Sensitive detection of loss of heterozygosity in the TP53 gene in pancreatic adenocarcinoma by fluorescence-based single-strand conformation polymorphism analysis using blunt-end DNA fragments.
We have developed a fluorescence-based single-strand conformation polymorphism analysis to detect Haelil-sensitive polymorphic sites in intron 1 of the TP53 gene. It is important to treat the PCR products with Klenow fragment to remove a 3'-protruding nucleotide from the amplified DNA fragments added during the reaction in order to obtain a single peak for each allele. A comparison of the signal profiles of two alleles with those of normal heterozygotes by data processing using computer software has enabled sensitive detection of loss of heterozygosity (LOH) from clinical materials with a fraction of tumor cells below 10%. In analysis of 14 pancreatic carcinomas in which the proportion of the tumor cells is usually low due to the abundance of the stromal component, 7 samples (50%) were informative and 5 of the 7 (71.4 %) were positive for LOH at the TP53 locus. This approach would be useful for allelotyping tumors with low cellularity, as well as other clinical samples such as biopsied specimens and paraffin embedded tissues.
Sugano
K
K
Division of Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan.
Nakashima
Y
Y
Yamaguchi
K
K
Fukayama
N
N
Maekawa
M
M
Ohkura
H
H
Kakizoe
T
T
Sekiya
T
T
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D000483:Alleles; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology / Q000601:surgery; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; D017353:Gene Deletion; D016158:Genes, p53; D006580:Genetic Carrier Screening; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017384:Sequence Deletion; D012984:Software; D019914:Taq Polymerase
D017931:DNA Primers; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases