Tailoring of the p53 Gene for an Electrode-Based Mutational Detection Assay.

Abstract

In order to analyze mutations in the mutational "hot spot" regions of the human p53 gene using an electrode-based assay, it is necessary to produce sufficient quantities of properly tailored DNA oligonucleotides representing pre-selected, "hot spot," regions of the p53 gene. A 5'-biotinylated primer is used in standard PCR to produce amplified double-stranded DNA in which one strand is modified with biotin. Incubation of the amplified product with streptavidin-coated magnetic beads allows for separation of the strands to yield a single-stranded DNA sequence that can serve as a template for hybridization. Short DNA oligonucleotides possessing a biotin molecule a the 5' end are used to protect the desired "hot spot" regions from nuclease digestion using mung bean nuclease, a single-strand specific endonuclease. Properly tailored single-stranded DNA fragments will be produced following denaturation and binding of the biotinylated strand to the streptavidin-coated beads. Preliminary data demonstrates that small conductivity differences characteristic of non-Watson-Crick base pairing, a mutation, can be detected following hybridization of the DNA oligomer to the wild-type DNA bound to the electrode.