TY - JOUR
T1 - Validation studies on the analysis of the HLA DQα locus using the polymerase chain reaction
AU - Comey, C. T.
AU - Budowle, B.
PY - 1991
Y1 - 1991
N2 - A series of experiments has been performed to evaluate typing of the HLA DQα gene by polymerase chain reaction (PCR) amplification of the gene and subsequent hybridization with sequence-specific oligonucleotide probes. These experiments were designed to evaluate DQα typing for analysis of evidentiary specimens. Bloodstains were exposed to a variety of conditions and environmental insults. These conditions included exposure to many different types of substrates, various microorganisms that could be encountered in evidentiary stains, sunlight, and a variety of chemical contaminants. Varying amounts of genomic deoxyribonucleic acid (DNA) were amplified to test the sensitivity of DQα typing. The sensitivity of the PCR technique raises the concern that DNA from sources other than the evidentiary material could be detected. A series of experiments was done to evaluate the question of DNA contamination. Purified DNA samples with different DQα types were mixed in different ratios to determine the ratio at which it could not be determined whether an allele was from the sample or the contaminant. Samples were exposed to a variety of situations that could lead to contamination, such as extensive handling and exposure to coughing or sweaty clothing, to other wet bloodstains, and to saliva. The DQα types were determined from 469 individuals from three sample populations (Caucasian, black, and Hispanic), and the genotype frequencies were compared with frequencies previously reported by others. DNA samples from old cases [which had previously been analyzed by restriction fragment length polymorphism (RFLP) typing of variable number of tandem repeat sequences] were typed. All samples that were excluded by DQα typing were also excluded by RFLP analysis, and all samples that were included by RFLP analysis were included by DQα typing. Finally, the problem of allele dropout, or the failure to detect particular alleles, was noted and alleviated by performing the typing under appropriate conditions. The results of these validation experiments indicate that typing of the DQα gene by PCR and detection of specific alleles can be accomplished, when the typing is done using proper protocols, without producing false positive or false negative results.
AB - A series of experiments has been performed to evaluate typing of the HLA DQα gene by polymerase chain reaction (PCR) amplification of the gene and subsequent hybridization with sequence-specific oligonucleotide probes. These experiments were designed to evaluate DQα typing for analysis of evidentiary specimens. Bloodstains were exposed to a variety of conditions and environmental insults. These conditions included exposure to many different types of substrates, various microorganisms that could be encountered in evidentiary stains, sunlight, and a variety of chemical contaminants. Varying amounts of genomic deoxyribonucleic acid (DNA) were amplified to test the sensitivity of DQα typing. The sensitivity of the PCR technique raises the concern that DNA from sources other than the evidentiary material could be detected. A series of experiments was done to evaluate the question of DNA contamination. Purified DNA samples with different DQα types were mixed in different ratios to determine the ratio at which it could not be determined whether an allele was from the sample or the contaminant. Samples were exposed to a variety of situations that could lead to contamination, such as extensive handling and exposure to coughing or sweaty clothing, to other wet bloodstains, and to saliva. The DQα types were determined from 469 individuals from three sample populations (Caucasian, black, and Hispanic), and the genotype frequencies were compared with frequencies previously reported by others. DNA samples from old cases [which had previously been analyzed by restriction fragment length polymorphism (RFLP) typing of variable number of tandem repeat sequences] were typed. All samples that were excluded by DQα typing were also excluded by RFLP analysis, and all samples that were included by RFLP analysis were included by DQα typing. Finally, the problem of allele dropout, or the failure to detect particular alleles, was noted and alleviated by performing the typing under appropriate conditions. The results of these validation experiments indicate that typing of the DQα gene by PCR and detection of specific alleles can be accomplished, when the typing is done using proper protocols, without producing false positive or false negative results.
KW - deoxyribonucleic acid (DNA)
KW - genetic typing
KW - pathology and biology
KW - polymerase chain reaction (PCR)
UR - http://www.scopus.com/inward/record.url?scp=0025720058&partnerID=8YFLogxK
U2 - 10.1520/jfs13188j
DO - 10.1520/jfs13188j
M3 - Article
C2 - 1685163
AN - SCOPUS:0025720058
SN - 0022-1198
VL - 36
SP - 1633
EP - 1648
JO - Journal of forensic sciences
JF - Journal of forensic sciences
IS - 6
ER -