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A simple and rapid genotyping method for beta‐2 receptor (β2 AR) gene using allele specific multiplex PCR
Author(s) -
Romaino S. M. N.,
Teh L. K.,
Zilfalil B. A.,
Thong C. P.,
Ismail A. A.,
Amir J.,
Salleh M. Z.,
Noor A. R.,
Ismail R.
Publication year - 2004
Publication title -
journal of clinical pharmacy and therapeutics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.622
H-Index - 73
eISSN - 1365-2710
pISSN - 0269-4727
DOI - 10.1046/j.1365-2710.2003.00535.x
Subject(s) - genotyping , multiplex polymerase chain reaction , multiplex , allele , biology , variants of pcr , gene , genetics , genotype , polymerase chain reaction , microbiology and biotechnology
Summary Background: Polymorphism of the beta2‐adrenergic receptor ( β 2 AR) gene is an important determinant of the function of this receptor. It affects receptor down‐regulation and β 2 ‐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 β 2 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). Objectives: To develop a simple and rapid PCR based method for the simultaneous detection of five β 2 AR alleles. Method: 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 MgCl 2 , Taq polymerase, primers and annealing temperature, to produce specific bands of interest. DNA samples were selected at random and submitted for direct PCR sequencing. Result: 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. Conclusion: The simple and rapid method for the simultaneous detection of the five β 2 AR alleles is suitable for the study of β 2 polymorphism and its clinical consequences.