Reliability of non-invasively acquired human genomic DNA as a substrate for real-time PCR-assisted analysis of genetic polymorphisms.
Molecular epidemiological studies require high numbers of participants. The combination of an non-invasive access to human DNA with a rapid genotyping analysis, e.g. by use of LightCycler assisted real-time polymerase chain reaction (PCR), can be helpful in conducting such trials. The aim of our study was to define, for the first time, the use of LightCycler technology in analysis of non-invasively derived DNA. DNA extracted from blood, mouthwash and buccal cytobrush samples from 100 volunteers was analyzed for the genotypes of cytochrome P450 CYP1B1, and glutathione S-transferases GSTT1, GSTM1 and GSTP1. The median amounts of DNA isolated from blood, mouthwash and buccal cytobrush samples were 95, 11 and 8 microg, respectively. While genotyping for CYP1B1 codon 432 polymorphism and GSTP1 codon 105 polymorphism resulted in a complete correspondence for all three modes of sampling, the identification of individuals with null-genotype for GSTT1 or GSTM1 failed in some cases due to atypical courses of the corresponding melting curves, leading to high false-positive rates in the group of non-invasively derived samples. Thus, the results presented here call for caution in using LightCycler assisted real-time PCR in non-invasively collected samples, at least when appropriate control strategies are not implemented.
Medizinische Universitäts-Poliklinik Bonn, Wilhelmstr. 35-37, 53111, Germany.
T
T
Neuhaus
G
G
Geisen
H M
HM
Bolt
V
V
Janzen
A
A
Kraemer
H
H
Vetter
Y
Y
Ko
D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D005260:Female; D005838:Genotype; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008297:Male; D009061:Mouth Mucosa / Q000166:cytology; D009067:Mouthwashes / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D013696:Temperature
D007527:Isoenzymes; D009067:Mouthwashes; D004247:DNA; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D005982:Glutathione Transferase