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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, 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
289:. 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.
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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.
1308:
55:
237:, 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,
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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
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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
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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
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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
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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).
324:. 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.
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507:. 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
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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
263:
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
268:, 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
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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
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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
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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
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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
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in 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
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205:. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as
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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
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Wheeler, David A.; Srinivasan, Maithreyan; Egholm, Michael; Shen, Yufeng; Chen, Lei; McGuire, Amy; He, Wen; Chen, Yi-Ju; Makhijani, Vinod (2008-04-17).
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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
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therefore indicates cellular activity, particularly desired in the studies of diseases, cellular behaviour, responses to reagents or stimuli.
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is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different
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Carlson, Robert H. Biology Is
Technology: The Promise, Peril, and New Business of Engineering Life. Cambridge, MA: Harvard UP, 2010. Print
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molecules. While sequencing DNA gives a genetic profile of an organism, sequencing RNA reflects only the sequences that are actively
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This article is about the genetics definition of "sequencing". For the sense of "sequencing" used in electronic music, see
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is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by
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from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to
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International
Conference on Computational Intelligence Methods for Bioinformatics and Biostatistics
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which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
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is a burgeoning discipline, with the potential for many useful products and services.
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sequence (often one end) by one of the above methods may be sufficient to identify a
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In genetics and biochemistry, determining the structure of an unbranched biopolymer
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Carlson, Robert (2003). "The Pace and
Proliferation of Biological Technologies".
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Biosecurity and
Bioterrorism: Biodefense Strategy, Practice, and Science
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View of the start of an example dye-terminator read (click to expand)
167:(sometimes incorrectly called the primary sequence) of an unbranched
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fragment. So far, most DNA sequencing has been performed using the
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343:(a subset of all DNA across all chromosomes that encode genes) or
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171:. Sequencing results in a symbolic linear depiction known as a
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38:. For other uses of the terms "sequencer" and "sequence", see
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African
Society for Bioinformatics and Computational Biology
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in the cells. To sequence RNA, the usual method is first to
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Max Planck
Institute of Molecular Cell Biology and Genetics
696:"A practical guide to structural analysis of carbohydrates"
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International
Nucleotide Sequence Database Collaboration
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79:. Unsourced material may be challenged and removed.
783:, database of protein sequences grouping together
210:technique was also used to sequence the genome of
34:. For sequence learning in cognitive science, see
189:DNA sequencing is the process of determining the
1018:US National Center for Biotechnology Information
402:that support particular cellular functions. The
347:(sequencing of the all nuclear DNA of a human).
222:may lead to treatments for contagious diseases.
1103:International Society for Computational Biology
259:Part of a radioactively labelled sequencing gel
233:to describe the biotechnological equivalent of
1170:ISCB Africa ASBCB Conference on Bioinformatics
1117:Institute of Genomics and Integrative Biology
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1146:European Conference on Computational Biology
1181:Research in Computational Molecular Biology
1158:International Conference on Bioinformatics
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712:https://www.nature.com/subjects/sequencing
641:Life 2.0. (2006, August 31). The Economist
1152:Intelligent Systems for Molecular Biology
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139:Learn how and when to remove this message
1140:Basel Computational Biology Conference
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316:which produce light in the presence of
229:The Carlson curve is a term coined by
1097:International Society for Biocuration
995:European Molecular Biology Laboratory
7:
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77:adding citations to reliable sources
1123:Japanese Society for Bioinformatics
1085:European Molecular Biology network
410:RNA molecules are not necessarily
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1175:Pacific Symposium on Biocomputing
1079:Australia Bioinformatics Resource
1046:Swiss Institute of Bioinformatics
1029:Netherlands Bioinformatics Centre
989:European Bioinformatics Institute
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977:Database Center for Life Science
965:Computational Biology Department
853:Arabidopsis Information Resource
53:
823:Specialised genomic databases:
330:True single molecule sequencing
64:needs additional citations for
1024:Japanese Institute of Genetics
390:of interest. Derived from the
1:
944:Rosalind (education platform)
861:Zebrafish Information Network
829:Saccharomyces Genome Database
1274:List of biological databases
793:Protein Information Resource
414:with their DNA template, as
394:these mRNAs are to be later
767:European Nucleotide Archive
459:Peptide mass fingerprinting
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674:10.1089/153871303769201851
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40:Sequencer (disambiguation)
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1052:Wellcome Sanger Institute
1006:J. Craig Venter Institute
486:Polysaccharide sequencing
44:Sequence (disambiguation)
1035:Philippine Genome Center
199:chain termination method
1279:Molecular phylogenetics
775:China National GeneBank
509:structure determination
445:Methods for performing
345:whole genome sequencing
163:means to determine the
983:DNA Data Bank of Japan
771:DNA Data Bank of Japan
542:Full genome sequencing
335:Large-scale sequencing
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1264:Computational biology
779:Secondary databases:
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1347:Biochemistry methods
761:Sequence databases:
525:methylation analysis
482:carrying this gene.
