Analysis of TP53 gene mutations in human lung cancer: Comparison of capillary electrophoresis single strand conformation polymorphism assay with denaturing gradient gel electrophoresis and direct sequencing
Introduction
The tumor suppressor gene TP53 is one the most studied genes in cancer research. The p53 protein mediates many different cellular processes that control cell growth, including effects on cell cycle, DNA repair, apoptosis, differentiation and angiogenesis [1]. It plays a central role in the cell's response to various kinds of stress situations, including damage to DNA. Mutation of the TP53-gene is the most common genetic alteration in the human cancers [2], [3]. TP53 is a fairly large gene, and mutations discovered in human cancer are scattered all over the 10 encoding exons. Still, majority of the mutations occur in exons 5–8, which is the conserved area of the gene. Mutations of the TP53 gene are typically of diverse types, including base substitutions, small deletions, and insertions [4]. Therefore, investigation of human tumor DNAs for presence of unknown mutations must rely on an efficient screening system with high sensitivity.
Various methods based on gel electrophoresis have traditionally been used in mutation detection to separate the wildtype sequence from the mutated ones. The methods based on gel separation are simple to run and do not require large investments in equipment. Denaturant gradient gel electrophoresis (DGGE) and single strand conformation polymorphism (SSCP) are both commonly used methods for mutation screening. They enable rather quick and reliable detection of variations in DNA sequence, but neither of them provides information about the type or exact location of the variation. Generally, DGGE is considered to be more sensitive than the standard SSCP assay [5], whereas the latter is considered simpler to run. These methods are, however, typically time consuming and they cannot be easily automated. Application of the capillary electrophoresis (CE) technique, among other fairly recently developed methods such as denaturing high performance liquid chromatography (DHPLC), has made it possible to achieve faster analysis and greater automation [6], [7]. With the use of CE technique, SSCP has become more readily applicable for investigating large collections of DNA samples [8]. Ultimately, for identification of the alterations detected in screening, the DNA fragments must be sequenced.
In the present study, capillary electrophoresis SSCP (CE-SSCP) was compared with the traditional slab gel DGGE, and direct sequencing to find out the benefits and relative sensitivities of the three methods for detection of somatic mutations of the TP53 gene in human tumor samples.
Section snippets
Tumor samples and DNA extraction
Twenty lung cancer samples were analysed for TP53 mutations. DNA samples were prepared from fresh lung tumor specimens from which non-tumor tissue was carefully excluded macroscopically by pathologist before storage at −80 °C, as described earlier [9]. For the present work, new DNAs were prepared from samples cut from the same tumor tissue specimens, except for the cases for which DNA samples but no tumor specimens were available (samples # T174, T182, T213, T246, and T316). The DNA was purified
Results
In the present analysis, altogether 17 out of 20 samples of the lung tumor DNA analyzed were found to carry a mutation. For three tumors, the mutation discovered in the previous analyses was not detected by any of the assays used. Since new DNA needed to be extracted from the tissue specimens for the present work, it may be that the newly cut tissue sections did not contain large enough proportions of the mutated cells to appear positive in the analysis. Of the total of 17 mutations, 15 were
Discussion
In the present study, all the mutation detection methods used were able to recognize the majority of the pre-known TP53 mutations present in the lung tumors examined. CE-SSCP assay had a high efficacy in detecting mutations (16 out of 17, 94%), DGGE assay detected one mutation less than CE-SSCP, but the mutation was situated in the intronic area and was thus not properly within the area efficiently screened with DGGE. Even though the sequences of the primers were the same for both of the
Acknowledgements
We thank Ms. Tuula Suitiala, chief technician, Finnish Institute of Occupational Health, Helsinki, for skillful technical assistance. The work was financially supported by the EU 5th FP project no. QLK4-2000-00573 (WOOD-RISK).
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