Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting

Abstract

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

A guide to nucleic acid detection by single-molecule kinetic fingerprinting

“…If more than ~500 molecules are present in a FOV, the diffraction-limited analysis presented here will result in a sub-linear increase and eventually a decrease in the accepted counts due to the inability to resolve closely spaced molecules. If it is desired to extend the dynamic range beyond this ~2.5 orders of magnitude into the range of thousands of molecules per FOV or more, it will likely be necessary to switch to a more conventional quantification scheme based on fluorescence intensity, or to implement super-resolution methods to analyze the kinetics of FP binding with sub-pixel accuracy [29]. Indeed, one recent paper describes the use of super-resolution imaging and kinetic analysis of dissociation kinetics to discriminate single-nucleotide variants in DNA with 95% accuracy [46].…”

“…It thus has a much broader scope than amplification-based approaches. To date, SiMREPS has been successfully applied to the identification and counting of short nucleic acids such as miRNAs (miR-16, miR-21, let-7a, let-7c, miR-141, cel-miR-139) [23] and ~22-160 bp fragments of singlestranded or double-stranded DNA [27][28][29] such as cancer-related EFFR mutations (see Results). Since the assay is typically performed at ambient room temperature, to ensure maximal sensitivity for double-stranded or highly structured analytes, care must be taken to fully denature and sequester any interfering secondary structure that might interfere with surface capture or fluorescent probe binding, e.g., by brief heating in an excess (e.g., 1-2 μM) of a carrier oligonucleotide or sequence-specific oligonucleotides that prevent the formation of interfering secondary structure.…”

“…Regardless of the type of CP used, the choice of capture region should be chosen such that it minimizes any interfering secondary structure in the CP and target. Such optimization can be carried out using prediction tools such as Exiqon's OligoAnalyzer, Integrated DNA Technology's T m prediction tool, or NUPACK [27][28][29].…”

Clinical validation of qPCR Target Selector™ assays using highly specific switch-blockers for rare mutation detection

“…At the same time, cytosine deamination poses a significant obstacle to reducing the performance of other steric blocking assays. It has gone largely unappreciated that even relatively short periods of heating of nucleic acids can deaminate cytosine 17. Once deaminated, cytosine residues become deoxyuridine that are ‘read’ by most polymerases as thymidine, and, as a result, create cytosine to thymine transversions that were not present in the original biological material.…”

Single-molecule dynamic DNA junctions for engineering robust molecular switches

“…[21][22][23] Transient binding mode is not only an essential component for super-resolution imaging but also useful for highly sensitive and specic target detection. 24,25 Due to the distinct single-molecule kinetics, targets can be arbitrarily distinguished from the background. We developed a series of analytical tools based on single-molecule DNA transient binding; however, the simple two-strand mode is not suitable for scalable and complex molecular switches or circuits.…”