Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology.

ABSTRACT: CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amp

SUBMITTER: Grossmann V 

PROVIDER: S-EPMC3128616 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature