Publication: Development of PCR Method for the Detection of mu Opiate Receptor Polymorphism.
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Date
2009
Authors
Talib, Nazila
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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 (PCRl ), exon 1, 2, 3 and intron 2 of OPRMl gene were amplified. There were two set of reaction involved in PCRl; 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 PCRl 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 ofPCR mixture. Conclusion: We were partially successful in developing and optimizing a multiplex PCR method which is suitable for use in population studies of OPRMl polymorphism.
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Mu opiate receptor serving as primary target for opiates drug. It plays a key role in addiction and pain perception.