Gold Nanoparticles Speed Mutation Detection

Gold and Disease
Enzyme-free system works without pre-purification
Gold nanoparticles are at the heart of a new system for rapidly, sensitively and specifically detecting single nucleotide polymorphisms (SNPs), the most common type of genetic variation in humans. Intended as competition for assays based on polymerase chain reaction (PCR) technology, this nanotechnology-enabled system can detect known SNPs in human blood samples with minimum preparation and without the use of enzymes.

"We have now shown that we can extract DNA from blood and with no other preparation, we can use gold nanoparticle DNA probes to identify SNPs within about two hours," explains Uwe M ' ller, Ph.D., vice president for applied science at Nanosphere in Northbrook, IL. Details of the system, along with results from a demonstration experiment, appeared late last month in the journal Nucleic Acid Research. Nanosphere scientist, Y. Paul Bao, Ph.D., was the lead author on the paper.
SNPs, one of the fruits of decoding the human genome, are abundant in human genes. Many represent the normal genetic variation that occurs among humans, but some SNPs are signposts of gene mutations that cause cancer and other genetic disorders. The presence of a particular SNP can also predict an individual's response to certain anti-cancer therapies.
The main problem in using SNPs in the clinic is that they can get lost among the more than one billion base pairs present in human DNA. One way to find these needles in a haystack is to selectively enrich, or amplify, pieces of DNA known to contain SNPs of interest. Today, this enrichment is most commonly done using PCR, a powerful technology but one that requires exacting and time consuming sample preparation. Once amplified by PCR, SNPs are then detected in an additional analytical step using synthetic DNA probes that specifically recognize a given SNP's DNA sequence. The exact sequence of the SNP and surrounding DNA is now known, thanks to the availability of the human genome sequence.
Nanosphere's technology, developed originally by Chad Mirkin, Ph.D., and Robert Letzinger, Ph.D., and their colleagues at Northwestern University, skips the amplification step and relies instead on the unique properties of gold nanoparticles to find these needles in the haystack. In the assay, DNA is extracted from blood and broken into small pieces using powerful sound pulses, a process known as sonication. The resulting mixture of DNA fragments, each about 500 base pairs long, is applied to a chip containing attached synthetic DNA sequences that will bind specifically to particular SNPs. These sequences are called capture probes. After an hour, the chip is washed extensively, leaving the test DNA sequences that match perfectly with the capture probes along with only a few DNA sequences that are not perfect matches.
Next come the gold nanoparticles, each programmed with an attached DNA sequence that complements another stretch of the human DNA that lies adjacent to a particular SNP. This probe provides a second high-specificity binding step that compensates for the few stray sequences that remain after the first wash. If the assay is designed to look for more than one SNP, each bound to its complementary capture probe, then multiple capture probes would be added. Each probe would consist of a gold nanoparticle attached to the appropriate DNA sequence designed to match one of the SNPs.
The presence of the nanoparticle on this detection probe changes the way that the probe binds to the human DNA, strengthening the connection, or hybridization, between the pair. This tight hybridization allows for a 30- minute second round of extensive washing to again eliminate any capture probes that might have bound inadvertently, and thus weakly, to the human DNA. Finally, a thin layer of silver is deposited around the gold nanoparticle, using much the same technology used in silver-based camera film. Silver helps boost the optical signal generated by the gold nanoparticle, increasing the sensitivity of the assay.
This system is capable of detecting a few SNPs simultaneously. Scientists at Nanosphere have, however, developed a slight modification that Dr. M ' ller says will allow the assay to be used to detect as many as 30 probes simultaneously on automated equipment. He is confident that the technique will be useful with at least 100 SNPs. Beyond that level, as is the case with SNPs that characterize mutations in the p53 oncogene, PCR will still be the technique of choice. A paper based on this latest work has been submitted for publication.