Enhanced Detection of TP53 Mutations Using a GC-clamp in Denaturing High Performance Liquid Chromatography

Abstract

Although denaturing gradient gel electrophoresis (DGGE) is a highly effective technique for screening for TP53 mutations, the use of denaturing high performance liquid chromatography (DHPLC) is a growing methodology. This report describes a comparison between DHPLC and DGGE in the detection of TP53 mutations in hematopoietic cell lines and lymphomas. In addition, the improved effectiveness of guanine cytosine (GC)-clamped DHPLC for TP53 screening is detailed. Thirty DNA samples with known TP53 mutations in the hotspot region of codons 5-8, previously identified by DGGE, were analyzed by DHPLC. We found 100% concordance in mutation detection by DHPLC with DGGE. Similar to the improved efficacy observed in DGGE, the addition of 40 nucleotide GC-clamps composed of guanine and cytosine bases at one end of the product enhanced the detection of a mutation pattern by DHPLC. DHPLC of GC-clamped products is a viable and faster alternative method for screening TP53 mutations.