Misplaced Pages

#127872

52-470: This method should not be confused with "sequencing by synthesis," a principle used by Roche-454 pyrosequencing (introduced in 2005, generating millions of 200-400bp reads in 2009), and the Solexa system (now owned by Illumina) (introduced in 2006, generating hundreds of millions of 50-100bp reads in 2009) These methods have reduced the cost from $ 0.01/base in 2004 to nearly $ 0.0001/base in 2006 and increased

104-659: A nucleobase , a five-carbon sugar ( ribose or deoxyribose ), and a phosphate group consisting of one to three phosphates . The four nucleobases in DNA are guanine , adenine , cytosine , and thymine ; in RNA, uracil is used in place of thymine. Nucleotides also play a central role in metabolism at a fundamental, cellular level. They provide chemical energy—in the form of the nucleoside triphosphates , adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP)—throughout

156-419: A (d5SICS–dNaM) complex or base pair in DNA. E. coli have been induced to replicate a plasmid containing UBPs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. The applications of synthetic nucleotides vary widely and include disease diagnosis, treatment, or precision medicine. Nucleotide (abbreviated "nt")

208-410: A chain reaction when pyrophosphate is released. Hence, the name pyrosequencing. The principle of pyrosequencing was first described in 1993 by, Bertil Pettersson, Mathias Uhlen and Pål Nyren by combining the solid phase sequencing method using streptavidin coated magnetic beads with recombinant DNA polymerase lacking 3´to 5´exonuclease activity (proof-reading) and luminescence detection using

260-466: A double helix, the two strands are oriented in opposite directions, which permits base pairing and complementarity between the base-pairs, all which is essential for replicating or transcribing the encoded information found in DNA. Nucleic acids then are polymeric macromolecules assembled from nucleotides, the monomer-units of nucleic acids . The purine bases adenine and guanine and pyrimidine base cytosine occur in both DNA and RNA, while

312-488: A genome wide scale is a valuable resource and could teach us much about disease and molecular biology in general. 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 . Pyrosequencing relies on light detection based on

364-540: A large amount of repetitive DNA . Lack of proof-reading activity limits accuracy of this method. The company Pyrosequencing AB in Uppsala, Sweden was founded with venture capital provided by HealthCap in order to commercialize machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique. Pyrosequencing AB was listed on the Stockholm Stock Exchange in 1999. It

416-421: A nitrogenous base, a pentose sugar 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 biomolecules within all life-forms on Earth . Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver . Nucleotides are composed of three subunit molecules:

468-483: A phosphorylated ribosyl unit. The covalent linkage between the ribose and pyrimidine occurs at position C 1 of the ribose unit, which contains a pyrophosphate , and N 1 of the pyrimidine ring. Orotate phosphoribosyltransferase (PRPP transferase) catalyzes the net reaction yielding orotidine monophosphate (OMP): Orotidine 5'-monophosphate is decarboxylated by orotidine-5'-phosphate decarboxylase to form uridine monophosphate (UMP). PRPP transferase catalyzes both

520-399: A second one-carbon unit from formyl-THF is added to the nitrogen group and the ring is covalently closed to form the common purine precursor inosine monophosphate (IMP). Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the addition of aspartate to IMP by adenylosuccinate synthase, substituting the carbonyl oxygen for a nitrogen and forming

572-432: Is a common unit of length for single-stranded nucleic acids, similar to how base pair is a unit of length for double-stranded nucleic acids. The IUPAC has designated the symbols for nucleotides. Apart from the five (A, G, C, T/U) bases, often degenerate bases are used especially for designing PCR primers . These nucleotide codes are listed here. Some primer sequences may also include the character "I", which codes for

SECTION 10

#1732779938128

624-443: Is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme . Essentially, the method allows sequencing a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from

676-411: Is fueled by ATP hydrolysis. In humans, pyrimidine rings (C, T, U) can be degraded completely to CO 2 and NH 3 (urea excretion). That having been said, purine rings (G, A) cannot. Instead, they are degraded to the metabolically inert uric acid which is then excreted from the body. Uric acid is formed when GMP is split into the base guanine and ribose. Guanine is deaminated to xanthine which in turn

728-508: Is introduced to remove nucleotides that are not incorporated by the DNA polymerase. This enabled the enzyme mixture including the DNA polymerase , the luciferase and the apyrase to be added at the start and kept throughout the procedure, thus providing a simple set-up suitable for automation. An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing. A third microfluidic variant of

