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

Abstract

Introduction: Automated capillary electrophoresis single strand conformation polymorphism (CE-SSCP), denaturant gradient gel electrophoresis (DGGE), and direct sequencing were compared to investigate the benefits and sensitivity of each of the methods for detection of unknown TP53 mutations in human lung cancer. Methods: Twenty previously analyzed DNA samples from lung tumors were examined under dummy laboratory codes for occurrence of mutations of the TP53 gene. Results: Mutations were found in 17 samples; 15 (88%) of them were detected by DGGE, 16 (94%) by CE-SSCP and 12 (71%) by direct sequencing. One of the two mutations that remained undetected in DGGE was in fact outside the sequence area covered by DGGE screening, thus rendering DGGE and CE-SSCP equally efficient in mutation detection. Direct sequencing performed less well in finding mutations than the two other assays, as also shown previously. Conclusion: The study showed that CE-SSCP is a fast and highly reproducible method, which is considerably less laborious compared to DGGE, for screening of unknown TP53 mutations.

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.