CFLP Up Close
Introduction (Back to Top)
Just as bar codes have helped speed up supermarket trips, a new technology developed with funding from the National Institute of Standards and Technology's Advanced Technology Program is helping scientists speed up identification of genetic variations.
Developed by Third Wave Technologies Inc. (Madison, WI), the novel genetic screening technique provides a fast, inexpensive way to turn DNA samples into individualized bar code patterns that can be scanned quickly for specific mutations. The technique costs up to 80% less per sample than other methods, including automated DNA sequencing.
DNA sequencing laboriously identifies the precise order in which four chemical bases appear in a gene fragment. In contrast, Third Wave's CFLP (cleavage fragment length polymorphism) method uses patented processing methods and enzymes to create and identify tell-tale folds in single-stranded DNA that indicate specific chemical sequences. If DNA sequencing is equivalent to reading every word of an organism's assembly instructions, then CFLP is a speed reading method that concentrates on critical junctures to infer the rest of the book.
Pattern Comparison (Back to Top)
CFLP generates a distinct bar code for every unique DNA sequence. Thus, mutations can be detected by comparing a sample to a normal coding pattern. The method should have a broad range of applications for research, diagnosing and treating infections and hereditary diseases, and accelerating drug development.
CFLP identifies mutations and other variations that affect physical structure of DNA, making it an alternative to conventional methods based on chemical sequence. When analyzed by CFLP, each DNA fragment produces a unique "fingerprint" that looks like a bar code. The ATP funding enabled Third Wave to expand on its early discoveries of bacterial enzymes involved in the identification process, design practical, reliable method, develop reagents, and conduct extensive testing.
"What we have is linear signal amplifacion for DNA and RNA," stated Third Wave marketing director Sheldon Clark, "that circumvents the need for PCR. Our technique goes straight to the genetic material, interrogating and detecting it." According to Clark, target markets include diagnostics, basic research, drug discovery, determination of drug resistance, and post-therapeutic patient monitoring applications. "The same technology used to detect cytomegalovirus, for example, can also measure drug resistance or viral load. And it's quantitative as well as qualitative."
"Traditional molecular assays depended on PCR, where you take a sample, extract DNA, use primers to amplify a portion of it, cleave with restriction enzymes at specific locations, then run the gel. Our method uses hybridization and cleavage: DNA cleaves only if the known or desired hybridizations have occurred. Cleavage, in turn, sets off a cascade of secondary reactions based on FRETfluorescence energy transferthe result of which is we can detect as few as 60 molecules. The method is very sensitive but very forgivingit's all based on structure, not sequence, so we don't get false positives."
The first product kit based on ATP-funded research was commercialized by Third Wave for the research market in mid-1996 and brought about $300,000 in sales that year. A variety of clinical applications have been validated by scientific studies, including one by Italian scientists who concluded that CFLP scans longer DNA fragments and detects mutations faster than conventional methods. When used to identify different strains of the hepatitis C virus (a factor in resistance to interferon therapy), CFLP costs much less than two standard competing methods (including automated DNA sequencing), according to tests performed by an independent contract research firm.
CFLP Up Close (Back to Top)
CFLP technology is based on the observation that when single strands of DNA fold on themselves, they assume higher order structures that are highly individual to the precise sequence of the DNA molecule. These secondary structures involve partially duplexed regions of DNA such that single stranded regions are juxtaposed with double stranded DNA hairpins.
The Cleavase I enzyme, at the heart of CFLP, is a structure-specific, thermostable nuclease that recognizes and cleaves the junctions between these single-stranded and double-stranded regions. DNA strands treated with Cleavase I yield collections of fragments that, when resolved, create unique "barcodes" that highlight the conformational characteristics of the DNA. Because these conformations can be altered by as little as a change in a single nucleotide, this approach can be used to generate a distinct barcode for each different DNA sequence examined.
UPC barcode (left). The gel lanes represent a PCR analysis (1), wild-type version of a gene (2), and a CFLP-generated genetic barcode of a single variant in the same genetic region as the wild type analysis (3).
The CFLP method comprises the CFLP reaction, followed by the separation and visualization of the resultant barcode, which may be completed in as little as 30 minutes.
The reactions themselves include: separation of DNA strands by heating; formation of intrastrand secondary structures on cooling; rapid enzymatic cleavage of these structures by the Cleavase I enzyme
Because these secondary structures are detected by enzymatic cleavage, rather than by electrophoretic mobility (as is the case with single stranded conformational polymorphism, or SSCP), CFLP analysis can be used to identify sequence polymorphisms in large DNA fragments (i.e. >1 kilobase). Unlike other mutation detection techniques, many of which simply provide a "yes or no" answer regarding the presence of sequence polymorphisms, CFLP method provides a means of uniquely identifying each variant. The CFLP technology therefore affords an amount of information comparable to that obtained by DNA sequencing for a fraction of the cost and effort.
Third Wave has formed alliances with five major distributors in four product markets. In addition, the company recently received a second ATP award to develop generic, enzyme-based technologies suitable for healthcare applications, such as large-scale treatment monitoring and point-of-care testing.
For more information: Sheldon Clark, Director of Marketing, Third Wave Technologies, 502 South Rosa Rd., Madison, WI 53719. Tel: 608-273-8933. Fax: 608-273-6989. Email: email@example.com.
By Angelo DePalma