780-432: Is oxidized to uric acid. This last reaction is irreversible. Similarly, uric acid can be formed when AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine. Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH 3 donor). Theories about

832-416: Is prepared from the sample to be sequenced, and is used to prepare clonal bead populations. That is, only one species of fragment will be present on the surface of each magnetic bead. The fragments attached to the magnetic beads will have a universal P1 adapter sequence attached so that the starting sequence of every fragment is both known and identical. Emulsion PCR takes place in microreactors containing all

884-543: Is protected to create a phosphoramidite , which can then be used to obtain analogues not found in nature and/or to synthesize an oligonucleotide . In vivo, nucleotides can be synthesized de novo or recycled through salvage pathways . The components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, and from ammonia and carbon dioxide. Recently it has been also demonstrated that cellular bicarbonate metabolism can be regulated by mTORC1 signaling. The liver

936-530: Is subsequently formed by the amination of UTP by the catalytic activity of CTP synthetase . Glutamine is the NH 3 donor and the reaction is fueled by ATP hydrolysis, too: Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two phosphates. The atoms that are used to build the purine nucleotides come from a variety of sources: The de novo synthesis of purine nucleotides by which these precursors are incorporated into

988-435: Is the committed step in purine synthesis. The reaction occurs with the inversion of configuration about ribose C 1 , thereby forming β - 5-phosphorybosylamine (5-PRA) and establishing the anomeric form of the future nucleotide. Next, a glycine is incorporated fueled by ATP hydrolysis, and the carboxyl group forms an amine bond to the NH 2 previously introduced. A one-carbon unit from folic acid coenzyme N 10 -formyl-THF

1040-421: Is the major organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto which

1092-413: Is then added to the amino group of the substituted glycine followed by the closure of the imidazole ring. Next, a second NH 2 group is transferred from glutamine to the first carbon of the glycine unit. A carboxylation of the second carbon of the glycin unit is concomitantly added. This new carbon is modified by the addition of a third NH 2 unit, this time transferred from an aspartate residue. Finally,

SECTION 20

#1732779938128

1144-434: The firefly luciferase enzyme. A mixture of three enzymes ( DNA polymerase , ATP sulfurylase and firefly luciferase ) and a nucleotide ( dNTP ) are added to single stranded DNA to be sequenced and the incorporation of nucleotide is followed by measuring the light emitted. The intensity of the light determines if 0, 1 or more nucleotides have been incorporated, thus showing how many complementary nucleotides are present on

1196-407: The origin of life require knowledge of chemical pathways that permit formation of life's key building blocks under plausible prebiotic conditions. The RNA world hypothesis holds that in the primordial soup there existed free-floating ribonucleotides , the fundamental molecules that combine in series to form RNA . Complex molecules like RNA must have arisen from small molecules whose reactivity

1248-406: The pyrimidine nucleotides . Being on a major metabolic crossroad and requiring much energy, this reaction is highly regulated. In the first reaction unique to purine nucleotide biosynthesis, PPAT catalyzes the displacement of PRPP's pyrophosphate group (PP i ) by an amide nitrogen donated from either glutamine (N), glycine (N&C), aspartate (N), folic acid (C 1 ), or CO 2 . This

1300-441: The umami taste, often in the form of a yeast extract. A nucleo tide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nucleobase (the two of which together are called a nucleo side ), and one phosphate group . With all three joined, a nucleotide is also termed a "nucleo side mono phosphate", "nucleoside di phosphate" or "nucleoside tri phosphate", depending on how many phosphates make up

1352-611: The Roche 454 FLX system and so large and difficult homopolymer repeat regions are no longer a problem to sequence. Naturally the technology will be used to sequence DNA, but because of the high parallel nature of all next generation technologies they also have applications in transcriptomics and epigenomics . Microarrays was once the mainstay of the transcriptomics the last ten years and array based technology has subsequently branched out to other areas. However, they are limited in that only information can be obtained for probes that are on

1404-453: The activity of proteins and other signaling molecules, and as enzymatic cofactors , often carrying out redox reactions. Signaling cyclic nucleotides are formed by binding the phosphate group twice to the same sugar molecule , bridging the 5'- and 3'- hydroxyl groups of the sugar. Some signaling nucleotides differ from the standard single-phosphate group configuration, in having multiple phosphate groups attached to different positions on

