Title

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

Authors

Cassia A. Aho

Document Type

Article

Publication Date

2000

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.

Advisor

Michael Hill

Department

chem

Support

Caltech-SURF Program



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