Nonradioactive PCR Sequencing Using Digoxigenin

Abstract

Techniques for direct sequencing of polymerase chain reaction (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 used to sequence mitochondrial and nuclear genes in Parkinson’s and Alzheimer’s disease brains using DNA extracted from formalin-fixed and paraffin-embedded neuropathological material (1–3). This method, which allows sequence information of PCR products to be obtained within a single day, can be performed in a research or clinical laboratory using relatively inexpensive equipment. After initial PCR amplification, amplicons are purified using spin columns for affinity chromatography or ultrafiltration. Subsequently, cycle sequencing (4) is performed using 5′-digoxigenin end-labeled oligonucleotide primers. Because the nucleotide sequences of the PCR and sequencing primers can be identical, both reactions may be performed using the same thermalcycling protocol. This obviates the need for time-consuming optimization procedures. For visualization of sequencing results, sequencing reactions are separated on a standard sequencing gel, the gel is contact-blotted to a nylon membrane, and sequencing bands are visualized using alkaline phosphatase-conjugated antibodies (Fig. 1).