Meller Group : Single Molecule Biophysics & Nano-biotechnologyTechnion Logo

DNA 'bar-coding' using PNA probes

The ability to rapidly tag and identify trace amounts of DNA without the need for costly and time-consuming amplification can lead to cheaper and faster pathogen and mutation diagnostics platforms. Low-cost and high-speed platforms are essential for an effective response to emerging infection threats and will ultimately result in a more accurate treatment, as well as an overall decrease in morbidity and mortality.

To this end we have developed a nanopore-based DNA bar-coding method, which employs a purely electrical detection of individual Peptide Nucleic Acid (PNA)-tagged double-stranded DNA molecules. The PNA probes are designed to hybridize to specific sequences in the target genomes, thus creating unique 'bar-code' pattern for the target, as shown schematically in Figure 1. Then single DNA molecules are threaded through a sub-5 nm solid-state pores, which reads that pattern, as illustrated in Figure 2. Sensing of individual DNA molecules tagged with PNA probes, proceeds with high-throughput (>1 molecule/second at sub-nM DNA concentrations), enabling rapid single-molecule identification of specific dsDNA sequences.

Our current result already demonstrated the ability to probe one or two PNA probes hybridized specifically to a ~3,000bp DNA, as shown in Figure 3. The bare DNA result in a reduction of ~1nA in the open-pore current. In contrast, the PNA/DNA hybrid results in a reduction of 1.5nA in the current, which is readily detectable using our system. Read more about this method in our Nano Letters paper.

References:

  1. Singer A, Wanunu M, Morrison W, Kuhn H, Frank-Kamenetskii M and A. Meller.(2010) Nanopore-based sequence-specific detection of duplex DNA for genomic profiling. Nano Letters. 10(2), 738-42.

 

DNA barcoding

Figure 1

PNA-Nnopore

Figure 2

2 PNA signal

Figure 3