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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_ligasa8/10/2019 Secuenciacin Por Ligacin
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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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