杏吧原创

AN INGENIOUS new technique based on a molecular hula-hoop will add a powerful
tool to geneticists鈥� kit. Its inventors say it could outperform the polymerase
chain reaction, the standard technique for identifying and replicating chunks of
DNA.

The hula-hoop technique, also known as rolling circle amplification, uses a
loop of DNA that sticks to either side of a target sequence and churns out
millions of copies of it until there is enough to be detected. The vast number
of sequences churned out make the test highly sensitive, so it is ideal for
tasks such as detecting a few fragments of DNA in criminal evidence, picking out
tiny traces of genetically modified foods and screening for mutations.

鈥淢illions of identical copies of the target sequence get reeled off like a
long strand of ticker tape,鈥� says Mike Evans, the vice-president of drug
discovery at Amersham Pharmacia Biotech in Buckinghamshire. Last month, Amersham
won a licence to develop the technology commercially with Molecular Staging, a
company set up by Yale University in Connecticut, where biochemists David Ward
and Paul Lizardi developed and patented the technique (Nature Genetics,
vol 19, p 225).

They found a way to 鈥減adlock鈥� strands of DNA to target sequences in genes and
chromosomes. An enzyme added to the mixture goes round and round the loop, using
it as a template to make multiple copies of the target DNA. 鈥淓very time you go
round the loop you replicate a section of the target you鈥檙e looking at,鈥� says
Evans.

The enzyme used, phi29 polymerase, is different to the Taq polymerase that
does the job in PCR鈥攕o Amersham believes that rolling circle amplification
will not infringe the PCR patents held by Hoffman-La Roche, the Swiss
pharmaceuticals giant.

Evans says that whole genes can be replicated. And unlike PCR, which churns
out millions of discrete copies of the target DNA, the multiple 鈥渢ickertape鈥�
copies produced remain anchored to the target sequence. 鈥淚t鈥檚 left flailing
there, but is conveniently localised to a specific spot in the sample,鈥� says
Evans.

So by incorporating fluorescent substances into the tickertape, geneticists
can use a microscope to see where the target gene is. 鈥淚t means you can home in
on individual mutations in DNA,鈥� says Evans. What鈥檚 more, functional genes can
be identified in cells by making probes that bind directly to the messenger RNA
produced by any active gene.

Evans says that the amplification technique can even be used indirectly to
detect when proteins are present in cells, a feat impossible with PCR. First,
antibodies are developed which bind to target proteins. Next, the antibody is
attached to a predetermined DNA sequence to which a loop will bind. If the
protein is present in a sample, the antibody will capture it. When the loop is
added, it will bind to the DNA sequence ready to churn out the usual strand of
tickertape copies.