Mary Ann D. Brow, Mary C. Oldenburg, Victor Lyamichev, Laura M. Heisler, Natasha Lyamicheva, Jeffrey Groteluechen, Richard Handrow, Sergei Kozyavkin, D. Michael Olive, Lance Fors, Lloyd M. Smith* and James E. Dahlberg**
Third Wave Technologies, Inc., 2800 S. Fish Hatchery Rd., Madison, WI 53711
Methods for the detection of genetic mutations resulting in human diseases such as cancer or infection with drug resistant Mycobacterium tuberculosis have become a critical need for effective healthcare. While some of these mutations may cause significant changes in the responsible genes, many of these mutations, such as those found associated with the p53 gene, are changes in single nucleotides. Methods such as single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) have yielded limited success in detecting mutations in short DNA fragments. Under the best conditions, these methods are limited to indicating the presence or absence of base changes, some of which may not result in phenotypic change. Until now, DNA sequencing has been the only method which can successfully identify phenotypically significant mutations, yet the cost and complexity of DNA sequencing has prevented its general use by the clinical community. We report here the use of Cleavase[TM], a thermostable structure-specific endonuclease, for detection of mutations in clinically significant genes including b-globin, p53, and the M. tuberculosis rpoB and katG genes. Cleavase[TM] is capable of recognizing and cutting secondary structures formed in the single strands of a DNA fragment following denaturation by heating and subsequent cooling. The digested single stranded DNA fragments are rapidly resolved by electrophoresis on denaturing acrylamide gels followed by detection by means of either a radioactive or a nonradioactive label. The presence of a mutation is indicated by a change in the fragment pattern near the region of the mutation. Thus the Cleavase[TM] reaction, referred to as Cleavase Fragment length Polymorphisms (CFLP), can be used for both detection and localization of mutations. We have been able to detect 102 of 103 polymorphisms in a total of 15 genetic systems. Point mutations in cDNA clones spanning exons 5 through 8 of the human p53 gene were reproducibly detected and differentiated. Using CFLP analysis, mutations resulting in a drug resistance phenotype could be reliably distinguished from phenotypically silet mutations in M. tuberculosis isolates. The simplicity, and rapidity of CFLP should facilitate the use of mutation analysis as a routine technique in the analysis of human disease processes.
Supported by a grant from the Department of Commerce, National Institutes of Standards and Technology Advanced Technology Program under Proposal Number 94-05-0012
* University of Wisconsin, - Madison, Department of Chemistry
** University of Wisconsin, - Madison, Department of Bimolecular Chemistry
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