Dna Sequencing

The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in 'pure' research into why and how organisms live, as well as in applied subjects. Because DNA is key to all living things, knowledge of DNA sequence may be useful in almost any biological subject area. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, research into Pathogens may lead to treatments for contagious diseases.

CHAIN TERMINATION METHOD

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). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. 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.

The classical chain termination method or Sanger method first involves preparing the DNA to be sequenced as a single strand. The DNA sample is divided into four separate samples. Each of the four samples have a Primer , the four normal Deoxynucleotides (dATP, dGTP, dCTP and dTTP), DNA Polymerase , and only one of the four Dideoxynucleotides (ddATP, ddGTP, ddCTP and ddTTP) added to it. The dideoxynucleotides are added in limited quantities. The primer or the dideoxynucleotides are either Radiolabeled or have a Fluorescent tag.

As the DNA strand is elongated the DNA polymerase catalyses the joining of deoxynucleotides to the corresponding bases. However, if a dideoxynucleotide is joined to a base, then that fragment of DNA can no longer be elongated since a dideoxynucleotide lacks a crucial 3'-OH group. Fragments of all sizes should be obtained due to the randomness of when a dideoxynucleotide is added. However, to make sure that all different lengths will occur, only short stretches of DNA can be sequenced in one test.

The DNA is then denatured and the resulting fragments are separated (with a resolution of just one nucleotide) by Gel Electrophoresis , from longest to shortest. Each of the four DNA samples is run on one of four individual lanes (lanes A, T, G, C) depending on which dideoxynucleotide was added. Depending on the whether the primers or dideoxynucleotides were radiolabeled or fluorescently labeled, the DNA bands can be detected by exposure to X-rays or UV-light and the DNA sequence can be directly read off the gel. In the image on the right, X-ray film was exposed to the dried gel, and the dark bands indicate the positions of the DNA molecules of different lengths. A dark band in a lane indicates a chain termination for that particular DNA subunit and the DNA sequence can be read off as indicated.

There can be various problems with sequencing through the Sanger Method. The primer used can also be annealed to a second site. This would cause two sequences to be interpreted at the same time. This can be solved by higher annealing temperatures and higher G and C content in the primer. Another problem can occur when RNA contaminates the reaction, which can act like a primer and leads to bands in all lanes at all positions due to non specific priming. Other contaminants can be from other plasmids, inhibitors of DNA pol, and low concentrations in general. Secondary structure of DNA being read by DNA pol can lead to reading problems and will be visualized on the readout by bands in all lanes of only a few positions.

There are two sub-types of chain-termination sequencing. In the original method, the nucleotide order of a particular DNA template can be inferred by performing four parallel extension reactions using one of the four chain-terminating bases in each reaction. The DNA fragments are detected by labelling the primer with radioactive phosphorous prior to performing the sequencing reaction. The four reactions would then be run out in four adjacent lanes on a slab polyacrylamide gel.

A development of this method used four different fluorescent dye-labelled primers. This has the advantage of avoiding the need for radioactivity; increasing safety and speed, and also that the four reactions can be combined and run in a single gel lane, if they can be distinguished. This approach is known as 'dye primer sequencing'.

Dye terminator sequencing

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 that 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.

Automation and sample preparation

Modern automated DNA sequencing instruments are able to sequence as many as 384 fluoresecently labelled samples in a batch (run) and perform as many as 24 runs a day. These perform only the size separation and peak reading; the actual sequencing reaction(s), cleanup and resuspension in a suitable Buffer must be performed separately.

To produce detectable labelled products from the template DNA, 'cycle sequencing' is most commonly performed. This approach uses repeated (25 - 40) rounds of primer annealing, DNA polymerase extension and disassociation (melting) of the template DNA strands. The major advantages of cycle sequencing is the more efficient use of the expensive sequencing reagent (BigDye) and the ability to sequence templates with strong secondary structures such as hairpins or GC-rich regions. The different stages of cycle sequencing are performed by altering the temperature of the reaction using a PCR thermal cycler. This relies on the fact that complementary DNA will anneal at a lower temperatures and disassociate at higher temperatures. An important part of making this possible is the use of DNA polymerase from a Thermophillic organism, which is not rapidly denatured at the high (>95C) temperatures involved. In the past, new DNA polymerase had to be added individually every cycle of PCR.

MAXAM-GILBERT SEQUENCING

At around the same time that the Sanger sequencing method was introduced, Maxam and Gilbert developed a method of DNA sequencing based on chemical modification of DNA followed by its subsequent cleavage {Link without Title} .
This method was initially popular since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. As the chain termination method has been developed and improved, Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity, the need for use of hazardous chemicals, and difficulties with scale-up.

OTHER DNA SEQUENCING METHODS

Other sequencing techniques which are under development, and may offer benefits over the conventional methods, include:

LARGE-SCALE SEQUENCING STRATEGIES

Current methods can directly sequence only short lengths of DNA at a time. For example, modern sequencing machines using the Sanger method can achieve a maximum of around 1000 base pairs {Link without Title} . This limitation is due to the geometrically decreasing probability of chain termination at increasing lengths, as well as physical limitations on gel size and resolution.

It is often necessary to obtain the sequence of much larger regions. For example, even simple bacterial Genomes contain millions of base pairs, and the Human Genome has more than 3 billion. Several strategies have been devised for large-scale DNA sequencing, including Primer Walking (see also Chromosome Walking ) and Shotgun Sequencing . These involve taking many small ''reads'' of the DNA through the Sanger method and subsequently assembling them into a contiguous sequence. The different strategies have different tradeoffs in speed and accuracy; for example, the shotgun method is the most practical for sequencing large genomes, but its assembly process is complex and potentially error-prone.

It is easier to obtain high quality sequence data when the desired DNA is purified and amplified from any contaminants that may be in the original sample. This can be achieved through PCR if it is practical to design primers that cover the entire desired region. Alternatively, the sample can be Cloned using a bacterial Vector , harnessing bacteria to "grow" copies of the desired DNA a few thousand base pairs at a time. Most large-scale sequencing efforts involve the preparation of a large ''library'' of such clones. The advantage of sequencing clones over PCR-products is that the possibility of the presence of non-specific PCR products that may cause signal noise is virtually eliminated.

SEE ALSO

Sequencing

Human Genome Project

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Dna Sequencing