Using Ultrafast EMCCD Cameras for Faster DNA Sequencing
- Friday, 01 October 2010
To decode the human genome and gain a greater understanding of diseases and their aetiology, advances need to be made in the rate at which the nucleotides that comprise DNA can be sequenced. Approaches to DNA sequencing have moved on considerably since the development of “chain-terminator” methods by Maxam and Gilbert, and by Sanger, in the1970s, and are now evolving beyond the so-called “next generation” toward today’s “third generation” systems.
The driving force behind these is a desire to:
- increase the read length and accelerate the read times, both of which were sacrificed in next generation systems in order to improve the throughput delivered by the traditional Sanger-based sequencers;
- reduce the price of the required instrumentation;
- cut the cost per sequenced DNA base.
Increasing the read length is vital since the longer the sequence, the more accurate the genomic map that is delivered.
The latest systems claim read lengths of up to 1,000 bases, increasing to 3,000 in certain instances.
Nanopores – A “Hole” New Approach
A single-molecule DNA detection using high-speed optical identification of individual converted DNA bases as they translocate through solid state nanopores with high temporal resolution (1000 frames per second), has been developed by Amit Meller, Associate Professor of Biomedical Engineering and Physics, Boston University1.
Translocation is a promising new approach since it can analyze extremely long DNA chains with a precision which is superior to current third generation systems, and competes very effectively with them on cost, speed, and accuracy. However, to be viable, it needs to be able to differentiate between the four nucleotides that comprise DNA on a single molecule level, and be capable of parallel readout.
The Technology Explained
Meller’s team is developing a novel single molecule DNA sequencing technique – called Optipore – based on the optical readout of DNA molecules as they translocate through nanometer scale pores.
A key to Meller’s approach to sequencing is the use of a custom total internal reflection fluorescence (TIRF) microscopy set-up, incorporating an ultra-sensitive Andor iXon 860 EMCCD camera to rapidly record fluorescence images from the nanopore membrane2.
To increase the contrast between the nucleotides, the DNA is first converted to an expanded, digitized form by systematically substituting each and every base in the DNA sequence with a specific ordered pair of concentrated oligonucleotides. The converted DNA is then hybridized with complementary molecular beacons labeled with two different colors. Nanopores are then used to sequentially ‘unzip’ the beacons. With each unzipping event a new fluorophore is un-quenched, giving rise to a series of photon flashes in two colors, which are recorded by the EMCCD camera. This unzipping process slows down the translocation of the DNA through the pore in a voltage-dependent manner, to a rate compatible with single molecule optical probing.