Dataset Information

Rapid detection of SNP (c.309T>G) in the MDM2 gene by the Duplex SmartAmp method.

ABSTRACT:

Background

Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans.

Methodology

In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms.

Results and conclusions

By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

SUBMITTER: Enokida Y

PROVIDER: S-EPMC3614994 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Rapid detection of SNP (c.309T>G) in the MDM2 gene by the Duplex SmartAmp method.

Enokida Yasuaki Y Shimizu Kimihiro K Atsumi Jun J Lezhava Alexander A Tanaka Yuki Y Kimura Yasumasa Y Soma Takahiro T Hanami Takeshi T Kawai Yuki Y Usui Kengo K Okano Yasuko Y Kakegawa Seiichi S Ogawa Hiroomi H Miyamae Yohei Y Miyagi Yohei Y Nakayama Haruhiko H Ishikawa Toshihisa T Hayashizaki Yoshihide Y Takeyoshi Izumi I

PloS one 20130402 4

<h4>Background</h4>Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans.<h4>Methodology</h4>In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us t ...[more]

PMID: 23565197

Similar Datasets

Project description:Background and Purpose- The E3 ubiquitin ligase MDM2 (murine double minute 2) is the main negative regulator of the p53 protein-a key player in neuronal apoptosis after ischemia. A functional single-nucleotide polymorphism in the human MDM2 gene promoter (rs2279744) regulates MDM2 protein expression. We investigated whether the MDM2 SNP309, by controlling p53-mediated apoptosis, determines functional outcome after stroke. Methods- Primary cortical neurons were subjected to oxygen and glucose deprivation. Mice were subjected to ischemic (transient middle cerebral artery occlusion) or hemorrhagic (collagenase injection) stroke models. Protein and mRNA levels of MDM2 and p53 were measured in both neuronal and brain extracts. The interaction of MDM2 with p53 was disrupted by neuronal treatment with nutlin-3a. siRNA was used to knockdown MDM2 expression. We analyzed the link between the MDM2 SNP309 and functional outcome, measured by the modified Rankin Scale scores, in 2 independent hospital-based stroke cohorts: ischemic stroke cohort (408 patients) and intracerebral hemorrhage cohort (128 patients). Results- Experimental stroke and oxygen and glucose deprivation induced the expression of MDM2 in the brain and neurons, respectively. Moreover, oxygen and glucose deprivation promoted MDM2 binding with p53 in neurons. Disruption of the MDM2-p53 interaction with nutlin-3a, or MDM2 knockdown by siRNA, triggered p53 accumulation, which increased neuronal susceptibility to oxygen and glucose deprivation-induced apoptosis. Finally, we showed that patients harboring the G allele in the MDM2 promoter had higher MDM2 protein levels and showed better functional outcome after stroke than those harboring the T/T genotype. The T/T genotype was also associated with large infarct volume in ischemic stroke and increased lesion volume in patients with intracerebral hemorrhage. Conclusions- Our results reveal a novel role for the MDM2-p53 interaction in neuronal apoptosis after ischemia and show that the MDM2 SNP309 determines the functional outcome of patients after stroke.

Dataset Information

Rapid detection of SNP (c.309T>G) in the MDM2 gene by the Duplex SmartAmp method.

Background

Methodology

Results and conclusions

Publications

Rapid detection of SNP (c.309T>G) in the MDM2 gene by the Duplex SmartAmp method.

Similar Datasets

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