S80 systems. However, the NaOH protocol is not advised when the quantity of DNA is limited since the treatment results in significant loss of DNA.

Jordanian population data on the PCR-based loci: LDLR, GYPA, HBGG, D7S8 and GC

Genotype and allele frequency distributions for PM polymerase chain reaction (PCR)-based genetic markers were determined in a Jordanian sample population. Results were obtained using the AmpliType PM PCR Amplification and typing kit. All loci were in agreement with the Hardy–Weinberg equilibrium expectations. The predominant alleles for LDLR, GYPA, HBGG, D7S8 and GC loci were B, A, B, A and C respectively. No statistically significant variation was detected in allele frequencies of these loci in Jordanians compared to that in Israeli Arab, U.S Caucasian and Japanese populations. Data presented here can be used to estimate the frequency of a specific DNA profile in the Jordanian population for forensic analyses and paternity testing.

Diagnosis of zygosity by questionnaire and polymarker polymerase chain reaction in young twins.

We developed a zygosity questionnaire for use in young twins and assessed its validity using the results of DNA diagnosis. The participants were divided into two groups: 105 pairs of adolescent twins (12-16 years old), 47 pairs of child twins (2-12 years old), and their respective parents. The DNA diagnosis of zygosity was made with polymarker polymerase chain reaction (PCR) amplification of five loci, using the AmpliType PM PCR Amplification and Typing Kit; this method has an accuracy rate of 99.0%. A parsimonious model for each sample was established using stepwise logistic regression analysis of the 20 items of the questionnaire. The total accuracy rate of the model was satisfactory for both parental reports (three items) and self-reports (three items) of adolescent twins (97.4 and 95.6%, respectively), while that for parental reports on child twins (two items) was less satisfactory (92.5%). For adolescent twins, if DNA diagnostic workups were limited to those with discordant reports either from themselves or from their parents, the accuracy rate increased to 100% for parental reports and 98% for self-reports.

The Effect of Pathologic Substances and Adulterants on the DNA Typing of Urine

Human urine has not been adequately investigated as a potential source of DNA for forensic identity testing. The advent of polymerase chain reaction technology has made possible the analysis of previously undetectable levels of nucleic acids from human urine and other body fluids lacking nucleated cells. In this study, we evaluated the ability to genotype DNA extracted from adulterated urine specimens using the AmpliType PM + DQA1 PCR amplification and typing system. Fresh, first-void male urine specimens were contaminated with household bleach, E. coli, human serum albumin, glucose and saponin (a strong detergent). All of the adulterated samples were typed without difficulty. Frozen male urine specimens were split into equal volumes; one aliquot was adulterated with either E. coli or saponin, and the other was left free of contaminants. Seventy-one percent of all frozen urine specimens tested (adulterated and unadulterated) were successfully typed using this amplification and typing system. Our data, therefore, suggest that the AmpliType PM + DQA1 PCR amplification and typing system described is suitable for genotype analysis of adulterated fresh and frozen urine specimens.

Italian Population Data on the Polymarker System and on the Five Short Tandem Repeat Loci CSF1PO, TPOX, TH01, F13B, and vWA

A population study on five short tandem repeat (STR) loci and five sequence specific polymorphism loci was performed on unrelated Italian Caucasians. Separation and detection of the amplified STR fragments were carried out by high resolution vertical denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining, respectively. The sequence specific loci were analyzed using the AmpliType PM Typing Kit (Perkin Elmer, Foster City, CA). All loci, except Gc (p = 0.031), meet Hardy-Wienberg expectations. In addition, there is no evidence for association of alleles between pairs of loci. The combined power of discrimination for the five STR loci is 0.9999862 and for the PM loci is 0.99503. The results suggest that these loci may be useful for human identification cases in Italy.

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.

An introduction to forensic DNA analysis

TABLE OF CASE HISTORIES (SIDEBARS) To Catch a Cougar The Bungled Burglary (Or, the Burglar that Couldn't) Quintanilla The Czar's Bones (Or, What Difference a Base Pair?) A Case of Victim's Evidence (Or, One Bled and the Other Pled) Minisatellite Variable Repeat Analysis (MVR) The Simpson Saga The Case of the Disappearing Sperm (Or, Whose Type is it Anyway?) The Botanical Witness (Or, What the Seed Pod Saw) A Cold Hit in Minnesota (Or, the Database in Winter) The First RFLP Case (State of Florida v. Tommy Lee Andrews) INTRODUCTION THE NATURE OF PHYSICAL EVIDENCE Science and the Law From Identification to Individualization Fingerprints and DNA Conventional Blood Typing THE COLLECTION AND PRESERVATION OF PHYSICAL EVIDENCE Contamination Collection of Evidence Preservation of Evidence Evaluation of Evidence RFLP PCR A SHORT HISTORY OF DNA TYPING THE SCIENTIFIC BASIS OF DNA TYPING Why DNA? An Introduction to Human Genetics The Physical Basis of Heredity Alleles - Variations on a Theme Population Genetics An Introduction to the Molecular Biology of DNA Two Kinds of Variation Enzymes, the Workhorses of the Biological World AN OVERVIEW OF FORENSIC DNA TYPING SYSTEMS RFLP Analysis PCR Amplification HLA DQa/HLA DQA1 AmpliType (R) PM - Polymarker D1S80 Gender ID Mitochondrial DNA What Kinds of Samples Can Be Analyzed? How Much Sample Do You Need? PROCEDURES FOR FORENSIC DNA ANALYSIS Isolation of DNA Chelex Extraction Organic Extraction Differential Extraction Determining Quality and Quantity of DNA RFLP Analysis PCR Amplification Analysis of PCR Product Sequence of Polymorphisms Length Polymorphisms (D1S80, STRs, Gender ID) Automated Analysis Systems SIGNIFICANCE OF RESULTS Determination of Similarity Evaluation of Results Frequency Estimate Calculations Population Substructure INTERPRETATION OF DNA TYPING RESULTS Complicating Factors System Specific Interpretational Issues DNA AND THE DATABASE QUALITY CONTROL AND REGULATION Certification and Accreditation TWGDAM NRC I and II NRC I NRC II Federal DNA Advisory Committee ADMISSIBILITY STANDARDS - SCIENCE ON TRIAL IN THE COURTROOM The Frye Standard and the Federal Rules of Evidence RFLP PCR The State of the Debate EPILOGUE - MOVING INTO THE NEXT MILLENNIUM GLOSSARY APPENDICES DNA - Some Key Phrases Slot Blot Yield Gel Digest Gel Product Gel Chromosomal Location of Some Forensic Loci Sample Frequency Calculation Guidelines for a Quality Assurance Program for DNA Analysis NRC I and NRC II Recommendations State Statutes and Legislation Regarding the Admissibility of DNA Evidence Statutes and Legislation Regarding Mandatory Submission of Blood Samples for DNA Identification Summary of DNA Decisions Reported Court Decisions on Forensic DNA Evidence Amicus Brief Presented to the Supreme Court of the State of California by the California Association of Criminalists Forensic Science Internet Resources INDEX

