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genetics Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar work ...
and
biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
, sequencing means to determine the primary structure (sometimes incorrectly called the 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.


DNA sequencing

DNA sequencing is the process of determining the
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecu ...
order of a given DNA fragment. So far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as
pyrosequencing Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequ ...
are gaining an increasing share of the sequencing market. More genome data are now being produced by pyrosequencing than Sanger DNA sequencing. Pyrosequencing has enabled rapid genome sequencing. Bacterial genomes can be sequenced in a single run with several times coverage with this technique. This technique was also used to sequence the genome of James Watson recently. The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in fundamental research into why and how organisms live, as well as in applied subjects. Because of the key importance DNA has to living things, knowledge of DNA sequences is useful in practically any area of biological research. 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.
Biotechnology Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...
is a burgeoning discipline, with the potential for many useful products and services. The Carlson curve is a term coined by ''The Economist'' to describe the biotechnological equivalent of Moore's law, and is named after author Rob Carlson. Carlson accurately predicted the doubling time of DNA sequencing technologies (measured by cost and performance) would be at least as fast as Moore's law. Carlson curves illustrate the rapid (in some cases hyperexponential) decreases in cost, and increases in performance, of a variety of technologies, including DNA sequencing, DNA synthesis, and a range of physical and computational tools used in protein expression and in determining protein structures.


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 A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to crea ...
, 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. 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 In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats. It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tr ...
. 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.


Pyrosequencing

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 Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequ ...
, 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
megabase A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both ...
s ow up to 400 megabasesin 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.


True single molecule sequencing


Large-scale sequencing

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 Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing all of the protein-coding regions of genes in a genome (known as the exome). It consists of two steps: the first step is to select only the sub ...
(a subset of all DNA across all chromosomes that encode genes) or
whole genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...
(sequencing of the all nuclear DNA of a human).


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. While sequencing DNA gives a genetic profile of an organism, sequencing RNA reflects only the sequences that are actively expressed in the cells. To sequence RNA, the usual method is first to reverse transcribe the RNA extracted from the sample to generate cDNA fragments. This can then be sequenced as described above. The bulk of RNA expressed in cells are
ribosomal RNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from riboso ...
s or
small RNA Small RNA (sRNA) are polymeric RNA molecules that are less than 200 nucleotides in length, and are usually non-coding. RNA silencing is often a function of these molecules, with the most common and well-studied example being RNA interference ( ...
s, detrimental for cellular translation, but often not the focus of a study. This fraction can be removed ''in vitro'', however, to enrich for the messenger RNA, also included, that usually is of interest. Derived from the exons these mRNAs are to be later
translated Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
to
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s that support particular cellular functions. The expression profile therefore indicates cellular activity, particularly desired in the studies of diseases, cellular behaviour, responses to reagents or stimuli.
Eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. This gives a certain complexity to map the read sequences back to the genome and thereby identify their origin. For more information on the capabilities of next-generation sequencing applied to whole transcriptomes see: RNA-Seq and MicroRNA Sequencing.


Protein sequencing

Methods for performing
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
sequencing include: * Edman degradation *
Peptide mass fingerprinting Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately ...
* Mass spectrometry * Protease digests If the gene encoding the protein is known, 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 identify a clone carrying this 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-dependent' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
. 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. This is particularly true for plant polysaccharides. Methods for the structure determination of
oligosaccharide An oligosaccharide (/ˌɑlɪgoʊˈsækəˌɹaɪd/; from the Greek ὀλίγος ''olígos'', "a few", and σάκχαρ ''sácchar'', "sugar") is a saccharide polymer containing a small number (typically two to ten) of monosaccharides (simple sug ...
s and polysaccharides include NMR spectroscopy and methylation analysis.


See also

*
Exome sequencing Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing all of the protein-coding regions of genes in a genome (known as the exome). It consists of two steps: the first step is to select only the sub ...
*
Full genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...
* Genetic code *
Pathogenomics Pathogenomics is a field which uses high-throughput screening technology and bioinformatics to study encoded microbe resistance, as well as virulence factors (VFs), which enable a microorganism to infect a host and possibly cause disease. This inclu ...
* RNA-Seq * MicroRNA sequencing *
Sequence motif In biology, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and usually assumed to be related to biological function of the macromolecule. For example, an ''N''-glycosylation site motif can be defined as ' ...


References


Links

* https://www.nature.com/subjects/sequencing {{Bioinformatics Biochemistry methods Molecular biology