Talib, Nazila
(2009)
Development of PCR Method for the Detection of
mu Opiate Receptor Polymorphism.
Project Report.
Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia.
(Submitted)
Abstract
Background: Mu opiate receptor serving as primary target for opiates drug. It plays a key
role in addiction and pain perception. This receptor is highly polymorphic, but a simple
method was not available to study its genetic polymorphism. We developed and optimized
nested allele-specific multiplex PCR to detect twelve SNPs. Three of the SNPs; 118 A/G,
IVS +31 G/A and IVS +691 C/G are common SNPs and have implication to human
system. But others SNPs were rare SNPs and not widely studied. Objective: The objective
of our study was to develop a simple and rapid PCR method for detecting polymorphism
of mu opiate receptor (OPRM1 ), then, to validate the PCR method developed. Method:
Genomic DNA was extracted from blood using Spin Protocol :from QIAamp DNA mini
kit. A two step PCR method was developed to detect twelve SNPs of OPRM1 gene. In the
first PCR (PCR1 ), exon 1, 2, 3 and intron 2 of OPRM1 gene were amplified. There were
two set of reaction involved in PCR1; Set 1 amplifies exon 1, 2, and 3 simultaneously
while Set 2 applies for intron 2 only. The PCR products, then, were used as template in
parallel allele-specific PCR (PCR2). Afterwards sequencing was used to validate the test
results. Result: We have successfully developed and optimized PCR1 which amplified
exon 1 (420 bp), exon2 (483 bp), exon 3 (677 bp), and intron 2 (1020 bp). Fortunately,
only a few SNPs were able to be detected in PCR2. These SNPs consist of 24 G/ A (1 02
bp), 440 C/G (330 bp), 802 TIC (424 bp), 942 G/A (434 bp), IVS +310/A (162 bp), and
IVS +6910/C (240 bp). Other six SNPs; 17 err, 118 A/G, 454 A/G, 779 G/A, 794 G/A,
and 820 G/A failed to be amplified specifically. It might be due to contamination and also
technique during preparation of PCR mixture. Conclusion: We were partially successful in
developing and optimizing a multiplex PCR method which is suitable for use in population
studies of OPRM1 polymorphism.
Actions (login required)
 |
View Item |
