Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Accurate detection of miRNAs with complementary probes is challenging due to the short target size, and often high sequence similarity between isoforms belonging to the same miRNA family. Ligation based methods can provide powerful discrimination of subtle sequence variation among target sequences, but they have been difficult to implement for direct RNA analysis due to the sloppiness and inefficiency of most DNA ligases on RNA substrates. In this work, we have studied if RNA substitutions in padlock probes can provide higher catalytic efficiencies for PBCV-1 DNA ligase on RNA substrates. We also characterise end-joining fidelity for Chlorella virus DNA ligase (PBCV DNA ligase 1) and T4RNA ligase 2 (T4Rnl2) in RNA-templated 3'-OH RNA/5’-pDNA chimeric probe ligation. Although we observed considerable ligation efficiency improvement towards short miRNA targets for PBCV-1 ligated chimeric probes, it showed no sequence specificity towards mismatches at the ligation junction. T4Rnl2 showed some base discrimination, but not satisfactory for robust RNA sequence analysis. To increase end-joining fidelity in PBCV-1 DNA ligase catalysed direct RNA detection assays (iLock probes), we have recently introduced an alternative ligation assay design in which ligation probes first undergo sequence- specific 5’ FLAP removal in order to create ligatable substrates. We have tested various chimeric iLock probe designs where RNA substitutions were introduced at different positions in the FLAP and at the ligation junction. We defined two particular nucleotide positions in the iLock probe sequence that when substituted with RNA, significantly increased iLock probe activation and ligation. We further characterized the end-joining fidelity of PBCV-1 and T4Rnl2 catalysed iLock reactions. Both enzymes showed high ligation fidelities for single nucleotide polymorphisms on RNA and miRNA. Finally, we demonstrate a multiplexed chimeric iLock probe miRNA profiling assay using sequencing-by-ligation as readout. 

F29 (Phi29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real-time (SMRT) sequencing. Here we report the ability of F29 DNA polymerase to amplify partially RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions support a similar amplification rate as pure DNA substrates. We observed that increasing the number of consecutive RNA substitutions in the circular templates suppress replication, and cannot be recovered by addition of M-MuLV reverse-transcriptase. In summary, this novel ability of F29 to accept RNA-containing substrates broadens the spectrum of applications for F29 mediated RCA. Applications include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.