Division of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
PLoS One. 2013;8(4):e60151. doi: 10.1371/journal.pone.0060151. Epub 2013 Apr 2.
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.
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.
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.
人类 MDM2 基因中的遗传多态性被认为是肿瘤易感性标志物和癌症的预后因素。据报道，MDM2 基因中的单核苷酸多态性 (SNP) c.309T>G 减弱了 p53 的肿瘤抑制活性，并加速了人类肿瘤的形成。
在这项研究中，为了检测 MDM2 基因中的 SNP c.309T>G，我们开发了一种新的 SNP 检测方法，称为“双链 SmartAmp”，它使我们能够在一个管中同时检测 309T 和 309G 等位基因。为了开发这种新方法，我们引入了新的引物 nBP 和 oBPs，以及两种不同的荧光染料，分别检测这些遗传多态性。
通过双链 SmartAmp 方法，可以直接从少量基因组 DNA 或血液样本中检测 MDM2 基因的遗传多态性。我们使用 96 个基因组 DNA 和 24 个血液样本与传统 PCR-RFLP 方法的结果进行比较，验证了双链 SmartAmp；因此，双链 SmartAmp 结果与 PCR-RFLP 方法的结果完全一致。因此，这种新的 SNP 检测方法被认为可用于检测 MDM2 基因中的 SNP c.309T>G，以便在临床环境中判断对某些细胞应激的癌症易感性，同时还可以处理大量样本并实现快速临床诊断。
