Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA

Abstract

Polymerase chain reaction (PCR) is dependent on two key hybridization events during each cycle of amplification, primer annealing and product melting. To ensure that these hybridization events occur, current PCR approaches rely on temperature set points and reaction contents that are optimized and maintained using rigid thermal cycling programs and stringent sample preparation procedures. This report describes a fundamentally simpler and more robust PCR design that dynamically controls thermal cycling by more directly monitoring the two key hybridization events during the reaction. This is achieved by optically sensing the annealing and melting of mirror-image l-DNA analogs of the reaction's primers and targets. Because the properties of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. This hybridization-sensing approach adapts in real time to variations in reaction contents and conditions that impact primer annealing and product melting and eliminates the requirement for thermal calibrations and cycling programs. Adaptive PCR is demonstrated to amplify DNA targets with high efficiency and specificity under both controlled conditions and conditions that are known to cause traditional PCR to fail. The advantages of this approach promise to make PCR-based nucleic acid analysis simpler, more robust, and more accessible outside of well-controlled laboratory settings.

Mirror‐Image Oligonucleotides: History and Emerging Applications

“…In as econd example, Adams et al reportedt he use of an l-DNA-based hybridization sensor for real-time optimization of temperature settings during PCR, referred to as adaptive PCR (Figure 5b). [37] Here, l-DNA analogues of both the target duplexa nd template-primer complex were added directly to the PCR. Each of the l-DNA analogues were designed to pro- duce au nique fluorescent response during temperature cycling.…”

Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood

“…The previously described 77-mer sequence derived from Mycobacterium tuberculosis (labeled as TB77) was synthesized and used to monitor melting of the amplicon. 37…”

“…The latter uses fluorescently labeled L-DNA analogs of the primers and amplicon to identify heating and cooling switch points by optically monitoring the annealing of L-DNA primers and melting of the L-DNA amplicon, respectively. 37 For the Rotor-Gene instrument, each reaction was performed with the following conditions: an initial 95 C hold for 3 minutes, followed by 45 cycles at 95 C for 15 seconds and 62 C for 15 seconds. On the Adaptive PCR instrument, the 3-minute initial denaturation was performed by programming LabVIEW software version 2017 (National Instruments, Austin, TX) to monitor the fluorescence signal of labeled L-DNA analogs of the TB77 amplicon while heating and then maintaining the fluorescence signal once the melt plateau was reached.…”

“…Instead, it monitors the denaturation and annealing of fluorescently labeled, mirror-image L-DNA molecules to switch between heating and cooling steps, which significantly simplifies instrument programming and calibration. 37 To develop an SNP genotyping process appropriate for field deployment, next, the probes' discrimination potential was tested in the simpler Adaptive PCR device. Subsequent experiments were performed in the Adaptive PCR instrument.…”

“…The assay was also evaluated using Adaptive PCR, a previously described real-time PCR platform that uses left-handed DNA (L-DNA) additives to monitor the reaction for more reliable point-of-care performance. 37…”