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Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational
Bulbin Sunar-Reeder1, Donald H Atha, Songul Aydemir
1Department of Medical Biology and Genetics, Faculty of Medicine, Inönü University, Malatya, Turkey.
Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology
|November 6, 2004
Summary
A new TP53 reference panel was validated for quality assurance in clinical genetic testing. The panel successfully detected TP53 mutations and polymorphisms in gynecological specimens, confirming its utility for accurate genetic test results.
Area of Science:
- Molecular Biology
- Genetics
- Clinical Diagnostics
Background:
- The increasing use of genetic information in clinical settings necessitates reliable reference materials for genetic test quality assurance.
- A panel of 12 plasmid clones with wild-type or point mutations in TP53 exons 5-9 was developed as a reference material.
- This panel is crucial for ensuring the accuracy of TP53 mutation detection.
Purpose of the Study:
- To validate the efficacy of the developed TP53 reference materials for quality assurance in detecting TP53 mutations within clinical samples.
- To assess the panel's performance in identifying mutations and polymorphisms in real-world clinical specimens.
Main Methods:
- Analysis of 33 gynecological samples (11 normal, 22 malignant tumors).
- Mutation detection using single-strand conformational polymorphism (SSCP) analysis via slab gel and capillary electrophoresis.
- Amplification of TP53 exons 5-9 using fluorescently labeled PCR primers, followed by DNA sequence analysis for confirmation.
Main Results:
- TP53 mutations and polymorphisms were identified in six out of 33 clinical specimens across three scanned exons (exons 5, 6, and 8).
- No mutations were detected in exons 7 or 9.
- Both mutations and polymorphisms were observed in non-malignant control specimens, highlighting the panel's sensitivity.
Conclusions:
- The study successfully detected TP53 mutations and polymorphisms in clinical samples, validating the reference panel's utility.
- Most identified mutations were silent, with one non-conservative mutation found in exon 5, codon 181.
- The National Institute of Standards and Technology (NIST) TP53 reference panel proved effective for TP53 mutation detection in clinical tissue specimens, enhancing quality assurance.