Sequencing
Overview
 
For the sense of "sequencing" used in electronic music
Electronic music
Electronic music is music that employs electronic musical instruments and electronic music technology in its production. In general a distinction can be made between sound produced using electromechanical means and that produced using electronic technology. Examples of electromechanical sound...

, see the music sequencer
Music sequencer
The music sequencer is a device or computer software to record, edit, play back the music, by handling note and performance information in several forms, typically :...

 article. For sequence learning in cognitive science, see sequence learning
Sequence learning
Sequence learning is inherent to human ability because it is an integrated part of conscious and nonconscious learning as well as activities. Sequences of information or sequences of actions are used in various everyday tasks: "from sequencing sounds in speech, to sequencing movements in typing or...

.


In genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....

 and biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

, sequencing means to determine the primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...

 (sometimes falsely called primary sequence) of an unbranched biopolymer
Biopolymer
Biopolymers are polymers produced by living organisms. Since they are polymers, Biopolymers contain monomeric units that are covalently bonded to form larger structures. There are three main classes of biopolymers based on the differing monomeric units used and the structure of the biopolymer formed...

. 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 is the process of determining the nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...

 order of a given DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 fragment.
Encyclopedia
For the sense of "sequencing" used in electronic music
Electronic music
Electronic music is music that employs electronic musical instruments and electronic music technology in its production. In general a distinction can be made between sound produced using electromechanical means and that produced using electronic technology. Examples of electromechanical sound...

, see the music sequencer
Music sequencer
The music sequencer is a device or computer software to record, edit, play back the music, by handling note and performance information in several forms, typically :...

 article. For sequence learning in cognitive science, see sequence learning
Sequence learning
Sequence learning is inherent to human ability because it is an integrated part of conscious and nonconscious learning as well as activities. Sequences of information or sequences of actions are used in various everyday tasks: "from sequencing sounds in speech, to sequencing movements in typing or...

.


In genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....

 and biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

, sequencing means to determine the primary structure
Primary structure
The primary structure of peptides and proteins refers to the linear sequence of its amino acid structural units. The term "primary structure" was first coined by Linderstrøm-Lang in 1951...

 (sometimes falsely called primary sequence) of an unbranched biopolymer
Biopolymer
Biopolymers are polymers produced by living organisms. Since they are polymers, Biopolymers contain monomeric units that are covalently bonded to form larger structures. There are three main classes of biopolymers based on the differing monomeric units used and the structure of the biopolymer formed...

. 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
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...

 order of a given DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...

. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...

 are gaining an increasing share of the sequencing market. More genome data is 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 X coverage with this technique. This technique was also used to sequence the genome of James Watson
James D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...

 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 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 genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...

 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
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

, 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.
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
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

. 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 2nd and 3rd generation systems from Illumina, 454, ABI, Helicos & Dover.]

Pyrosequencing

Pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...

, which was developed by Pål Nyhren and Mostafa Ronaghi, 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 7-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
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

s. 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. http://www.454.com

RNA sequencing

RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

 is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

 from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in Eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

s RNA molecules are not necessarily co-linear with their DNA template, as intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...

s are excised. To sequence RNA, the usual method is first to reverse transcribe
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...

 the sample to generate DNA fragments. This can then be sequenced as described above.

For more information on the capabilities of next-generation sequencing applied to whole transcriptome
Transcriptome
The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.-Scope:...

s see: RNA-Seq
RNA-Seq
RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" and dubbed "a revolutionary tool for transcriptomics", refers to the use of high-throughput sequencing technologies to sequence cDNA in order to get information about a sample's RNA content, a technique that is quickly becoming...

.

Protein sequencing

Methods for performing protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 sequencing
include:
  • Edman degradation
    Edman degradation
    Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues....

  • Peptide mass fingerprinting
    Peptide mass fingerprinting
    Peptide mass fingerprinting is an analytical technique for protein identification that was developed in 1993 by several groups independently. In this method, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass...

  • Mass spectrometry
    Mass spectrometry
    Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

  • Protease digests


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 polysaccharide
Polysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...

s 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 monosaccharide
Monosaccharide
Monosaccharides are the most basic units of biologically important carbohydrates. They are the simplest form of sugar and are usually colorless, water-soluble, crystalline solids. Some monosaccharides have a sweet taste. Examples of monosaccharides include glucose , fructose , galactose, xylose...

s) can be used, and bonded
Chemical bond
A chemical bond is an attraction between atoms that allows the formation of chemical substances that contain two or more atoms. The bond is caused by the electromagnetic force attraction between opposite charges, either between electrons and nuclei, or as the result of a dipole attraction...

 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
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

. 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
Oligosaccharide
An oligosaccharide is a saccharide polymer containing a small number of component sugars, also known as simple sugars...

s and polysaccharide
Polysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...

s include NMR
NMR
NMR may refer to:Applications of Nuclear Magnetic Resonance:* Nuclear magnetic resonance* NMR spectroscopy* Solid-state nuclear magnetic resonance* Protein nuclear magnetic resonance spectroscopy* Proton NMR* Carbon-13 NMR...

 spectroscopy and methylation analysis.

See also

  • Genetic code
    Genetic code
    The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....

  • Full Genome Sequencing
    Full genome sequencing
    Full genome sequencing , also known as whole genome sequencing , complete genome sequencing, or entire genome sequencing, is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time...

  • Pathogenomics
    Pathogenomics
    Pathogen infections are among the leading causes of infirmity and mortality among humans and animals in the world. Until recently, it has been difficult to compile information to understand the generation of pathogen virulence factors as well as pathogen behaviour in a host environment...

  • RNA-Seq
    RNA-Seq
    RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" and dubbed "a revolutionary tool for transcriptomics", refers to the use of high-throughput sequencing technologies to sequence cDNA in order to get information about a sample's RNA content, a technique that is quickly becoming...

  • Sequence motif
    Sequence motif
    In genetics, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance...

The source of this article is wikipedia, the free encyclopedia.  The text of this article is licensed under the GFDL.
 
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