Enhanced detection of TP53 mutations using a GC-clamp in denaturing high performance liquid chromatography.
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.
Greiner
Timothy C
TC
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-3135, USA. tgreiner@unmc.edu
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003596:Cytosine / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D006147:Guanine / Q000737:chemistry; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016159:Tumor Suppressor Protein p53; D006147:Guanine; D003596:Cytosine