Abstract
BackgroundGenetic 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.MethodologyIn 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 ConclusionsBy 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.
Highlights
The p53 gene encodes a nuclear protein that plays a pivotal role of inducing growth arrest or apoptosis of cancer cells in response to cellular stress and such external stimuli as drugs and medical radiation exposure [1,2,3]
The new single nucleotide polymorphism (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 to handle a large number of samples and enable rapid clinical diagnosis
To achieve high fidelity of SNP typing, we introduced two novel primers, namely, the outer Boost Primer and the neutral Boost Primer, in addition to the standard SmartAmp primers (i.e., TP, FP, BP, and OP)
Summary
The p53 gene encodes a nuclear protein that plays a pivotal role of inducing growth arrest or apoptosis of cancer cells in response to cellular stress and such external stimuli as drugs and medical radiation exposure [1,2,3]. In an in vitro study, it was reported that cells harboring homozygous alleles of 309G/G express higher levels of MDM2 protein, thereby reducing the tumor suppressor activity of p53. The SNP 309T.G in the MDM2 gene was reported to be associated with an earlier onset of tumor formation in both hereditary and sporadic cancers [7]. It has recently been reported that this polymorphism in the MDM2 gene is associated with the prognosis for several types of tumors, such as esophageal, pancreatic, and lung cancers [15,16,17]. 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
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