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Encyclopedia > Pyrosequencing

Pyrosequencing, is a novel method of DNA sequencing developed initially by Mostafa Ronaghi and co-workers in the late 1990s, then further by Biotage. The method is based on a chemiluminescent enzymatic reaction, which is triggered when a molecular recognition event occurs. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it. Each time a nucleotide, A, C, G or T is incorporated into the growing chain a cascade of enzymatic reactions is triggered which results in a light signal. It is a method primarily used for sequencing of short stretches of DNA, SNP detection and methylation analysis. Such analyses are crucial for biological research, genetics and some medical and forensic applications. Pyrosequencing is fully automated, reliable and accurate, and large numbers of samples can be analysed in a short time. Pyrosequencing methods have been pursued to reduce costs relative to other automated sequencing methods. DNA sequencing is the process of determining the nucleotide order of a given DNA fragment, called the DNA sequence. ... The 1990s decade refers to the years from 1990 to 1999, inclusive. ... Lightsticks Chemoluminescence (sometimes chemiluminescence) is the emission of light (luminescence) as the result of a chemical reaction. ... Ribbon diagram of the enzyme TIM. TIM is catalytically perfect, meaning its conversion rate is limited, or nearly limited to its substrate diffusion rate. ... A reaction is the following: In physics, a reaction (physics) is defined by Newtons third law: For every action there is an equal and opposite reaction. The idea that any given force has a pair or opposite force. ... The general structure of a section of DNA Deoxyribonucleic acid (DNA) is a nucleic acid —usually in the form of a double helix— that contains the genetic instructions specifying the biological development of all cellular forms of life, and most viruses. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... In genetics and biochemistry, sequencing means to determine the primary structure (or primary sequence) of an unbranched biopolymer. ... The general structure of a section of DNA Deoxyribonucleic acid (DNA) is a nucleic acid —usually in the form of a double helix— that contains the genetic instructions specifying the biological development of all cellular forms of life, and most viruses. ... SNP may refer to: The Scottish National Party The Slovak National Uprising A single nucleotide polymorphism Sodium nitroprusside The Solar neutrino problem Sentinel node procedure (oncological surgery) A Microsoft Access Report Snapshot file This page expands a three-character combination which might be any or all of: an abbreviation, an... In biochemistry, methylation refers to the replacement of a hydrogen atom (H) with a methyl group (CH3), regardless of the substrate. ...



The technique of pyrosequencing is conducted in a five step process:


Step 1: PCR amplified ssDNA templates is hybridized to a sequencing primer and incubated with the enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin. Wikipedia does not yet have an article with this exact name. ... DNA replication Deoxyribonucleic acid (DNA) is a nucleic acid which carries genetic instructions for the biological development of all cellular forms of life and many viruses. ... Primer can refer to more than one thing: Look up Primer in Wiktionary, the free dictionary. ... 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. ... Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. ... This article may be too technical for most readers to understand. ... Luciferin is a generic name for light-emitting pigments found in organisms capable of bioluminescence, like fireflies, deep-sea fish and microbes. ...


Step 2: The addition of one of the four deoxynucleotide triphosphates (dNTPs) initiates the second step. DNA polymerase will catalyze the incorporation of dNTP onto the template if it is complementary. It is important to note that if there is an incorporation there will be a release of pyrophosphate (PPi) equivalent to the amount of dNTP incorporated. A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... In chemistry, pyrophosphate, or PPi is an anion observed in living systems, usually formed by the hydrolysis of ATP into AMP. The hydrolysis is called pyrophosphorolysis. ...


Step 3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and this can be analyzed in a program. Each light signal is proportional to the number of nucleotides incorporated.


Step 4: To continue the sequencing the degrading of nucleotides is essential. Apyrase is a nucleotide degrading enzyme and does in this step clean the solution from all dNTP.


Step 5: New nucleotides can be added and a new cycle can start.



The technique has been further developed for whole genome sequencing by the company 454. To date this is the fastest sequencing method. However, a serious limitiation of the method is that read lengths are currently much shorter than those obtained by di-deoxy nucleotide based chain termination methods, which makes the process of genome assembly much more complicated, particularly for genomes which contain a large amount of repetitive DNA. Pyrosequencing is therefore most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ... Addition polymerization, also called polyaddition or chain growth polymerization, is a polymerization technique where monomer molecules add on to a growing polymer chain one at a time. ... Genome assembly refers to the process of taking a large number of short DNA sequences, all of which were generated by a shotgun sequencing project, and putting them back together to create a representation of the original chromosomes from which the DNA originated. ... In the study of DNA sequences, one can distinguish two main types of repeated sequence: Tandem repeats: Satellite DNA, Minisatellite, Microsatellite; Interspersed repeats: SINE (Short INterspersed Elements), LINE (Long INterspersed Elements). ...


External links and references

http://www.biotage.com


http://www.454.com

  • Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms

http://www.pyrosequencing.com


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11156611&dopt=Citation


  Results from FactBites:
 
Journal of Translational Medicine | Full text | Pyrosequencing™ : A one-step method for high resolution HLA ... (3675 words)
Pyrosequencing™ [9-11] is a real-time, sequencing by synthesis method catalyzed by four kinetically well-balanced enzymes, DNA polymerase, ATP sulfurylase, luciferase, and apyrase.
In conclusion, Pyrosequencing can be used as a one-step method for high resolution DNA typing and could be applied in several settings spanning from HLA typing in support of donor/recipient selection to become a complement to comprehensive immunogenetic profiling in several clinical setting where other aspects of immune polymorphism need to be explored [8].
Constant improvements in the chemistry for sample preparation for Pyrosequencing and Pyrosequencing [25-34] and the implementation of automation devices http://www.pyrosequencing.com it may be possible in the future to apply this technology directly for routine typing of HLA and other immune related genes characterized by extensive polymorphisms [8].
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