1456-474: The base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1. According to ABI, the SOLiD 3plus platform yields 60 gigabases of usable DNA data per run. Due to the two base encoding system, an inherent accuracy check is built into the technology and offers 99.94% accuracy. The chemistry of the systems also means that it is not hindered by homopolymers unlike

1508-692: The cell for the many cellular functions that demand energy, including: amino acid , protein and cell membrane synthesis, moving the cell and cell parts (both internally and intercellularly), cell division, etc.. In addition, nucleotides participate in cell signaling ( cyclic guanosine monophosphate or cGMP and cyclic adenosine monophosphate or cAMP) and are incorporated into important cofactors of enzymatic reactions (e.g., coenzyme A , FAD , FMN , NAD , and NADP ). In experimental biochemistry , nucleotides can be radiolabeled using radionuclides to yield radionucleotides. 5-nucleotides are also used in flavour enhancers as food additive to enhance

1560-425: The chip. Only information for organisms for which chips are available can obtained, and they come with all the problems of hybridizing large numbers of molecules (differing hybridizing temperatures). RNA-Seq transcriptomics by next gen sequencing will mean these barriers no longer hold true. Any organism's entire transcriptome could be potentially sequenced in one run (for very small bacterial genomes) and not only would

1612-415: The formation of PRPP . PRPS1 is the enzyme that activates R5P , which is formed primarily by the pentose phosphate pathway , to PRPP by reacting it with ATP . The reaction is unusual in that a pyrophosphoryl group is directly transferred from ATP to C 1 of R5P and that the product has the α configuration about C1. This reaction is also shared with the pathways for the synthesis of Trp , His , and

ABI Solid Sequencing - Misplaced Pages Continue

1664-412: The formation of carbamoyl phosphate from glutamine and CO 2 . Next, aspartate carbamoyltransferase catalyzes a condensation reaction between aspartate and carbamoyl phosphate to form carbamoyl aspartic acid , which is cyclized into 4,5-dihydroorotic acid by dihydroorotase . The latter is converted to orotate by dihydroorotate oxidase . The net reaction is: Orotate is covalently linked with

1716-557: The identification of each transcript be available but expression profiling is possible as quantitative reads can also be achieved. Chromatin immunoprecipitation (ChIP) is a method for determining transcription factor binding sites and DNA-protein interactions. It has in the past been combined with array technology (ChIP-chip) with some success. Next gen sequencing can also be applied in this area. Methylation immunoprecipitation (MeDIP) can also be performed and also on arrays. The ability to learn more about methylation and TF binding sites on

1768-407: The intermediate adenylosuccinate. Fumarate is then cleaved off forming adenosine monophosphate. This step is catalyzed by adenylosuccinate lyase. Inosine monophosphate is converted to guanosine monophosphate by the oxidation of IMP forming xanthylate, followed by the insertion of an amino group at C 2 . NAD is the electron acceptor in the oxidation reaction. The amide group transfer from glutamine

1820-511: The necessary reagents for PCR. The beads with the resulting PCR products are deposited to a glass slide. Primers hybridize to the P1 adapter sequence within the library template. A set of four fluorescently labelled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. Multiple cycles of ligation, detection and cleavage are performed with

1872-422: The nucleotide monomers of a nucleic acid end-to-end into a long chain. These chain-joins of sugar and phosphate molecules create a 'backbone' strand for a single- or double helix . In any one strand, the chemical orientation ( directionality ) of the chain-joins runs from the 5'-end to the 3'-end ( read : 5 prime-end to 3 prime-end)—referring to the five carbon sites on sugar molecules in adjacent nucleotides. In

1924-448: The number of cycles determining the eventual read length. Following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles. Five rounds of primer reset are completed for each sequence tag. Through the primer reset process, each base is interrogated in two independent ligation reactions by two different primers. For example,

1976-415: The phosphate group. In nucleic acids , nucleotides contain either a purine or a pyrimidine base—i.e., the nucleobase molecule, also known as a nitrogenous base—and are termed ribo nucleotides if the sugar is ribose, or deoxyribo nucleotides if the sugar is deoxyribose. Individual phosphate molecules repetitively connect the sugar-ring molecules in two adjacent nucleotide monomers, thereby connecting

