Secuenciación do ADN: Diferenzas entre revisións

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== Métodos en desenvolvemento ==
DNAActualmente sequencinghai methodsvarios currentlymétodos underde developmentsecuenciación includedo labelingADN theque DNAestán en desenvolvemento, como o da ADN polimerase polymeraseetiquetada,<ref>{{cite web|url=http://visigenbio.com/technology_overview.html|title=VisiGen Biotechnologies Inc. – Technology Overview |publisher=Visigenbio.com |date= |accessdate=2009-11-15}}</ref> readinglectura thede sequencesecuencias ascomo atránsitos DNAde strandcadeas transitsde throughADN a través de [[nanoporesecuenciación por sequencingnanoporos|nanoporesnanoporos]],<ref>{{cite web|url=http://mcb.harvard.edu/branton/index.htm |title=The Harvard Nanopore Group |publisher=Mcb.harvard.edu|date= |accessdate=2009-11-15}}</ref><ref name="Physorg">{{cite web |url=http://www.physorg.com/news157378086.html |title=Nanopore Sequencing Could Slash DNA Analysis Costs |work= |accessdate=}}</ref> ande microscopy-basedtécnicas techniquesbaseadas en microscopía, suchcomo as que usan o [[Atomicmicroscopio forcede microscope|atomic forceforza microscopyatómica]] orou o [[Transmissionmicroscopio electronelectrónico microscopyde DNA sequencing|transmission electron microscopytransmisión]], thatque arese usedusan topara identifyidentificar theas positionsposicións ofde individualcada nucleotidesnucleótido withinen longfragmentos DNAde ADN fragmentslongos (&gt;5,.000 [[par de bases|bp]]) bypor nucleotidemedio labelingdun withetiquetado heavierdos elementsnucleótidos con elementos máis pesados (e.g.por exemplo, halogens[[halóxeno]]s) forpara visuala detectiondetección andvisual e recordinggravación.<ref> US patent 20060029957, ZS Genetics, "Systems and methods of analyzing nucleic acid polymers and related components", issued 2005-07-14</ref><ref>{{cite journal |author=Xu M, Fujita D, Hanagata N |title=Perspectives and challenges of emerging single-molecule DNA sequencing technologies|journal=Small |volume=5 |issue=23 |pages=2638–49 |year=2009 |month=December |pmid=19904762 |doi=10.1002/smll.200900976 |url=}}</ref>
ThirdAs generationtecnoloxías technologiesde aimterceira toxeración increasepretenden throughputincrementar ando decreaserendemento thee timediminuír too resulttempo andpara costobter byos eliminatingresultados thee needo forcusto excessiveao reagentseliminar anda harnessingnecesidade thede processivityusar ofexcesivos DNAreactivos polymerasee aprovitar a procesividade da [[ADN polimerase]].<ref>{{cite journal|last=Schadt|first=E.E.|coauthors=S. Turner, A. Kasarskis|title=A window into third-generation sequencing|journal=Human Molecular Genetics|year=2010|volume=19|issue=R2|pages=R227–40|doi=10.1093/hmg/ddq416|pmid=20858600}}</ref>
 
=== NanoporeSecuenciación DNAde sequencingADN por nanoporos ===
{{main|Nanopore sequencing}}
This method is based on the readout of electrical signals occurring at nucleotides passing by alpha-[[hemolysin]] pores covalently bound with [[cyclodextrin]]. The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. The method has a potential of development as it does not require modified nucleotides, however single nucleotide resolution is not yet available.<ref>{{cite journal|last=Stoddart|first=D|coauthors=Heron, AJ; Mikhailova, E; Maglia, G; Bayley, H|title=Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=2009 May 12|volume=106|issue=19|pages=7702–7|pmid=19380741|doi=10.1073/pnas.0901054106|pmc=2683137}}</ref>
 
ThisO methodmétodo isde based[[secuenciación onde theADN readoutpor ofnanoporos]] electricalestá signalsbaseado occurringna atlectura nucleotidesde passingsinais byeléctricos alphaque se producen ao paseren os nucleótidos por poros de alfa-[[hemolysinhemolisina]] pores covalentlyunida boundcovalentemente withcon [[cyclodextrinciclodextrina]]. TheO DNApaso passingdo throughADN thea nanoporetravés changesdo itsnanoporo ioncambia current.a Thissúa changecorrente isde dependentións. onEste thecambio shapeé dependente da forma, sizetamaño ande lengthlonxitude ofda the[[secuencia DNAde sequenceADN]]. EachCada typetipo ofde thenucleótido nucleotidebloquea blockso thefluxo ionde flowións througha thetravés poredo forporo adurante differentun periodperíodo ofde timetempo diferente. TheO methodmétodo hasten aun potentialgrande ofpotencial developmentde asdesenvolvemento, itxa doesque notnon requireprecisa modifiednucleótidos nucleotidesmodificados, howeverpero aínda non se singlepode nucleotideobter resolutionunha isresolución notdun yet availablenucleótido.<ref>{{cite journal|last=Stoddart|first=D|coauthors=Heron, AJ; Mikhailova, E; Maglia, G; Bayley, H|title=Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=2009 May 12|volume=106|issue=19|pages=7702–7|pmid=19380741|doi=10.1073/pnas.0901054106|pmc=2683137}}</ref>
=== Sequencing by hybridization ===
 
''[[Sequencing by hybridization]]'' is a non-enzymatic method that uses a [[DNA microarray]]. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced.<ref>{{cite journal |author=Hanna GJ |title=Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase |journal=J. Clin. Microbiol. |volume=38 |issue=7 |pages=2715–21 |date=1 July 2000|pmid=10878069 |pmc=87006 |url=http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=10878069 |author-separator=, |author2=Johnson VA |author3=Kuritzkes DR |display-authors=3 |last4=Richman |first4=DD |last5=Martinez-Picado |first5=J |last6=Sutton |first6=L |last7=Hazelwood |first7=JD |last8=d'Aquila |first8=RT }}</ref>
=== Sequenciación por hibridación ===
A ''[[Sequencingsecuenciación bypor hybridizationhibridación]]'' isé aun método non-enzymatic methodencimático thatque usesusa aunha [[DNAmicromatriz de microarrayADN]]. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced.<ref>{{cite journal |author=Hanna GJ |title=Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase |journal=J. Clin. Microbiol. |volume=38 |issue=7 |pages=2715–21 |date=1 July 2000|pmid=10878069 |pmc=87006 |url=http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=10878069 |author-separator=, |author2=Johnson VA |author3=Kuritzkes DR |display-authors=3 |last4=Richman |first4=DD |last5=Martinez-Picado |first5=J |last6=Sutton |first6=L |last7=Hazelwood |first7=JD |last8=d'Aquila |first8=RT }}</ref>
 
=== Sequencing with mass spectrometry ===