TWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis.

Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.

Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA.

The PCR amplification of tetranucleotide short tandem repeat (STR) loci typically produces a minor product band 4 bp shorter than the corresponding main allele band; this is referred to as the stutter band. Sequence analysis of the main and stutter bands for two sample alleles of the STR locus vWA reveals that the stutter band lacks one repeat unit relative to the main allele. Sequencing results also indicate that the number and location of the different 4 bp repeat units vary between samples containing a typical verses low proportion of stutter product. The results also suggest that the proportion of stutter product relative to the main allele increases as the number of uninterrupted core repeat units increases. The sequence analysis and results obtained using various DNA polymerases appear to support the slipped strand displacement model as a potential explanation for how these stutter products are generated.

Consistency and reproducibility of AmpliType PM results between seven laboratories: field trial results.

The AmpliType PM Field Trial was designed to assess the ability of forensic laboratories to obtain the correct results from samples commonly encountered in forensic casework. The seven forensic laboratory participants of the AmpliType PM Field Trial each performed four studies. Samples were analyzed using components of the AmpliType PM PCR Amplification and Typing Kit. Laboratories were also provided with DNA probe strips to type the DQA1 locus. Of the 381 PM and 325 DQA1 DNA probe strip results obtained from DNA-containing and non-DNA-containing samples, 98.2% and 95.7% showed the correct result for PM and DQA1 types, respectively. No samples were typed incorrectly. The remaining small percentage of samples were either uninterpretable due to the presence of a mixture, or no result was obtained due to insufficient DNA. The Field Trial demonstrated that laboratories can easily implement the AmpliType PM system to analyze DNA-containing samples and controls successfully for forensic casework applications.

Localization of predisposition to Hodgkin disease in the HLA class II region.

Molecular typing of HLA class II loci has been performed on a sample of 196 patients with Hodgkin lymphoma. Division of patients into two histological categories--nodular sclerosing Hodgkin disease versus all other types--shows significant overall association of the nodular sclerosing group with the HLA class II region. Haplotypes and alleles defined for the four loci typed--DRB1, DQA1, DQB1, and DPB1--were present in both excess and deficit in the nodular sclerosing sample. Some of the effects are attributable to particular DRB1 and DQB1 alleles, while other effects are best explained by haplotypes marking the entire class II region. The latter effects might be due to variation in additional, as-yet-unexamined loci in the class II region or to particular combinations of alleles from two or more loci. These data also explain why earlier studies showed HLA linkage but not association, and they substantiate the specific involvement of the immune system in certain neoplastic diseases.

Polymorphism, recombination, and linkage disequilibrium within the HLA class II region.

Thirty-nine CEPH (Centre d'Etude du Polymorphisme Humain) families, comprised of 502 individuals, have been typed for the HLA class II genes DRB1, DQA1, DQB1, and DPB1 using nonradioactive sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA. This population, which consists of 266 independent chromosomes, contains 27 DRB1, 7 DQA1, 12 DQB1, and 17 DPB1 alleles. Analysis of the distribution of allele frequencies using the homozygosity statistic, which gives an indication of past selection pressures, suggests that balancing selection has acted on the DRB1, DQA1, and DQB1 loci. The distribution of DPB1 alleles, however, suggests a different evolutionary past. Family data permits the estimation of recombination rates and the unambiguous assignment of haplotypes. No recombinants were found between DRB1, DQA1, and DQB1; however, recombinants were detected between DQB1 and DPB1, resulting in an estimated recombination fraction of greater than or equal to 0.008 +/- 0.004. Only 33 distinct DRB1-DQA1-DQB1 haplotypes were found in this population which illustrates the extreme nonrandom haplotypic association of alleles at these loci. A few of these haplotypes are unusual (previously unreported) for a Caucasian population and most likely result from past recombination events between the DR and DQ subregions. Examination of disequilibrium across the HLA region using these data and the available serologic HLA-A and HLA-B types of these samples shows that global disequilibrium between these loci declines with the recombination fraction, approaching statistic nonsignificance at the most distant interval, HLA-A to HLA-DP.DR-DQ haplotypes in linkage disequilibrium with DPB1 and B are noted and, finally, the evolutionary origin of certain class II haplotypes is addressed.

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit.

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.

HLA-DQ alpha allele and genotype frequencies in various human populations, determined by using enzymatic amplification and oligonucleotide probes.

Allele and genotype frequencies at the HLA-DQ alpha locus have been determined by the use of polymerase chain reaction (PCR) amplification and nonradioactive oligonucleotide probes. The probes define six alleles and 21 genotypes in a dot-blot format. A total of over 1,400 individuals from 11 populations has been typed by two different laboratories using this method. In contrast to some variable-number-of-tandem-repeat markers that have been used for identity determination, DQ alpha genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium in all populations studied. The distribution of alleles varies significantly between most of these populations. In Caucasians, the allele frequencies range from 4.3% to 28.5%. In this population, the power of discrimination is .94, and, for paternity determination, the power of exclusion is .642. These population data will allow the use of the HLA-DQ alpha marker in paternity determination, the analysis of individual identity in forensic samples, and anthropological studies.

Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV-1.

To determine whether apparently healthy persons who have had repeatedly reactive enzyme immunoassays and an indeterminate Western blot assay for antibody to the human immunodeficiency virus type 1 (HIV-1) are infected with HIV-1 or HIV-2, we studied 99 such volunteer blood donors in a low-risk area of the country. The subjects were interviewed about HIV risk factors. Coded blood specimens were tested again for HIV-1 antibody (by two different enzyme immunoassays, a Western blot assay and a radioimmunoprecipitation assay) and for HIV-2 antibody by enzyme immunoassay, for HIV-1 by the serum antigen test, for HIV-1 by culture, for human T-cell leukemia virus Type I or II antibody by enzyme immunoassay, and for sequences of HIV DNA by the polymerase chain reaction. Of the 99 blood donors, 98 reported no risk factors for HIV-1 infection; 1 donor had used intravenous drugs. After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66 percent) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92 percent) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2.