Nonradioactive PCR Sequencing Using Digoxigenin

Abstract

Techniques for direct sequencing of PCR products are of central importance to contemporary research in molecular biology and genetics. The rapidly growing number of cloned human disease genes increasingly allows sequencing of PCR amplicons for diagnostic purposes. Nonradioactive sequencing protocols are of particular use because health, environmental, and administrative risks are minimized compared with conventional isotopic methods. The PCR-based nonradioactive cycle sequencing protocol described in this chapter has been successfully employed to sequence mitochondrial and nuclear genes in Parkinson’s and Alzheimer’s disease brains using DNA extracted from formalin-fixed and paraffiembedded neuropathological material (1-3). This method, which allows sequence information of PCR products to be obtained within a single day, can be carried out in a research or clinical laboratory using relatively inexpensive equipment.