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.
Highlights
Single-molecule fluorescence techniques have substantially advanced our understanding of molecular and cellular processes over the last two decades[9,10]
A short fluorescent DNA probe that is fully complementary to the SNV, but which forms a single mismatch with wild type (WT) DNA, is added
The single-nucleotide selectivity of hybridization-based probes was greatly improved by utilizing the transient binding of short probes and by determining the binding kinetics
Summary
Single-molecule fluorescence techniques have substantially advanced our understanding of molecular and cellular processes over the last two decades[9,10]. By taking advantage of the fluorescence excitation geometry of the evanescent field in total internal reflection fluorescence microscopy (TIRFM)[11], researchers can capture DNA targets on slides and detect single molecules of nucleic acid in vitro[12,13] These methods still discriminate poorly between SNVs and wild type (WT) sequences, due to poor hybridization specificity. Compared to previous work[18], the single-nucleotide discrimination capability is further improved by optimizing the assay conditions which is guided by statistical modeling of kinetics with a gamma distribution This kinetic approach can detect synthetic DNA with an SNV at an allelic frequency as low as 0.01% in the presence of WT. This method was successfully used to detect mutations in single-stranded DNA that was reverse-transcribed from cellular mRNA from two cancer cell lines
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