10 - Analysis of PCR Products by Covalent Reverse Dot Blot Hybridization

Abstract

This chapter illustrates the reverse dot blot (RDB) method that utilizes the immobilization of a multitude of oligonucleotide probes onto a solid support and subsequent hybridization to biotin-amplified DNA from an individual. The hybrids are then detected via a streptavidin–horseradish peroxidase (HRP) conjugate and chromogenic substrate. The chapter also describes the general laboratory protocol, which is a reliable technique for detecting subtle known mutations in amplified DNA. Once the technique is established, a change of PCR primers and oligonucleotide probes permits switching from one gene to another or from one type of organism to the next. The use of positive and negative controls every time the assay is performed is a prerequisite of this method. The positive controls are changed frequently, and it is preferable to use as positive control DNA a sample homozygous for a single mutation. Such a control lacks a hybridization signal at the normal probe site, and therefore, PCR contamination from a previously amplified sample is likely to be normal at this site. It will thus show hybridization at the normal probe site, turning the homozygous control into a heterozygote.