For the pipette-jockeys out there who have been toiling away with tedious bisulfite sequencing runs, MS-PCR, or Me-DIP protocols: Hang in there! It looks like help might soon be on the way. A group of Oxford researchers just published a method that uses nanopore technology to sequence single molecules of DNA, including 5-methylcytosine, all without the use of fluorescent labeling or chemical modifications.
The team used an adapted staphylococcal α-haemolysin (αHL) protein pore to create a nanopore. Using an applied potential, individual nucleotides that have just been liberated by an exonuclease, are channeled through the nanopore where the ionic current is measured. Since ionic conductivity varies for each base, it’s possible to accurate sequence the nucleotides passing by, as well as detection of 5-methylcytosine, the elusive “5th base”.
The advantages of this single molecule nanopore DNA sequencing technology are many, and were outlined in this paper, but for starters:
- Target DNA no longer requires amplification, reducing time, cost and amplification errors.
- Modified nucleotides like 5-methylcytosine, can be directly observed.
- There is no need for fluorescent labels, chemical treatments or expensive imaging equipment.
- The method produces long read lengths, simple alignments, and the simple signal output requires much less resources to process and store the data.
- The use of established, inexpensive technology (electrical signal readout) allows the system to be easily manufactured and scalable.
Faster, cheaper, easier and more accurate than traditional or 2nd generation sequencing methods? The only thing more you could ask for is the paper reference, so check out all the details at Nature Nanotechnology, February 2009