Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR.
The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.
Grenett
H E
HE
Department of Medicine, University of Alabama at Birmingham, 35294-2170, USA. hgrenett@cardio.dom.uab.edu
Khan
N
N
Jiang
W
W
Booyse
F M
FM
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015247:Deoxyribonuclease HindIII / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005069:Evaluation Studies as Topic; D005312:Fetal Blood / Q000737:chemistry; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013997:Time Factors
D020413:3' Untranslated Regions; D017395:Plasminogen Activator Inhibitor 1; D004247:DNA; D015247:Deoxyribonuclease HindIII