Sequencing

Part of a radioactively labelled sequencing gel

In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). The deoxynucleotides lack in the OH group both at the 2' and at the 3' position of the ribose molecule, therefore once they are inserted within a DNA molecule they prevent it from being further elongated. In this sequencer four different vessels are employed, each containing only of the four dideoxyribonucleotides; the incorporation of the chain terminating nucleotides by the DNA polymerase in a random position results in a series of related DNA fragments, of different sizes, that terminate with a given dideoxiribonucleotide. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

View of the start of an example dye-terminator read (click to expand)

An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability. This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers. This is changing rapidly due to the increasing cost-effectiveness of second- and third-generation systems from Illumina, 454, ABI, Helicos, and Dover.

The pyrosequencing method is based on the detection of the pyrophosphate release on nucleotide incorporation. Before performing pyrosequencing, the DNA strand to sequence has to be amplified by PCR. Then the order in which the nucleotides have to be added in the sequencer is chosen (i.e. G-A-T-C). When a specific nucleotide is added, if the DNA polymerase incorporates it in the growing chain, the pyrophosphate is released and converted into ATP by ATP sulfurylase. ATP powers the oxidation of luciferase through the luciferase; this reaction generates a light signal recorded as a pyrogram peak. In this way, the nucleotide incorporation is correlated to a signal. The light signal is proportional to the amount of nucleotides incorporated during the synthesis of the DNA strand (i.e. two nucleotides incorporated correspond to two pyrogram peaks). When the added nucleotides aren't incorporated in the DNA molecule, no signal is recorded; the enzyme apyrase removes any unincorporated nucleotide remaining in the reaction. This method requires neither fluorescently-labelled nucleotides nor gel electrophoresis. Pyrosequencing, which was developed by Pål Nyrén and Mostafa Ronaghi DNA, has been commercialized by Biotage (for low-throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases [now up to 400 megabases] in a seven-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow.

Whereas the methods above describe various sequencing methods, separate related terms are used when a large portion of a genome is sequenced. Several platforms were developed to perform exome sequencing (a subset of all DNA across all chromosomes that encode genes) or whole genome sequencing (sequencing of the all nuclear DNA of a human).