449:sequencing include:
73:improve this article
1057:Whitehead Institute
845:Rat Genome Database
612:10.1038/nature06884
603:2008Natur.452..872W
562:MicroRNA sequencing
498:) can be used, and
429:MicroRNA Sequencing
1294:Sequence alignment
1001:Flatiron Institute
441:protein sequencing
435:Protein sequencing
404:expression profile
372:reverse transcribe
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597:(7189): 872–876.
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464:Mass spectrometry
454:Edman degradation
251:Sanger sequencing
245:Sanger sequencing
193:order of a given
165:primary structure
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36:sequence learning
16:(Redirected from
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62:This article
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1218:Nexus format
1213:NeXML format
1208:FASTQ format
1203:FASTA format
1191:File formats
953:Institutions
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547:Genetic code
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157:biochemistry
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88:"Sequencing"
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71:Please help
66:verification
63:
1198:CRAM format
1119:(CSIR-IGIB)
235:Moore's law
1341:Categories
1284:Sequencing
1248:GTF format
1243:GFF format
1238:VCF format
1228:SAM format
991:(EMBL-EBI)
917:SOAP suite
837:VectorBase
799:BioNumbers
785:Swiss-Prot
573:References
476:amino-acid
408:Eukaryotic
396:translated
380:small RNAs
322:base pairs
287:wavelength
214:recently.
191:nucleotide
169:biopolymer
161:sequencing
129:April 2008
99:newspapers
1111:(ISCB-SC)
1081:(EMBL-AR)
1014:(MPI-CBG)
755:Databases
621:0028-0836
412:co-linear
368:expressed
306:megabases
220:pathogens
1313:Category
1183:(RECOMB)
1133:Meetings
1087:(EMBnet)
937:Server:
912:SAMtools
907:PANGOLIN
870:Software
849:PHI-base
841:WormBase
811:InterPro
682:15040198
629:18421352
531:See also
519:include
400:proteins
384:in vitro
173:sequence
153:genetics
1325:Commons
1160:(InCoB)
1105:(ISCB)
1093:(INSDC)
1075:(ASBCB)
979:(DBCLS)
973:(COSBI)
887:Clustal
833:FlyBase
807:Ensembl
781:UniProt
763:GenBank
599:Bibcode
557:RNA-Seq
490:Though
447:protein
425:RNA-Seq
416:introns
314:enzymes
113:scholar
1166:(CIBB)
1154:(ISMB)
1148:(ECCB)
1125:(JSBi)
1031:(NBIC)
1020:(NCBI)
1008:(JCVI)
997:(EMBL)
985:(DDBJ)
939:ExPASy
922:TopHat
902:MUSCLE
892:EMBOSS
882:Bowtie
857:GISAID
817:, and
789:TrEMBL
680:
627:
619:
591:Nature
505:enzyme
500:bonded
115:
108:
101:
94:
86:
1177:(PSB)
1099:(ISB)
1048:(SIB)
1037:(PGC)
967:(CBD)
931:Other
897:HMMER
877:BLAST
706:Links
480:clone
423:see:
392:exons
310:EmPCR
120:JSTOR
106:books
859:and
825:BOLD
815:KEGG
791:and
773:and
678:PMID
625:PMID
617:ISSN
515:and
427:and
155:and
92:news
42:and
670:doi
607:doi
595:452
521:NMR
511:of
398:to
378:or
356:RNA
318:ATP
270:di-
195:DNA
151:In
75:by
1343::
1142:()
855:,
851:,
847:,
843:,
839:,
835:,
831:,
827:,
813:,
809:,
805:,
801:,
787:,
769:,
765:,
676:.
664:.
623:.
615:.
605:.
593:.
589:.
527:.
431:.
388:is
159:,
740:e
733:t
726:v
698:.
684:.
672::
666:1
631:.
609::
601::
142:)
136:(
131:)
127:(
117:·
110:·
103:·
96:·
69:.
46:.
20:)
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