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Encyclopedia > Sequencing

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. DNA, the molecular basis for inheritance. ... Biochemistry is the study of the chemical processes and transformations in living organisms. ... A protein primary structure is a chain of amino acids. ... This article or section does not adequately cite its references or sources. ...

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DNA sequencing

Main article: DNA sequencing

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus 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 is gaining an increasing share of the sequencing market. Majority of genome data are being produced by Pyrosequencing than Sanger DNA sequencing these days. Pyrosequencing has enabled rapid genome sequencing. Bacterial genome 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 recently. DNA sequencing is the process of determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... The structure of part of a DNA double helix Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living organisms. ... The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases of) DNA. It is named after Frederick Sanger who developed the process in 1975. ... Frederick Sanger, OM, CH, CBE, FRS (born 13 August 1918) is an English biochemist and a two time Nobel laureate in Chemistry. ... The introduction to this article provides insufficient context for those unfamiliar with the subject matter. ... There is more than one person with the name James Watson: James Watson, participant in the Battle of the Little Bighorn James Watson, author of the novel Talking in Whispers James Watson, U.S. Senator from New York (1797-1801) James Watson, painter of 77 portraits held by the U...


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 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. A pathogen (literally birth of pain from the Greek παθογένεια) is a biological agent that can cause disease to its host. ... The structure of insulin Biological technology is technology based on biology, especially when used in agriculture, food science, and medicine. ...


Sanger sequencing

Part of a radioactively labelled sequencing gel
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). 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. DNA sequencing gel. ... DNA sequencing gel. ... 3D structure of the DNA-binding helix-hairpin-helix motifs in human DNA polymerase beta A DNA polymerase is an enzyme that assists in DNA replication. ...

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. Image File history File links Download high resolution version (994x230, 19 KB)Example of (the start of) a Sanger sequencing read. ... Image File history File links Download high resolution version (994x230, 19 KB)Example of (the start of) a Sanger sequencing read. ... The wavelength is the distance between repeating units of a wave pattern. ...


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 is less of a concern with frequently used 'universal' primers.


Pyrosequencing

Pyrosequencing, which was originally developed by 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 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. 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. [1] The introduction to this article provides insufficient context for those unfamiliar with the subject matter. ... Wikipedia does not yet have an article with this exact name. ... Neuraminidase ribbon diagram An enzyme (in Greek en = in and zyme = blend) is a protein, or protein complex, that catalyzes a chemical reaction and also controls the 3D orientation of the catalyzed substrates. ... Adenosine 5-triphosphate (ATP) is a multifunctional nucleotide that is most important as a molecular currency of intracellular energy transfer. ...


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. Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions... A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary transcripts. ... Kingdoms Animalia - Animals Fungi Plantae - Plants Protista Alternative Phylogeny Unikonta    Opisthokonta    Amoebozoa Bikonta    Apusozoa    Cabozoa       Rhizaria       Excavata    Corticata       Archaeplastida       Chromalveolata Animals, plants, fungi, and protists are eukaryotes (IPA: ), organisms with a complex cell or cells, where the genetic material is organized into a membrane-bound nucleus or nuclei. ... ... Diagram of the location of introns and exons within a gene. ... In biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. Normal transcription involves the synthesis of RNA from DNA, hence reverse transcription is the reverse of this. ...


Protein sequencing

Main article: protein sequencing

Methods for performing protein sequencing include: Proteins are found in every cell and are essential to every biological process, protein structure is very complex: determining a proteins structure involves first protein sequencing - determining the amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ...

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. Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. ... Peptide mass fingerprinting (also known as protein fingerprinting) (PMF) is an analytical technique for protein identification that was developed by John Yates and colleagues (3). ... Mass spectrometry (also known as mass spectroscopy (deprecated)[1] or informally, mass-spec and MS) is an analytical technique used to measure the mass-to-charge ratio of ions. ... Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. ...


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. 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 oligosaccharides and polysaccharides include NMR spectroscopy and methylation analysis[1]. Polysaccharides (sometimes called glycans) are relatively complex carbohydrates. ... Monosaccharides are the simplest form of carbohydrates. ... A chemical bond is the physical process responsible for the attractive interactions between atoms and molecules, and that which confers stability to diatomic and polyatomic chemical compounds. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... An oligosaccharide is a saccharide polymer containing a small number (typically three to six) of component sugars, also known as simple sugars. ... Polysaccharides (sometimes called glycans) are relatively complex carbohydrates. ... NMR may refer to: Nuclear magnetic resonance, a phenomenon involving the interaction of atomic nuclei and external magnetic fields Nielsen Media Research, a U.S. company which measures TV, radio and newspaper audiences This is a disambiguation page — a navigational aid which lists other pages that might otherwise share...


See also

A series of codons in part of a mRNA molecule. ... 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. ...

References

  1. ^ A practical guide to structural analysis of carbohydrates

  Results from FactBites:
 
DNA Sequencing | Nucleics (1667 words)
In addition to being able to detect sequencing problems independent of the base caller used, the QualTrace™ analysis software also offers DNA sequencing core facilities a reproducible and cost efficient means of providing independent sequencing troubleshooting advice to their customers as to the cause of any sequencing problems.
The CounterTrace sequencing kit is compatible with both the ABI 3700 and 3730 DNA sequencers and has been designed to be used with the phred base caller.
The ASIN genome sequencing technology was sold to Takara Biomedicals, a division of Takara Shuzo (Japan) in February 2000.
Davis Sequencing, an Automated DNA Sequencing Facility: Home Page (77 words)
Davis Sequencing provides high-throughput automated DNA sequencing services to university, commercial and government customers worldwide.
Our goal is to provide a high quality DNA sequencing service at a low price with a fast turn-around time.
Davis Sequencing will be closed on Labor Day on Monday, September 3rd.
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