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Methods Mol Biol. 2019;1881:63-81. doi: 10.1007/978-1-4939-8876-1_6.

Detection and Functional Analysis of TP53 Mutations in CLL.

Author information

1
Department of Internal Medicine, Hematology and Oncology, University Hospital Brno, Brno, Czech Republic.
2
Faculty of Medicine, Masaryk University, Brno, Czech Republic.
3
Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
4
Department of Pathology, University Hospital Brno, Brno, Czech Republic.
5
Department of Internal Medicine, Hematology and Oncology, University Hospital Brno, Brno, Czech Republic. Trbusek.Martin@fnbrno.cz.
6
Faculty of Medicine, Masaryk University, Brno, Czech Republic. Trbusek.Martin@fnbrno.cz.

Abstract

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

KEYWORDS:

FASAY; Low-burden mutations; Next-generation sequencing; Subclonal mutations; TP53

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