Tp53

The TP53 tumor suppressor gene is associated with numerous pathways that maintain genetic stability and control cell life and death. Normally, p53 protein levels are very low; however, the levels of p53 change over time. P53 levels fluctuate as cells adapt to pathway-initiated stimuli and p53 feedback systems.[9] Activation of p53 occurs when cells are stressed or damaged. For example, DNA single-strand breaks caused by ionizing radiation are sufficient to trigger up-regulation of p53. Damaged cells have a greater risk of becoming cancerous, thus p53 protein inhibits the cell cycle resulting in programmed cell death. The normal p53 protein does not function in human cancers when the gene is inactivated as a result of mutations. Hence p53 appears to be a critical element in the protection against tumorigenesis.[10]

Almost all mutations in the p53 gene reduce the p53 protein's capability to activate transcription. The majority of p53 mutations that occur in human tumors are located in the sequence-specific DNA binding domain.[11] Hence mutations in the p53 gene disrupt basic cellular function. PCR-based cancer diagnosis of the status of the p53 gene requires detection of both common and rare mutation(s) that occur throughout the gene. Moreover, amplification efficiencies of mutant and wild-type sequences are unequal and associated with the distance from the primer to the point mutation.[12] For sensitive detection of TP53 mutations, reliable detection technologies and reference materials for PCR mutation detection systems are needed to monitor both the accuracy of detection and the possible introduction of errors during PCR. Renewable reference materials, such as the NIST TP53 mutation panel,[2] serve to normalize the data collected in different laboratories using different primers and analytical platforms.

The renewable reference panel contains the single-base substitutions most commonly found in human cancers, as well as three that proved difficult to detect by scanning technologies.[2] The panel consists of 12 plasmid clones derived from human genomic DNA containing a wild-type TP53 gene, 11 of which contain a specific mutation in exons 5-9; the 12th is wild type. This panel accounts for 30% of reported TP53 cancer-associated mutations.[13] We evaluated the panel using single strand conformation polymorphism (SSCP), denaturing gradient gel electro-phoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and exon sequencing, four of the most commonly used methods for mutation detection. Following exon-specific amplification, the amplicons of all 12 clones were analyzed by SSCP, DGGE, DHPLC, and sequencing. SSCP detected 75% of the mutations in the reference panel, as expected. The heteroduplex technologies, DGGE and DHPLC, as well as fluorescent sequencing, positively identified 100% of the reference mutations.

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