cascode sequencing

Rapid DNA sequencing is an essential part of modern biotechnology, it has also become routine for patients to have their genetic sequence taken down as to better subscribe treatments, it has also become important in xenobiology where new species are waiting to be sequenced.

One method of sequencing has offered extremely rapid sequencing if not completely reliable and trouble free. Previous sequencing methods generally use artificial reactions that do not occur in nature. Though these methods are very useful, such as chain termination PCR. The rate of sequencing is far slower than the rate of natural polymerises in vivo. Cascoding uses the same mechanisms used in vivo, exploiting the very enzymes used to unwrap the double helix.

Cascoding works on a simple principle, the bases on DNA are structurally different from each, because of these structural differences the different bases can be observed spectrochemically. The other effect is that the observed spectrochemical effect is changed by its immediate environment. Because each base can be resolved by spectrochemical techniques and a base hidden within a protein is distinct from a base in solution this allowed a way for processing a DNA sequence.

The cascode method uses a family of proteins called monomeric helicases, these proteins crawl along the DNA strand splitting the duplex part of this proteins mechanism is to feed one strand of the duplex through the central core of the protein. Because these enzymes display a very selective specificity a DNA strand can be prepared by restriction to have differently cut ends, because the helicases can only work on one type of end, this ensures all the helicases to work on the same strand. However to get all the helicases to split the strand at the same rate the solution of DNA, helicases and SSB protein (to prevent re-annealing) has to be cooled well below physiological temperatures, often in eutectic mixtures well below 273’K, this reduces background rate of helix splitting was essentially nil. However for the helix to be split at all energy needs to be supplied in the form of ATP, it was found that at low enough temperatures the chilled helicases were unable to split the helix even at high ATP concentrations. However a brief blast of laser light was enough to prompt catalysis of ATP and to move the enzyme one base along the helix.

By this mechanism each flash of laser light buries one new nucleotide, the identity of which could be revealed from the spectroscopic differences before and after the laser light. Using the cold and laser light specific, processive sequencing of the helix could be made, by careful manipulation of the conditions you can approximate the speeds to which these enzymes work in their natural environment, which is to unravel hundreds of bases a second, this is also the rate to which the strand is sequenced, this rate is in the order of thousand times faster than the chain termination techniques.

Though very fast this method is somewhat unreliable, and although the enzymes are very processive and can process very long sequences, the gradual de-synchronization of the helicases in solution slowly introduces error in sequence.