Optimized polymerase chain reaction-based single-strand conformation polymorphism analysis of p53 gene applied to Bulgarian patients with invasive breast cancer

Abstract

During the last few decades a substantial amount of evidence has accumulated proving that the abrogation of the normal p53 pathway is a critical step in the initiation and progression of tumors. Decoding the genetic mechanisms involved in carcinogenesis requires screening for consistent genetic tumor alterations, including those concerning the p53 gene. Thus, practical, efficient, and inexpensive techniques for accurate determination of p53 mutational status are needed. Polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis is considered to be a useful tool to investigate the role of the p53 gene in the development and progression of human cancers. The sensitivity of the method can be increased considerably by varying the experimental conditions. Here we demonstrate a scheme of PCR-SSCP optimization for detection of p53 gene mutations of patients with various cancers. Optimal conditions for PCRSSCP of p53 exons 4-9 are reported. Such PCR-SSCP optimization could allow an increase in the sensitivity and reproducibility of the technique and facilitates screening of large series of patients to assess the clinical significance of p53 mutations in human cancers. Using the optimized PCR-SSCP analysis we screened Bulgarian patients with invasive breast cancer for p53 gene mutations and registered a 33.33% frequency of mutations. To date, there are no data concerning the p53 status of Bulgarian breast cancer patients. Screening for p53 gene mutations enables an accurate and routine determination of the p53 status of patients with cancer and may be applied in clinical oncology to cancer diagnosis, prediction of prognosis and response to treatment.

Alterations in p53, BRCA1, ATM, PIK3CA, and HER2 genes and their effect in modifying clinicopathological characteristics and overall survival of Bulgarian patients with breast cancer

“…Until now, BC research in Bulgaria is limited mainly to clinical, morphological, and epidemiological aspects of the disease. Except one previous investigation of our research group (Krasteva et al 2003) and another study including 20 patients (MarkoV et al 1998), there are no other data on the genetic status of Fig. 1 Kaplan-Meier survival curves.…”

“…To amplify exons 4 to 9 of p53 gene, PCRs were performed in a total volume of 25 l containing 150 ng of template DNA, 1£ PCR buVer, 1.75 mM MgCl2, 0.2 mM of each dNTP, 2.5 pmol of each primer, and 0.3 U of Taq DNA polymerase (Krasteva et al 2003). The cycling reactions included 32 cycles of denaturation at 94°C for 30 s. (Wrst cycle-5 min.…”

Analysis of the K-ras/B-raf/Erk signal cascade, p53 and CMAP as markers for tumor progression in colorectal cancer patients

“…To detect p53 mutations, a previously optimized PCR-SSCP method was applied (18). PCR reactions (MJ Research and Techne Cyclogen thermal cyclers) were conducted in a total volume of 25 μl containing 150 ng of template DNA, 1X PCR buffer, 1.75 mM MgCl 2 , 0.2 mM of each dNTP, 2.5 pmol of each primer and 0.5 units of Taq DNA polymerase.…”