Genetic mutations turn healthy cells into cancer cells, so detecting these mutations as early on as possible would greatly improve patient diagnosis and therapy. One such mutation in the "BRAF" gene is known to be involved in various cancers, including thyroid cancer. At the moment, such mutations are detected using real-time PCR, in which mutant DNA sequences are selectively "amplified" and copied. However, this technique is not accurate enough to be completely reliable.

Recently, researchers have developed methods to sequence DNA bases using nanopores. These techniques typically involve electrically detecting each base pair as the DNA molecule translocates through the pore, by measuring either the voltage across the membrane or the current through the pore. Again, however, such approaches are not accurate enough to be used on their own.

Detecting changes at the single-molecule level

A team led by Li-Qun Gu has now developed a new nanolock–nanopore sensor made from alpha-haemolysin (αHL), a protein that is especially well suited to detecting individual bases of DNA (and RNA). The new sensor can detect changes in DNA sequences at the single-molecule level and distinguish between mutant and normal DNA.

"The nanolock is a special structure that can stabilize base pairs of the DNA at the mutation site of the molecule as it passes through a nanopores," explains Gu. "We found that mutant DNA carrying a nanolock undergoes a unique type of unzipping when it moves through the pore. This produces a highly accurate and sensitive nanopore fingerprint for the BRAF mutation in thyroid-cancer patient tissue samples that we analysed. "In the future, the device might be integrated with a miniature, high-throughput device for accurate and PCR-free detection of this mutation," he adds.

Precision medicine applications

"We foresee applications in precision medicine," he explains. "Since we can determine if a single DNA molecule is mutant or normal, our technology has low false positive and low false negative results – for a simple yes/no cancer diagnostic. What is more, since we can also quantify the percentage of mutant DNA in a sample, the technique might be used to monitor the efficiency of a treatment by measuring the percentage change before and after administration of a given drug. This would greatly help doctors in choosing the right therapy for their patients."

The researchers, reporting their work in ACS Sensors doi: 10.1021/acssensors.7b00235, say that they are now looking to improve their nanolock–nanopore so that it can detect a broader range of clinically important cancer driver mutations, such as mutations on KRAS and EGFR. "Since clinical diagnostics requires the accurate detection of a group of mutations rather than only single ones, we will also be working on developing multiplex mutation sensors using a 'nanopore barcode' approach that we previously developed in our lab for detecting microRNA," reveals Gu. "We will be collaborating with clinical investigators to validate this approach."

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