Sequencing
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- For the sense of "sequencing" used in electronic music, see the music sequencer article.
In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
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DNA sequencing
Overview
In genetics terminology, DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Currently, almost all DNA sequencing is performed using the chain termination method[1], developed by Frederick Sanger. This technique uses sequence-specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates.
Why sequence DNA?
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 of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for both genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.
Sanger sequencing
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 original Sanger sequencing method
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 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 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 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.
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 [2]. 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 [3]. 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.
RNA sequencing
RNA is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in Eukaryotes RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. To sequence RNA, the usual method is first to reverse transcribe the sample to generate DNA fragments. This can then be sequenced as described above.
Protein Sequencing
Methods for performing protein sequencing include:
If the gene encoding the protein can be identified it is currently much easier to sequence the DNA and infer the protein sequence. Determining part of a protein's amino-acid sequence (often one end) by one of the above methods may be sufficient to enable the identification of a clone carrying the gene.
Polysaccharide Sequencing
Though polysaccharides are also biopolymers, it is not so common to talk of 'sequencing' a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual monosaccharides) can be used, and bonded in different ways. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependant' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different enzyme. In many cases the assembly is not uniquely specified; depending on which enzyme acts, one of several different units may be incorporated. This can lead to a family of similar molecules being formed.
See also
External links
- Information on genome projects, and the data they have produced at the National Center for Biotechnology Information
- (2005) Genome Sequencing: Using Models to Predict Who's Next. PLoS Biol 3(1): e25.
