Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR.
The G-->A transition at nucleotide 21881 of the human catechol-O-methyltransferase (COMT) gene represents a functional genetic polymorphism (Val158Met), rendering an enzyme with reduced activity that has been associated with psychiatric disorders and estrogen-related cancers. A new method for the detection of this polymorphism is described, based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), with a single PCR to discriminate both alleles. Two primers amplify a common amplicon independently of the allele considered. At the same time, two primers are used, differing in the 3' base. In the Val/Val or Met/Met conditions, amplification occurs both in the general amplicon and in the specific allele; in the Val/Met condition three different amplicons are produced. Direct DNA sequencing of a COMT region containing the G/A polymorphism demonstrates the validity of this tetra-primer ARMS-PCR method. Reevaluation by PCR-RFLP revealed 100% accordance for genotype adscription. Subjects carrying the COMT(HH) genotype in a Spanish population comprised 28%, and the COMT(LL) homozygotes amounted to 21%. The described method provides a fast and reliable approach for determining COMT polymorphism that can be useful in large clinical studies using minimal quantity of DNA, avoiding the timely and costly use of restriction enzymes.
Ruiz-Sanz
José Ignacio
JI
Department of Physiology, Medicine School, University of the Basque Country, 48080 Bilbao, Spain.
Aurrekoetxea
Igor
I
Ruiz del Agua
Ainhoa
A
Ruiz-Larrea
María Begoña
MB
D019943:Amino Acid Substitution; D001483:Base Sequence; D002394:Catechol O-Methyltransferase / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005260:Female; D005787:Gene Frequency; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D013030:Spain; D014633:Valine / Q000235:genetics
D017931:DNA Primers; D008715:Methionine; D002394:Catechol O-Methyltransferase; D014633:Valine