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Clinical Chemistry 47: 821-828, 2001;
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(Clinical Chemistry. 2001;47:821-828.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Enzymatic Mutation Detection Method Evaluated for Detection of p53 Mutations in cDNA from Breast Cancers

Torbjörn Norberg1a, Sigrid Klaar1, Lena Lindqvist1, Thomas Lindahl1, Johan Ahlgren2 and Jonas Bergh1

1 Cancer Centre Karolinska, Radiumhemmet, Karolinska Institute & Hospital, SE-171 76 Stockholm, Sweden.

2 Department of Oncology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.

aAuthor for correspondence. Fax 46-8-339031; e-mail torbjorn.norberg{at}cck.ki.se.

Background: Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMDTM) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53.

Methods: The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products were analyzed and scored using an ALFTM automated DNA sequencer and ALFwin Fragment Analyzer software (Ver. 1.02).

Results: The EMD technique had sensitivities of 45% and 64% and specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47).

Conclusions: EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.




The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
B. Lumbreras-Lacarra, J. M. Ramos-Rincon, and I. Hernandez-Aguado
Methodology in Diagnostic Laboratory Test Research in Clinical Chemistry and Clinical Chemistry and Laboratory Medicine
Clin. Chem., March 1, 2004; 50(3): 530 - 536.
[Abstract] [Full Text] [PDF]


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