Multifunctional molecules aid mutation analysis

Over the past decade, DNA sequencing technology has become faster and more powerful while also growing more affordable and accessible. As a result, researchers have been able to chart a multitude of genomic variations that could potentially contribute to the development of various cancers as well as a host of hereditary diseases.

Identifying a candidate mutation is only the beginning, however, as independent assays are required to confirm the link between gene and disease. Matthias Harbers and Kengo Usui from the Division of Genomic Technologies at the RIKEN Center for Life Science Technologies have now developed a special class of probes that could accelerate the development of such assays by allowing scientists to simultaneously quantify target DNA sequences while also detecting the presence of mutations of interest¹.

Scientists have used variations of a technique called real-time polymerase chain reaction (PCR) to amplify target DNA while monitoring the progress of the amplification. These data are used to determine the original amount of starting genetic material, which can be valuable information in a diagnostic context. Real-time PCR is performed using short stretches of DNA called ‘primers’, which bracket the target sequence of interest and initiate amplification. Harbers and Usui previously worked with a special class of fluorescently labeled molecules called ‘exciton-controlled hybridization-sensitive fluorescent oligonucleotides’ (ECHOs), which feature a pair of dye molecules that quench each other’s fluorescence when in close proximity. When the ECHO binds DNA, the dye molecules become separated as they insert themselves into the double-helix structure, generating a signal that is proportional to the target DNA’s amplification. To efficiently couple this quantitation with the ability to directly detect the presence or absence of mutations, the researchers set out to devise a modified ECHO variant that combines both functions into a single assay.

DNA strands that are directly complementary to each other bind more tightly than those that contain mismatches. Scientists can use targeted probes to detect the presence or absence of mutations based on this principle through a technique called melting curve analysis. In contrast, PCR primers generally need to be a perfect match for their target sequence to work reliably. Harbers, Usui and their colleagues therefore opted to modify ECHOs so that they serve as detection probes that act in parallel with a separate set of real-time PCR primers.

The ECHO-based ‘Eprobes’ developed by the research team are designed to bind to potential mutation-containing sites within the region being amplified, allowing amplification detection in real time (Fig. 2). However, Eprobes also contain a chemical group that prevents them from being incorporated into the amplified DNA and instead causes them to dissociate from the target as the sample is heated during the PCR process. Once the PCR has concluded, the Eprobes go back to detecting mutations. “Since their fluorescence activity is still maintained after PCR, we can carry out melting curve analysis as a post-PCR assay in the same tube without additional modification of the reaction mix,” says Usui.

As an initial demonstration, the researchers designed a series of Eprobes to recognize a segment of the gene encoding the epidermal growth factor receptor (EGFR) protein, targeting a mutation associated with cancer risk. The Eprobes performed well in initial experiments, achieving sensitive detection of amplification over the course of the real-time PCR process. Importantly, these Eprobes also proved to be capable of distinguishing between samples that contain only mutant target DNA from those that contain both normal and mutated sequences. Such discrimination is important for many diseases, where a patient’s prognosis can differ depending on whether one or both copies of a chromosome carry a particular mutation. Furthermore, unlike other fluorescent detection tools, Eprobes are completely dependent on DNA binding to generate a signal and are therefore far less likely to produce false positive results that could confound diagnosis.