2028-478: The purine and pyrimidine bases. Thus a reaction network towards the purine and pyrimidine RNA building blocks can be established starting from simple atmospheric or volcanic molecules. An unnatural base pair (UBP) is a designed subunit (or nucleobase ) of DNA which is created in a laboratory and does not occur in nature. Examples include d5SICS and dNaM . These artificial nucleotides bearing hydrophobic nucleobases , feature two fused aromatic rings that form

2080-488: The purine ring proceeds by a 10-step pathway to the branch-point intermediate IMP , the nucleotide of the base hypoxanthine . AMP and GMP are subsequently synthesized from this intermediate via separate, two-step pathways. Thus, purine moieties are initially formed as part of the ribonucleotides rather than as free bases . Six enzymes take part in IMP synthesis. Three of them are multifunctional: The pathway starts with

2132-485: The pyrimidine bases thymine (in DNA) and uracil (in RNA) occur in just one. Adenine forms a base pair with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds. In addition to being building blocks for the construction of nucleic acid polymers, singular nucleotides play roles in cellular energy storage and provision, cellular signaling, as a source of phosphate groups used to modulate

ABI Solid Sequencing - Misplaced Pages Continue

2184-454: The pyrosequencing method was described in 2005 by Jonathan Rothberg and co-workers at the company 454 Life Sciences . This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support and they showed that sequencing could be performed in a highly parallel manner using a microfabricated microarray . This allowed for high-throughput DNA sequencing and an automated instrument

2236-481: The reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template. For the solution-based version of pyrosequencing, the single-strand DNA ( ssDNA ) template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase , ATP sulfurylase , luciferase and apyrase , and with

2288-403: The ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP. First, the diphosphate from UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis: CTP

2340-407: The ring synthesis occurs. For reference, the syntheses of the purine and pyrimidine nucleotides are carried out by several enzymes in the cytoplasm of the cell, not within a specific organelle . Nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides. The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with

2392-563: The sequencing capacity from 1,000,000 bases/machine/day in 2004 to more than 5,000,000,000 bases/machine/day in 2009. Over 30 publications exist describing its use first for nucleosome positioning from Valouev et al., transcriptional profiling or strand sensitive RNA-Seq with Cloonan et al., single cell transcriptional profiling with Tang et al. and ultimately human resequencing with McKernan et al. The method used by this machine (sequencing-by-ligation) has been reported to have some issue sequencing palindromic sequences. A library of DNA fragments

2444-479: The substrates adenosine 5´ phosphosulfate (APS) and luciferin . The process can be represented by the following equations: where: Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequences containing

2496-471: The sugar. Nucleotide cofactors include a wider range of chemical groups attached to the sugar via the glycosidic bond , including nicotinamide and flavin , and in the latter case, the ribose sugar is linear rather than forming the ring seen in other nucleotides. Nucleotides can be synthesized by a variety of means, both in vitro and in vivo . In vitro, protecting groups may be used during laboratory production of nucleotides. A purified nucleoside

2548-416: The template strand. The nucleotide mixture is removed before the next nucleotide mixture is added. This process is repeated with each of the four nucleotides until the DNA sequence of the single stranded template is determined. A second solution-based method for pyrosequencing was described in 1998 by Mostafa Ronaghi , Mathias Uhlen and Pål Nyren . In this alternative method, an additional enzyme apyrase

2600-548: Was governed by physico-chemical processes. RNA is composed of purine and pyrimidine nucleotides, both of which are necessary for reliable information transfer, and thus Darwinian evolution . Becker et al. showed how pyrimidine nucleosides can be synthesized from small molecules and ribose , driven solely by wet-dry cycles. Purine nucleosides can be synthesized by a similar pathway. 5'-mono- and di-phosphates also form selectively from phosphate-containing minerals, allowing concurrent formation of polyribonucleotides with both

2652-413: Was introduced to the market. This became the first next generation sequencing instrument starting a new era in genomics research, with rapidly falling prices for DNA sequencing allowing whole genome sequencing at affordable prices. "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method

SECTION 50

#1732779938128

2704-495: Was renamed to Biotage in 2003. The pyrosequencing business line was acquired by Qiagen in 2008. Pyrosequencing technology was further licensed to 454 Life Sciences . 454 developed an array-based pyrosequencing technology which emerged as a platform for large-scale DNA sequencing , including genome sequencing and metagenomics . Roche announced the discontinuation of the 454 sequencing platform in 2013. Nucleotides Nucleotides are organic molecules composed of

#127872