Chapter 6 - Direct Genome Sequencing in Diagnostic Pathology

Abstract

Molecular diagnostics identify or confirm genetic variants associated with diseases or that can serve as surrogate markers of disease. DNA is the primary target for molecular diagnostics because of its simplicity, relative stability, and wide applicability. DNA variants associated with diagnostic or prognostic value can include simple abnormalities such as point mutations, single nucleotide polymorphisms (SNPs), small additions and deletions, repeat sequences of varying length, translocations, and gross chromosomal abnormalities. Molecular methodologic approaches routinely used for diagnostics include polymerase chain reaction (PCR) coupled with multiple detection systems, real-time PCR, fluorescence in situ hybridization, restriction fragment length polymorphism analysis (RFLP), denaturing gradient gel electrophoresis, single-stranded conformation polymorphism (for insertions and deletions), hybrid capture, and the hybridization protection assay. However, only direct genome sequencing using the template DNA generated from genome by PCR will be sufficiently robust for some applications.