Determination of optimal conditions for analysis of p53 status in leukemic cells using functional analysis of separated alleles in yeast.
Tumor suppressor p53 is transcription factor that participates in control of many cellular functions. Somatic mutations of the p53 gene are frequently detected in human cancers. Several methods can be used for identification of p53 mutations, including FASAY - functional analysis of separated alleles in yeast. FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. The validity of the method depends on a low background level. There are several sources of background as PCR-induced point mutations, low quality of RNA and alternative splicing of intron 9 affecting the p53 carboxy-terminus. In the present work we show that FASAY can be successfully used for analysis of mRNA isolated from blood samples that were collected and stored for 24 hours at 0 degrees C without undesired increase of background. We also measured fidelity of several commonly used DNA polymerases and determined the most suitable kinds of Pfu DNA polymerases for FASAY. Reaction conditions described in this report allow routine analysis of p53 status in leukemic cells using FASAY.
Smardová
Jana
J
Department of Experimental Oncology, Masaryk Memorial Cancer Institute, Brno, 656 53, Czech Republic. smardova@mou.cz
Pavlová
Sárka
S
Koukalová
Hana
H
D000483:Alleles; D017398:Alternative Splicing; D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016158:Genes, p53; D005821:Genetic Techniques; D006801:Humans; D007438:Introns; D007938:Leukemia / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D012313:RNA; D004259:DNA-Directed DNA Polymerase