Molecular Diagnostics

By Jan Hodnett, MS, MT (ASCP)

It all began in a “Eureka” moment. Kary Mullis, Ph.D., has said that the concept of the polymerase chain reaction (PCR) came to him during a night drive to his cabin in California. Playing in his mind with a new way for analyzing mutations in DNA, he suddenly realized that he had thought up a method for amplifying any one chosen region of DNA. Before he arrived at his cabin, Mullis was savoring prospects of a Nobel Prize, which he was awarded in 1993. Cetus Corp., where Mullis worked, paid him $10,000 for his discovery and later sold it to Hoffmann-La Roche for $300 million.

Since Mullis discovered PCR in 1983, it has become a routine procedure in some research and clinical laboratories, leading to the development of other molecular diagnostics technologies. Mike Jones of Front Line Strategic Management Consulting, Inc., Foster City, Calif., estimates that the worldwide market for nucleic acid tests (NAT), which involves analysis of DNA or RNA for predisposition, diagnosis or prognosis of a disease or condition, is more than $1.5 billion, or 8 percent of the total in vitro diagnostic (IVD) market. By 2010, he estimates that nucleic acid testing will have grown at a compounded annual growth rate of 26.1 percent and increased to $16.7 billion.

The big players in this market today are Abbott, Bayer, BioMerieux, Chiron, Digene, Gen-Probe, Genzyme, ImPath, Roche and Vysis. Roche has the biggest market share, with Gen-Probe and Chiron second and third. There are many companies involved in NAT today, but not more than 20 produce substantial income. “When you predict 20 percent or more growth, then a lot more people will get into it, especially if they think they can get a patent on the gene and no one else can provide that test,” said Jones. The government is looking at patent laws now, but Jones believes that there has been enough of a precedent set that patents on genes are not going away. “You have to identify a use as well as a sequence, but if you can do that, you’ll have full use of that patent,” he explained.

While PCR started it all and is the major nucleic acid technology used in clinical laboratories, a variety of DNA and RNA technologies are available for diagnosing disease and determining genetic make-up.

PCR: Roche saw the value early
Roche acquired the rights to the PCR patent in 1991. The original patents expire in 2005. But it doesn’t end there. “We have taken the original patents and improved some of the primers and enzymes used to cleave the DNA,” said Ronnie Andrews, vice president of marketing for laboratory systems and molecular diagnostics at Roche Diagnostics in Indianapolis. These later patents are valid until 2017. “The older PCR patented material can still be used when we go off patent in 2005, but there have been so many improvements that laboratorians would be using a less sensitive and specific method that would not be as easy or as fast as the newer technologies,” added Andrews.

In PCR, the DNA is uncoiled and the bonds between two strands are severed. A segment of one strand is tagged and amplified many times over during repeated cycles. A billion copies of DNA material can be made in 20 cycles, creating titers high enough for analysis.

Roche Diagnostics molecular strategy is centered around PCR, but they realize its limitations in proteomics and other areas, so they have acquired or entered into relationships with nanotechnology companies to give them access to these technologies in the future.

In addition, Roche has worked with hundreds of research labs to realize the potential of PCR. “We have over 700 sublicenses for Roche PCR, giving us 700 R&D labs around the world. Because of that we have over 90 different kit configurations for ASRs. Our challenge is to recognize which ones have the greatest medical and commercial value,” said Andrews.

Infectious disease methods developed first
Infectious disease was among the first clinical areas to benefit from molecular diagnostics. Many organisms are difficult to culture, especially viruses, so molecular technology offered better performance, faster turnaround time and high sensitivity for detection of a small number of organisms. Over 25 companies now offer kits for infectious agents.

For example, Chlamydia trachomatis and/or Neisseria gonorrhoeae can now be detected by molecular techniques. Culture and EIA were not always effective for these organisms and sampling with a urethral swab was invasive. Gen-Probe recently introduced the Aptima Combo 2 Assay for detecting both organisms at once using either urine or urogenital swab samples. Sensitivity is high, however, one problem in evaluating performance characteristics of these sensitive assays is that they may be more sensitive than the current “gold standards.”

Craig Hill, Ph.D., manager of scientific affairs at Gen-Probe in San Diego said, “The Aptima Combo 2 is a second-generation amplified product.” This second-generation methodology ties together three pieces of their technology that isolate target rRNA from the sample and remove sample inhibitors. Transcription mediated amplification technology amplifies the target rRNA and the dual kinetic assay technology allows detection of two distinct organisms in the same test tube. “That puts Gen-Probe up a notch over existing amplification technology in that we have improved vastly on specimen processing to get rid of inhibitory factors that can cause decreases in sensitivity with other techniques, such as PCR and LCR,” said Hill.

Non-amplification techniques
Third Wave Technologies of Madison, Wis., and others have developed molecular technologies that do not use PCR or amplification. Lance Fors, Ph.D., CEO of Third Wave, said that their technology, “is the only one that gets around the PCR bottleneck.” The Invader System doesn’t require DNA amplification and can be used to make the hundreds of products needed to understand the genetics of disease. “Right now, Third Wave Technologies has about 65,000 unique products for analyzing genetic variations and the number is growing,” Fors said.

For example, Third Wave has developed a cardiovascular panel for patients undergoing major surgery. The panel of tests determines the patient’s predisposition for blood clotting, so that a blood thinner may be prescribed pre-operatively to prevent clotting peri-or post-operatively.

A key feature of the Invader System is that it uses routine instrumentation. “Often each technology requires a specific instrument, but our products can be used on instruments that are already in everyone’s lab. Our technology is only just emerging. PCR has been available since 1985, and we started commercializing the Invader system in 2001. We’ve only been out there for two quarters and already, we’ve reached $20 million in sales,” Fors said.

The Invader technology is based on enzyme substrate chemistry that is highly precise. “If you have a mutation, you see the mutation 100 percent of the time. If the mutation is there, we create this perfect structure that the enzyme can see and it opens the lock. If it is not perfect, and it is not in the right sequence, nothing happens and there is not much background. If a specific sequence is present, the signal is strong. Our background is about 1,000-fold less than conventional PCR-based approaches,” said Fors.