Novel polymerase chain reaction approach for full-coding p53 mutation detection in microdissected archival tumors.
Evidence suggests that up to 25% of p53 mutations are outside of exons 5-8 and that insertions, deletions, and polymorphic sites in the p53 gene may play a significant role in the process of carcinogenesis. A novel polymerase chain reaction (PCR) approach for the analysis of the entire p53 coding and splice site regions from microdissected, formalin-fixed, paraffin-embedded tumor tissues has been developed which allows multiple genetic analyses to be performed from one primary amplification reaction. The method was initially evaluated using well-characterized cell lines. In addition to confirming the published p53 mutations for HT29, Molt 4, A431, and HN5, a 16 base pair (bp) duplication within intron 3 was detected in both the A431 and HT29 cell lines. Analysis of archival samples of ovarian cancer identified the same 16-bp duplication and coding region variations. In all samples, using GenBank submission U94788 as a reference, a C-insertion was detected at nucleotide positions 11818 and 11874 within intron 2. At nucleotide position 14168, within intron 7, a T-to-G base change was found. This novel PCR approach has the potential to reduce the amount of clinical material required by up to 95%, thus facilitating retrospective studies on archival tumor collections. Furthermore, a wider analysis of the p53 gene, including splice sites and intronic regions, may yield additional information regarding cancer predisposition, response to therapy, and progression.
Thompson-Hehir
J
J
Clatterbridge Cancer Research Trust, J. K. Douglas Cancer Research Laboratories, Clatterbridge Hospital, Bebington, Cheshire, United Kingdom. joh@ccrt.co.uk
Davies
M P
MP
Green
J A
JA
Halliwell
N
N
Joyce
K A
KA
Salisbury
J
J
Sibson
D R
DR
Vergote
I
I
Walker
C
C
D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D008297:Male; D008846:Micromanipulation; D008969:Molecular Sequence Data; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53