Single-Molecule Counting of Point Mutations by Transient DNA Binding

Abstract

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

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

“…The apparent discrimination factor Q app for T790M ( Figure 4f, Table 1 ) is ~11 million in our optimized assay, which is approximately 2600 times greater than the maximum thermodynamic discrimination factor under our imaging conditions as estimated by NUPACK 38 . While kinetic fingerprinting has been shown to be capable of single-nucleotide discrimination 18,39 , the demonstrated specificity in prior studies was 2–4 orders of magnitude lower than that achieved here, and in one case required asymmetric PCR amplification 39 . The performance achieved in the present work suggests that arbitrarily high specificity in an amplification-free assay can indeed be achieved by single-molecule kinetic fingerprinting, given (1) suitable assay optimization and (2) treatment of samples to remove damage that would otherwise be read as false positives.…”

Enhancing the Performance of DNA Surface-Hybridization Biosensors through Target Depletion

“…where we used k PM , k 1MM and k 2MM to denote the off-rates, while K PM = k + /k PM , K 1MM = k + /k 1MM and K 2MM = k + /k 2MM . Equations (17) are shown in Fig. 2 as dotted lines and are in excellent agreement with numerics.…”

“…In particular, we focus on typical situations interesting for diagnostic purposes, where the sample to be analyzed contains a large amount of "wild type" sequence at concentration c 1 and a much smaller amount of "mutant" sequence, differing by a single nucleotide. [15][16][17][18] The latter is at a concentration c 2 c 1 . We discuss a minimal-design strategy ( Fig.…”

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

“…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].…”