Atomic Force Microscopic Detection Enabling Multiplexed Low-Cycle-Number Quantitative Polymerase Chain Reaction for Biomarker Assays

Abstract

Quantitative polymerase chain reaction is the current “golden standard” for quantification of nucleic acids; however, its utility is constrained by an inability to easily and reliably detect multiple targets in a single reaction. We have successfully overcome this problem with a novel combination of two widely used approaches: target-specific multiplex amplification with 15 cycles of polymerase chain reaction (PCR), followed by single-molecule detection of amplicons with atomic force microscopy (AFM). In test experiments comparing the relative expression of ten transcripts in two different human total RNA samples, we find good agreement between our single reaction, multiplexed PCR/AFM data, and data from 20 individual singleplex quantitative PCR reactions. This technique can be applied to virtually any analytical problem requiring sensitive measurement concentrations of multiple nucleic acid targets.

Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis of DNA Length Polymorphisms

“…HSAFM offers several benefits in the characterization of genetic variants . Single-molecule imaging requires less material, which minimizes invasive biopsies and allows for additional testing per sample, and there are statistical advantages to single-molecule resolution over bulk methods: for example, individual species can be identified and quantified after multiplexing with low-cycle-number PCR, and the Bayesian approach we applied here can be extended to sophisticated hierarchical models . In addition, unlike a narrow-range CE like that employed in the Leukostrat assay, HSAFM can image DNA of virtually any length by stitching together multiple overlapping HSAFM images, , and HSAFM images can convey more than length: as we previously showed, labeling a target motif with programmable Cas9 and imaging to reveal the Cas9 molecules bound to strands allows on-target DNA to be distinguished from off-target, improving specificity.…”

Using a PCR‐Based Method To Analyze and Model Large, Heterogeneous Populations of DNA

“…Interestingly, the X 16 sample produced the largest shift. We then analyzed the PCR products through AFM . After 20 PCR cycles, mainly small structures of DNA duplex were observed for both S 1 and X 20 (Figures E and S6).…”

Modelling Large, Dynamic, and Heterogeneous Populations Using DNA Libraries

“…Interestingly, the X 16 sample produced the largest shift. We then analyzed the PCR products using atomic force microscopy (AFM) 12 . After 20 PCR cycles, mainly small structures of DNA duplex were observed for both S 1 and X 20 (Fig.…”

“…AFM images of the RT-PCR products were deposited on freshly cleaved mica in the presence of the imaging buffer (300 mM spermidine trihydrochloride, 300 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane in analytical grade water 20 ). Optimal dilutions were deposited on the mica (Plano, Germany) and kept still for 90 s. The substrate was then rinsed with analytical grade water and dried with a steady flow of nitrogen 12 . The images were obtained on air, by contact mode, using a Nanowizard II AFM (JPK, Germany).…”