TP53 sequencing methods

The method we recommend to assess TP53 mutation status is to perform gene sequencing. Immuno-histochemical analysis is not a good method for assessing TP53 mutation status, as it cannot distinguish between the various types of mutations, does not detect truncating mutations, and gives false-positive results. Although most mutations cluster within exons 5-8, it is recommended to screen at least exons 4–10, including the splice junctions, so that less than 1% of mutations may be missed.

The current sequencing procedures used at IARC for human TP53 gene are direct sequencing or DHPLC/sequencing. Protocols can be downloaded below:

 IARC protocols

DHPLC prescreening and sequencing

Direct sequencing

SNP sequencing and haplotyping

Here are some links to publications that describe other methods to sequence the human TP53 gene:

  • Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus
    Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F, De Silva D, Lesueur F, Landi S, Tavtigian SV. BMC Genet. 2009 Feb 17;10:5. PubMed (Primers list)

  • APEX microarray: comparison with dHPLC and direct sequencing
    Le Calvez F, Ahman A, Tonisson N, Lambert J, Temam S, Brennan P, Zaridze DG, Metspalu A, Hainaut P. Clin Chem. 2005 Jul;51(7):1284-7. PubMed

  • Temporal Temperature Gradient Electrophoresis (TTGE)
    - Sorlie T, Johnsen H, Vu P, Lind GE, Lothe R, Borresen-Dale AL. Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis. Methods Mol Biol. 2005;291:207-16. Pdf
    - Taniere P, Martel-Planche G, Maurici D, Lombard-Bohas C, Scoazec JY, Montesano R, Berger F, Hainaut P. Molecular and clinical differences between adenocarcinomas of the esophagus and of the gastric cardia. Am J Pathol. 2001 Jan;158(1):33-40. PubMed

  • Single Strand Conformation Polymorphism (SSCP)
    Mazars R, Pujol P, Maudelonde T, Jeanteur P, Theillet C. p53 mutations in ovarian cancer: a late event? Oncogene. 1991 Sep;6(9):1685-90. PubMed

  • Constant Denaturant Gel Electrophoresis (CDGE)
    Borresen AL, Hovig E, Smith-Sorensen B, Malkin D, Lystad S, Andersen TI, Nesland JM, Isselbacher KJ, Friend SH. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8405-9. PubMed

  • Denaturing Gradiant Gel Electrophoresis (DGGE)
    Hamelin R, Jego N, Laurent-Puig P, Vidaud M, Thomas G. Efficient screening of p53 mutations by denaturing gradient gel electrophoresis in colorectal tumors. Oncogene. 1993 Aug;8(8):2213-20. PubMed

  • Cloning of genomic DNA and sequencing
    Baker SJ, Preisinger AC, Jessup JM, Paraskeva C, Markowitz S, Willson JK, Hamilton S, Vogelstein B. p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis. Cancer Res. 1990 Dec1;50(23):7717-22. PubMed

  • DHPLC
    - Keller G, Hartmann A, Mueller J, Hofler H. Denaturing high pressure liquid chromatography (DHPLC) for the analysis of somatic p53 mutations. Lab Invest. 2001 Dec;81(12):1735-7. PubMed
    - Gross E, Kiechle M, Arnold N. Mutation analysis of p53 in ovarian tumors by DHPLC. J Biochem Biophys Methods. 2001 Jan 30;47(1-2):73-81.PubMed

  • Dideoxy fingerprinting (ddF)
    Blaszyk H, Hartmann A, Schroeder JJ, McGovern RM, Sommer SS, Kovach JS. Rapid and efficient screening for p53 gene mutations by dideoxy fingerprinting. Biotechniques. 1995 Feb;18(2):256-60. PubMed

  • Yeast functional assay (FASAY)
    - Smardova J, Smarda J, Koptikova J. Functional analysis of p53 tumor suppressor in yeast. Differentiation. 2005 Jul;73(6):261-77. PubMed
    - Robert V, Michel P, Flaman JM, Chiron A, Martin C, Charbonnier F, Paillot B, Frebourg T. High frequency in esophageal cancers of p53 alterations inactivating the regulation of genes involved in cell cycle and apoptosis. Carcinogenesis. 2000 Apr;21(4):563-5. PubMed