"Rolling Circles" Provide New Way To Amplify And Detect DNA

A patent for the technology has been approved by the U.S. Patent Office and will issue soon. Kool has also presented the work at several scientific conferences, and has published results in the Journal of the American Chemical Society.

"This method of DNA production is very easy and inexpensive," Kool says. "You can just toss a rolling circle, some free DNA nucleotides, and DNA-copying enzymes into a test tube, and when you come back 12 hours later, the tube is full of DNA.

"It's now feasible to make bucketfuls of DNA, an amount we can't even begin to approach with other amplification techniques."

Unlike other DNA-copying techniques now in use, rolling circles don't require special enzymes or complex equipment like thermocyclers, so the method could reduce the cost of genetic research or routine laboratory testing. It might also open the door to new procedures requiring very high sensitivity -- such as detection of DNA sequences in organisms' genomes, for instance, or identification of specific cancer-related mutations.

The new method of DNA production uses as its template a single piece of circular DNA far smaller than that found in even the tiniest of organisms. Like a molecular fishing reel, this tiny ring of DNA unfurls a long strand of daughter DNA that repeats a single sequence hundreds of times. Using molecular scissors known as restriction endonucleases -- which always cut DNA strands at precisely the same array of neighboring bases -- scientists then cut this long strand of DNA into many smaller strands with sequences that exactly complement the template.

In the lab, Kool has produced strands of DNA more than 12,000 bases long from a 26-base rolling circle, yielding some 460 identical strands of DNA after clipping by enzymes. "We can easily control the length of the DNA strand created by a rolling circle simply by varying the enzyme or the duration of the reaction," he notes.

The technology relies on tricking the polymerases that copy DNA strands. These enzymes start at one end of a strand and work until they reach the other end. But Kool's strands are circular, not linear -- since there is no end, the polymerase keeps going until it runs out of raw material or spontaneously falls off.

"This is how some viruses replicate themselves," says Kool. "We tapped methods that have been proven over millions of years of evolutionary engineering."

Scientists currently use various other techniques to reproduce DNA. In PCR, or polymerase chain reaction, strands of DNA are amplified exponentially: Repeatedly, single strands are copied, yielding double-stranded DNA, and then heated to unzip the strands for more rounds of copying. But such thermal cycling is expensive, and the amount of DNA that scientists can prepare using PCR is limited by the need for a new DNA primer sequence for every strand copied.

Even though scientists often use PCR to amplify a strand of DNA into an amount that they can study, it's not a feasible means of reproducing even a thimbleful of DNA. The same is true of cloning, where scientists insert a piece of DNA into a bacterium so the organism's own replication machinery will copy it.

The third widely used option for making short strands of DNA, computerized chemical synthesis, relies on a machine to synthesize DNA sequences from free DNA nucleotides. But the reagents and solvents used in the synthesis create toxic byproducts, and machine-based synthesis is also not as accurate as rolling-circle synthesis.

While rolling-circle synthesis resembles natural virus replication in some ways, it is actually quite distinct. Kool's rolling circles are 100 times smaller than the smallest viruses -- in fact, they can be even smaller than the DNA-copying enzymes themselves. In addition, Kool has shown that both DNA and RNA can be synthesized using rolling circles.

The University has filed for additional patents on other applications of the technology. The process could provide a way to produce ultra-bright fluorescent labels to target individual DNA sequences in the body's cells, Kool says. He is also investigating a new kind of gene therapy, where rolling circles inserted into cells might produce RNAs that target cancer-related genes and viruses like HIV.

Kool's work is funded by the National Institutes of Health and the U.S. Army.