Protein based approaches to heteroduplex recognition for the high throughput detection of unknown mutations

Abstract

DNA sequencing is the ultimate method for detection and definition of mutations.  However, the need for scanning methods exists due to the expensive and time-consuming nature of DNA sequencing.  Scanning methods have arisen to avoid sequencing whole stretches of DNA, to reduce costs and increase throughput.  It is not feasible when trying to detect and characterise mutations in a population to sequence the DNA of all the samples.  Therefore, the need for cost-effective simple scanning methods exists.

Endonucleases, Resolvases and DNA binding proteins all provide potential tools for the screen for mutations.  Two such proteins MutS, a mismatch binding protein and T4 endonuclease VII, a resolvase with mismatch cleavage properties were studied to determine their suitability for mutation detection.  Both proteins were His-tag labelled to allow convenient and high yield production of protein via affinity chromatography.  The basis of a scanning technique using MutS or Endo VII for epidemiological studies is based upon heteroduplex analysis.  The mismatch recognition of both these proteins has been used in conjunction with the high throughput capabilities of (MADGE) in order to develop novel mutation scanning strategies.

The work presented here investigates the suitability of the two enzymes MutS and T4 endonuclease VII for the high throughput identification of unknown mutations in epidemiological studies using short track electrophoresis.  Following the initial characterisation of the proteins and application to short tract electrophoresis, the mutation detection capabilities of both proteins were tested using a variety of known SNPs and mutations.  Of the two proteins studied, T4 Endo VII demonstrated the greatest potential for high throughput mutation detection.  It was successfully utilised to detect a number of mutations using MADGE.  By combining the mismatch cleavage properties of Endo VII with the high throughput capabilities of MADGE, a cost effective method for the high throughput detection of unknown or rare mutations has been created.

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