Validity of PCR with emphasis on variable number of tandem repeat analysis.
OBJECTIVE
Variable number of tandem repeat polymorphisms (VNTR) are frequently analyzed by PCR in genetic, epidemiologic and forensic studies. We wanted to explore the validity of these PCR analyses.


METHODS
The amplification of the different alleles of the 17- and the 44-bp VNTR of the serotonin transporter gene and the 39-bp VNTR of the glycoprotein Ibalpha gene was analyzed. We studied the effects of the parameters magnesium, dimethylsulfoxide, 7-deaza-dGTP, formamide, betaine, PCR temperatures and different types of polymerases.


RESULTS
In all three VNTR polymorphisms selective amplification of one of the alleles of heterozygous individuals could be obtained by change of the magnesium concentration. This problem could be minimized by a combination of Taq- and Pwo-polymerases and by use of 7-deaza-dGTP.


CONCLUSIONS
PCR analysis of all of these VNTRs may give reproducibly wrong results in truly heterozygous subjects due to selective amplification of only one of the alleles.
Kaiser
Rolf
R
Institute of Clinical Pharmacology, Universitätsklinikum Charité, Humboldt Universität Berlin, D-10098, Berlin, Germany. rolf.kaiser@med.uni-goettingen.de
Tremblay
Pierre Benoit
PB
Roots
Ivar
I
Brockmöller
Jürgen
J
D001622:Betaine / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005559:Formamides / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D018598:Minisatellite Repeats / Q000235:genetics*; D009419:Nerve Tissue Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000592:standards*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D050486:Serotonin Plasma Membrane Transport Proteins; D013696:Temperature
D002352:Carrier Proteins; D003848:Deoxyguanine Nucleotides; D005559:Formamides; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C065361:2'-deoxy-7-deazaguanosine triphosphate; D001622:Betaine; C031066:formamide; D004247:DNA; D008274:Magnesium; D004121:Dimethyl Sulfoxide