High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling
✍ Scribed by Roy Bastien; Tracey B. Lewis; Jason E. Hawkes; John F. Quackenbush; Thomas C. Robbins; Juan Palazzo; Charles M. Perou; Philip S. Bernard
- Publisher
- John Wiley and Sons
- Year
- 2008
- Tongue
- English
- Weight
- 367 KB
- Volume
- 29
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
Communicated by Ian N.M. Day
Identifying mutations in the TP53 gene is important for cancer prognosis, predicting response to therapy, and determining genetic risk. We have developed a high-throughput scanning assay with automatic calling to detect TP53 mutations in DNA from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues. The coding region of the TP53 gene (exons 2-11) was PCR-amplified from breast cancer samples and scanned by high-resolution fluorescent melting curve analyses using a 384-well format in the LightCycler 480 instrument. Mutations were confirmed by direct sequencing. Sensitivity and specificity of scanning and automatic mutation calling was determined for FF tissue (whole genome amplified [WGA] and non-WGA) and FFPE tissue. Thresholds for automatic mutation calling were established for each preparation type. Overall, we confirmed 27 TP53 mutations in 68 primary breast cancers analyzed by high-resolution melting curve scanning and direct sequencing. Using scanning and automatic calling, there was high specificity (495%) across all DNA preparation methods. Sensitivities ranged from 100% in non-WGA DNA from fresh tissue to 86% in WGA DNA and DNA from formalin-fixed, paraffin-embedded tissue. Scanning could detect mutated DNA at a dilution of 1:200 in a background of wild-type DNA. Mutation scanning by high-resolution fluorescent melting curve analyses can be done in a high-throughput and automated fashion. The TP53 scanning assay can be performed from a variety of specimen types with high sensitivity/specificity and could be used for clinical and research purposes. Hum Mutat 29(5), 757-764, 2008.