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The Mutation Sensation: Cleavase Fragment Length Polymorphism from Third Wave Technologies

p53 GENE MUTTION: Exons 5 and 6 of the p53 gene of five individuals were amplified and examined by Cleavase® Fragment Length Polymorphism (CFLP®) Analysis. The pattern marked "WT" is from an individual known to have a normal p53 gene. For those of you reluctant to put a down payment on an automated sequencing apparatus, pay heed to an alternative that may fit your applications. Third Wave Technologies (Madison, Wis.) recently introduced a procedure for identifying generic sequences and

September 15, 1997


p53 GENE MUTTION: Exons 5 and 6 of the p53 gene of five individuals were amplified and examined by Cleavase® Fragment Length Polymorphism (CFLP®) Analysis. The pattern marked "WT" is from an individual known to have a normal p53 gene.
For those of you reluctant to put a down payment on an automated sequencing apparatus, pay heed to an alternative that may fit your applications. Third Wave Technologies (Madison, Wis.) recently introduced a procedure for identifying generic sequences and mutations. Dubbed Cleavase Fragment Length Polymorphism (CFLP"), the method employs the proprietary enzyme Clevase I to cleave DNA into a series of fragments which appear as bands on sequencing gels. Utilising this method, double-stranded DNA obtained via PCR is denatured and then cooled in low-salt buffer. Under these conditions the denatured DNA strands form hairpin-like structures. Cleavase I cuts at the 5' region of the hairpin secondary structure and yields fragments, which appear as bands on a gel. These bands serve as the fingerprint of the DNA, resembling a bar code pattern. Differences in the bar code patterns among DNA samples are indicative of sequence changes and can be specific for different mutations.

"CFLP is a method that summarizes the data that's in the DNA of the cell of an organism," explains James Dahlberg, Professor of Biomolecular Chemistry at the University of Wisconsin Madison and co-founder of Third Wave Technologies. "With CFLP we can determine whether two samples of DNA are the same or not without having to expend the tremendous amount of energy and resources to sequence them." This technology may help accelerate important research efforts such as defining genetic mutations associated with drug resistant strain of Mycobacterium tuberculosis (TB) and genotyping Hepatitis C Virus to determine responsiveness to therapy. Also, CFLP can be used to evaluate tradeoffs associated with partial or radical surgical intervention in cases of breast cancer on the basis of knowing which mutations in the p53 gene are associated with the most aggressive form of malignancy. According to company literature, CFLP allows for speedy, overnight analysis, whereas other methods can take weeks.

Tom Taggart, Associate Professor of Genetics at SUNY-Buffalo, uses radioactive and non- radioactive CFLP protocols (among other methods)to scan for genetic mutations. According to Taggart, an advantage of this technique as opposed to other protocols is that "it allows for scanning and detecting of larger fragments (100 bp to 1.5 kb) than can reliably be analyzed by single strand conformation polymorphism (SSCP; 100-150 bp) or dideoxyfingerprinting (ddF;100- 450 bp)"

The company reports the procedure is easily performed and uses instrumentation common to most labs. "It is a very robust assay, allowing flexibility in time of reaction, salinity, temperature, and reagent amounts without altering the results, " notes Lance Fors, President, CEO, and co- founder of Third Wave Technologies. Third Wave Technologies: (888) TWT-CFLP or www.TWT.com.

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