secuenciación por ligación

8/10/2019 Secuenciacin Por Ligacin

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Secuenciacin por Ligacin

Es un mtodo enzimtico de secuenciacin que

emplea una DNA ligasaen lugar de una

polimerasa para identificar la secuencia

objetivo.

Mtodo SOLiD.
http://es.wikipedia.org/wiki/ADN_ligasahttp://es.wikipedia.org/wiki/ADN_ligasa


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Metodologia:

Library Preparation

1.Prepare one of the two types of libraries

(Figure 1) for SOLiD System sequencing-

fragment or mate-paired.


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Emulsion PCR/Bead Enrichment

2.Prepare clonal bead populations (Figure 2).3.After PCR, denature the templates and

perform bead enrichment to separate beads

with extended templates from undesired

beads.


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Bead Deposition

4.Deposit 3 modified beads onto a glass slide

(Figure 3). During bead loading, deposition

chambers enable you to segment a slide into

one, four, or eight sections.


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Sequencing by Ligation

5.Primers hybridize to theP1 adapter sequence onthe templated beads(Figure 4).

6.A set of fourfluorescently labeled di-base probes compete forligation to the sequencing

primer.

7.Multiple cycles ofligation, with the numberof cycles determining theeventual read length.

8.Following a series ofligation cycles, theextension product isremoved and thetemplate is reset with a

primer complementary tothe n-1 position for asecond round of ligationcycles.


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Primer Reset

9.Five rounds of primer reset are completed

for each sequence tag (Figure 5). Through the

primer reset process, virtually every base is

interrogated in two independent ligation

reactions by two different primers.


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Exact Call Chemistry

10.Up to 99.99% accuracy is achieved with the

Exact Call Chemistry Module by sequencing

with an additional primer using a multi-base

encoding scheme.


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Secuenciacin por Sntesis

Esta tcnica fue desarrollada en la Universidad

de Cambridge por Shankar Balasubramanian

and David Klenerman.

Se basa en un mtodo similar al SANGER .


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Los fragmentos a secuenciar son preparados conadaptadores en los extremos, para permitir que

sus cadenas desnaturalizadas puedan unirse auna superficie slida y as poder ser enriquecidaspor medio de una amplificacin tipo puente,generando clster de cadenas del mismo

fragmento, con el fin de amplificar la sealgenerada por los nucletidos fluorescentesincorporados a la cadena en crecimiento y aspuedan ser detectados por una cmara, esto

durante un proceso cclico que permite alargar laslecturas dependiendo de la plataformaseleccionada.


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Referencias:

http://langebio.cinvestav.mx/labsergen/

http://nxseq.bitesizebio.com/articles/sequencing-by-synthesis-explaining-the-illumina-sequencing-technology/

http://www.appliedbiosystems.com/absite/us/en/home/ap

plications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http%3A%2F%2Fwww3.appliedbiosystems.com%2FAB_Home%2Fapplicationstechnologies%2FSOLiDSystemSequencing%2FOverviewofSOLiDSequencingChemistry%2F&ei=L5BRUo64K4vS9QT-

j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWU
http://langebio.cinvestav.mx/labsergen/http://nxseq.bitesizebio.com/articles/sequencing-by-synthesis-explaining-the-illumina-sequencing-technology/http://nxseq.bitesizebio.com/articles/sequencing-by-synthesis-explaining-the-illumina-sequencing-technology/http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-sequencing-chemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CF0QFjAI&url=http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/&ei=L5BRUo64K4vS9QT-j4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&sig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWUhttp://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing/next-generation-systems/solid-seque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