A simple and rapid genotyping method for beta-2 receptor (beta2 AR) gene using allele specific multiplex PCR.
BACKGROUND
Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164).


OBJECTIVE
To develop a simple and rapid PCR based method for the simultaneous detection of five beta2 AR alleles.


METHODS
DNA was extracted from whole blood using standard alkali lysis method. Primers specific at the 3' end for the polymorphic sites were designed. The nested allele specific PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, Taq polymerase, primers and annealing temperature, to produce specific bands of interest. DNA samples were selected at random and submitted for direct PCR sequencing.


RESULTS
The first PCR produced a fragment of size 710 bp, which was used as template for the subsequent duplex and triplex PCR to detect the mutation sites of the five alleles. The method was found reproducible and specific when used to genotype patients with bronchial asthma. The sequencing results confirmed the specificity of the PCR method.


CONCLUSIONS
The simple and rapid method for the simultaneous detection of the five beta2 AR alleles is suitable for the study of beta2 polymorphism and its clinical consequences.
Romaino
S M N
SM
Pharmacogenetics Study Group, Department of Pharmacology, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
Teh
L K
LK
Zilfalil
B A
BA
Thong
C P
CP
Ismail
A A
AA
Amir
J
J
Salleh
M Z
MZ
Noor
A R
AR
Ismail
R
R
D000483:Alleles; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*
D018343:Receptors, Adrenergic, beta-2