IDT_Logo

A novel non-enzymatic assay for SNP detection in cancer DNA

Miotke L, Maity A, et al. (2015)Enzyme-free detection of mutations in cancer DNA using synthetic oligonucleotide probes and fluorescence microscopy. PLoS One 10(8): e0136720.

Citation summary: Learn how scientists use a long, biotinylated Ultramer Oligo to capture specific gene fragments for the identification of SNPs with single-molecule resolution. Their method is not only cost-effective, but avoids enzyme-based technologies, such as PCR and NGS, that vary in fidelity and increase risk of introducing amplification bias.

Nov 6, 2015

Background

Single-nucleotide polymorphisms (SNPs) and variants (SNVs) are the leading source of genetic variation in the human genome, as well as in other species. Such DNA alterations, when present in certain genes, are often associated with disease, including cancers. Therefore, their rapid, reliable detection is highly desirable for clinical and translational research. Current SNP/SNV diagnostic techniques rely heavily on enzyme-based technologies, such as PCR and NGS, to achieve the required sensitivity and specificity of detection. This can be problematic, as the DNA polymerases used in these methods vary in their fidelity and increase the risk of introducing amplification bias. In this paper, the authors introduce an approach for rapid, reliable, enzyme-free detection of a melanoma-linked SNP in human DNA.

Methods

Miotke and colleagues set out to detect mutation V600E (T→A at gene position 1799) in BRAF, a human gene encoding the intracellular signaling protein B-Raf. Mutations in BRAF disrupt regulation of cell growth and are associated with multiple cancers, particularly melanoma.

They obtained DNA from 2 human cell lines that possessed the desired BRAF mutation, performed restriction digestion of the DNA, and isolated single-stranded BRAF gene fragments using a 120 nt, BRAF-specific, biotinylated Ultramer® Oligonucleotide from IDT. Using controlled-pore glass (CPG) for solid support, the researchers then annealed these fragments to LNA/DNA capture probes complementary to mutated BRAF (mBRAF). The resulting mBRAF:LNA/DNA probe duplexes had increased Tm, allowing them to be isolated and quantified with fluorescence microscopy using EvaGreen® Dye, which emits a signal that is proportional to the amount of dsDNA present in a sample.

Conclusions

The researchers showed that this novel assay could accurately detect SNPs at single-molecule resolution directly from human serum. The technique detected 17% and 67% mutations in the 2 cell lines, and the assay measurements were confirmed by digital PCR. In comparison to previously reported genotyping methods for the BRAF mutation, this assay is exceptionally low in cost and provides quick turnaround time. Importantly, the assay does not require enzymes other than the restriction enzymes used to fragment the DNA samples. Thus, the assay avoids errors that often occur with PCR and high throughput sequencing. Overall, the ability to quickly and effectively detect small DNA alterations using this method suggests it can be used for other clinically-relevant mutations. It might also serve as an important tool in translational research settings.

lockup_1
lockup_2