Achieving room temperature DNA amplification by dialling in destabilization

Abstract

The ability to amplify nucleic acid biomarkers at room temperature has remained elusive despite the great need of diagnostics suitable for the point of care. To exponentially amplify DNA within a wide range of ambient temperatures (18-26 °C), we explore combining two destabilizing elements in our isothermal lesion-induced DNA amplification system. We demonstrate rapid DNA amplification at the bench without a heat source.

Tuning Toehold Length and Temperature to Achieve Rapid, Colorimetric Detection of DNA from the Disassembly of DNA–Gold Nanoparticle Aggregates

“…A wide range of operational temperatures may also allow this method to be applicable at room temperature, circumventing the need for heating equipment, a goal which is desirable for point-of-care assays. [30][31][32] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 To accomplish this goal, at both ends of each linker strand we introduced differing lengths of overhangs, i.e. nucleotide extensions that would not participate in hybridization with the DNA-AuNP probes.…”

Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA

“…As mentioned, our previous work showed that the replication temperature in LIDA can be tuned by the introduction of a mismatch to the system, which is less destabilizing than an abasic group. [33] Thus, a T base was switched into a C base in one of the probes (DNA-Ia), which resulted in an A:C mismatch (square, Figure 1). This combination of destabilizing elements allowed us to lower the temperature from 37 °C to 28 °C (Figure S2B).…”

“…Previously we reported the development of lesion-induced DNA amplification (LIDA), which was capable of rapid, exponential amplification of different 18 nucleotide DNA target sequences by incorporating a destabilizing abasic lesion into one of the target-complementary primers, or probes, in a ligase chain reaction. [33][34][35] With this approach, as little as 140 fM (2.1 attomoles) of target DNA was detected following a two-step serial amplification procedure. [35] In more recent work, it was revealed that adding a second destabilizing lesion consisting of a mismatch or another abasic group allowed the replication temperature to be tuned from 18 -30 °C for a particular target sequence.…”

“…Using the optimal probe sequences, target DNA was amplified on the benchtop without any heating source or equipment. [33] Regarding the visualization step, our group has also developed a rapid, room-temperature method for detecting single-stranded DNA using DNA-modified gold nanoparticle aggregate disassembly. [36] Herein we combine these two approaches and add an additional step to facilitate both amplification and detection triggered by a RNA target sequence.…”

Thermodynamics and kinetics of Lesion Induced DNA Amplification (LIDA)

“…7 Product inhibition implies that a newly formed complementary template is very unlikely to dehybridize from the template. However, Gibbs-Davis and her team 1 have developed a method to circumvent product inhibition based on mismatches and lesions between the complementary templates that destabilize them and thus enables a continued replication process. In this work we explore in simulation the thermodynamic and kinetic properties of lesion induced DNA amplification processes.…”