Renewable standard reference material for the detection of TP53 mutations.
BACKGROUND
Numerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0 kb region of the TP53 gene, including exons 5-9. Eleven of these clones contain a single mutation within the mutational hot spots of the TP53 gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-type TP53 from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms.


RESULTS
The single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM.


CONCLUSIONS
The generation of a standard TP53 reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. coc@nist.gov
Catherine D
CD
O'Connell
Lois A
LA
Tully
Joseph M
JM
Devaney
Michael A
MA
Marino
John P
JP
Jakupciak
Donald H
DH
Atha
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000592:standards*; D017931:DNA Primers / Q000235:genetics / Q000592:standards; D019075:Electrophoresis, Capillary; D005091:Exons; D016158:Genes, p53; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000592:standards; D018807:Polymorphism, Single-Stranded Conformational; D012015:Reference Standards
D017931:DNA Primers