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High‐throughput amplicon scanning of the TP53 gene in breast cancer using high‐resolution fluorescent melting curve analyses and automatic mutation calling
Author(s) -
Bastien Roy,
Lewis Tracey B.,
Hawkes Jason E.,
Quackenbush John F.,
Robbins Thomas C.,
Palazzo Juan,
Perou Charles M.,
Bernard Philip S.
Publication year - 2008
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20726
Subject(s) - biology , amplicon , melting curve analysis , high resolution melt , microbiology and biotechnology , dna , mutation , dna sequencing , gene , polymerase chain reaction , genetics
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 (>95%) 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. © 2008 Wiley‐Liss, Inc.