Japanese Population DNA Typing Data for the Loci LDLR, GYPA, HBGG, D7S8, and GC

Population studies on the five loci LDLR, GYPA, HBGG, D7S8, and GC (PM loci) were carried out in a sample of 366 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using the AmpliType PM PCR Amplification and Typing kit (Perkin Elmer). For all loci, no significant deviations from Hardy-Weinberg equilibrium could be found in our Japanese population sample. The combined mean exclusion chance and power of discrimination for the PM loci were 0.68 and 0.993, respectively. The Japanese and Chinese population data were similar for the all loci. The Japanese and Korean population data were similar for all loci other than D7S8. Significant differences were observed between the Japanese population data and the 16 other population data compared for 3 loci or more.

Summary of Validation Studies from Twenty-Six Forensic Laboratories in the United States and Canada on the Use of the AmpliType® PM PCR Amplification and Typing Kit

A cooperative study was undertaken to collect and summarize the results of validation studies from forensic laboratories in the United States and Canada on the use of the AmpliType PM PCR amplification and typing kit for genetic typing of forensic biological evidence. This report compiles data from 26 laboratories on: 1) reproducibility studies on DNA extracted from various samples, 2) genetic typing of DNA extracted from a variety of biological samples on various substrates, 3) the effects of exogenous chemicals, materials, and environmental factors on test results, 4) sensitivity studies to determine the least detectable amount of extracted genomic DNA that can be reliably typed, 5) analysis of mixtures containing two sources of genomic DNA, 6) cross-hybridization with DNA extracted from various nonhuman species, and 7) evaluation of assay performance on parallel studies with other genetic typing systems on proficiency test panels, mock cases, and adjudicated/nonprobative casework. Equivalent results were obtained by each laboratory that supplied data, demonstrating the reliability and consistency of the test. Overall, it can be concluded from this study that the AmpliType PM PCR amplification and typing kit meets the guidelines of the Technical Working Group on DNA Analysis Methods (TWGDAM) and there is general scientific acceptability of this kit for forensic DNA testing.

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work

Experiments were performed to evaluate the Amplitype PolyMarker DNA typing system for application to forensic casework. DNA extraction using chelex was compared with phenol-chloroform extraction for various biological materials including postmortem blood, blood samples used for alcohol quantification, fresh urine, envelopes and cigarette butts. Different amounts of genomic DNA were amplified to test the sensitivity of the Amplitype PM. Mixed samples of two different bloods were typed to determine the dilution at which mixtures could be detected. Different storage conditions were evaluated using urine samples. Postmortem blood samples were typed during 4 months to determine the effects of natural degradation. A population sample of 105 unrelated individuals from South-West Switzerland was analyzed and the genotype frequencies were compared with those reported by others. Finally, practical usefulness of the Amplitype PM system is illustrated by analysing casework samples. The results of this validation proved the great usefulness and sensitivity of the Amplitype PM system using the appropriate extraction and typing method. However, mixed samples had to be interpreted with caution owing to the possibility of non-specific alleles with stored material such as urine and postmortem blood.

Post-Amplification Primer Extension of Heat-Denatured AmpliType® PCR Products: Effects on Typing Results

Alleles of the HLA, DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobilized on a nylon membrane strip. Using reagents supplied in AmpliType PCR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequently, the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensities. The relative probe signal intensities obtained from an undegraded DNA sample extracted from a single individual will be different from those obtained from degraded DNA and from samples containing DNA from more than one source. Because probe signal intensity is an essential consideration for interpretation, factors that can influence it need to be identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signal intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the selective loss of GC B and HLA DQA1 4.1 probe signals observed after shorter cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficient residual Taq DNA polymerase activity to extend primers as the solution cools and that this primer extension occurs at a more rapid rate than PCR product reannealing. Primer extension across probe binding sites will prevent hybridization of the PCR product to complementary probes on the strip. The extent of signal reduction is dependent on the position of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all probe binding sites will be available for hybridization to PM and HLA DQA1 DNA probe strips.