?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.title=Optimizing+DNA+isolation+from+recalcitrant+tree+root+material+for+successful+molecular+genetic+analysis&rft.creator=Blanquer+Valderas%2C+Laura&rft.contributor=Berrocal+Lobo%2C+Marta&rft.contributor=Fink%2C+Siegfried&rft.subject=Botany&rft.description=El+proyecto+tiene+como+objetivo+determinar+cu%C3%A1les+son+las+condiciones+m%C3%A1s+adecuadas+para+el+proceso+de+extracci%C3%B3n+de+ADN+de+material+le%C3%B1oso+procedente+de+ra%C3%ADces%2C+de+manera+que+se+realice+con+%C3%A9xito+la+PCR%2C+secuenciaci%C3%B3n+y+finalmente+identificaci%C3%B3n+de+las+especies+utilizadas.+El+estudio+se+centra+en+la+optimizaci%C3%B3n+del+procedimiento+llevado+a+cabo+en+el+laboratorio+de+Bot%C3%A1nica+Forestal+de+la+Universidad+de+Friburgo%2C+al+que+se+encuentra+asociado+un+servicio+de+identificaci%C3%B3n+de+especies+a+trav%C3%A9s+de+material+radical.+Por+lo+tanto%2C+se+busca+determinar+qu%C3%A9+tejidos+de+ra%C3%ADz+y+m%C3%A9todos+de+extracci%C3%B3n+proporcionan+los+mejores+resultados+en+t%C3%A9rminos+de+rendimiento+y+pureza+del+ADN+y+de+%C3%A9xito+en+la+PCR+y+en+la+identificaci%C3%B3n+final.+Para+el+estudio+4+pares+de+primers+fueron+utilizados+en+la+PCR+(ITS1-ITS4%2C+ITS5-ITS4%2C+trnL%2C+psbA-trnH)%2C+y+su+eficacia+fue+comparada.+Una+vez+obtenidas+las+muestras+de+ra%C3%ADces+le%C3%B1osas%2C+el+material+es+preparado+distinguiendo+dos+tejidos%3A+floema+(corteza)+y+xilema+(madera).+A+continuaci%C3%B3n%2C+se+realizan+extracciones+con+diferentes+m%C3%A9todos+basados+tanto+en+kits+comerciales+como+en+protocolos+tradicionales+CTAB%3B+distintas+modificaciones+son+realizadas+en+los+protocolos+buscando+maximizar+su+efectividad.+Tras+las+extracciones+se+recogen+los+datos+de+rendimiento+de+ADN+y+de+pureza+obtenida.+Seguidamente+se+realiza+la+PCR+con+los+extractos+obtenidos+y+se+determina+su+tasa+de+%C3%A9xito+mediante+una+electroforesis+en+gel+que+permite+identificar+el+tama%C3%B1o+de+los+fragmentos+logrados.+Por+%C3%BAltimo%2C+las+muestras+de+ADN+que+han+mostrado+una+suficiente+intensidad+de+bandas+en+el+gel%2C+son+limpiadas+y+secuenciadas.+Los+resultados+de+la+secuenciaci%C3%B3n+son+cotejados+con+los+contenidos+en+la+base+de+datos+online+GenBank+utilizando+el+algoritmo+de+alineamiento+BLAST+para+lograr+la+identificaci%C3%B3n+de+las+muestras.+El+estudio+analiza+paralelamente+otras+cuestiones+secundarias+como+el+efecto+de+sucesivos+ciclos+de+congelado+y+descongelado+en+la+concentraci%C3%B3n+de+ADN+y+la+relaci%C3%B3n+entre+la+cantidad+de+material+inicial+utilizado+y+el+rendimiento+de+ADN+de+la+muestra.+----------ABSTRACT----------+The+aim+of+the+present+study+is+to+define+the+most+suitable+conditions+during+the+process+of+DNA+extraction+from+plant+woody+root+material%2C+to+obtain+a+successful+PCR%2C+sequencing+and+finally+identification+of+the+species+used.+The+study+focuses+on+the+optimization+of+the+procedure+carried+out+in+the+laboratory+of+Forest+Botany+of+the+University+of+Freiburg%2C+which+offers+a+service+of+tree+identification+through+root+samples.+The+goal+is+to+determine+which+tissues+and+extraction+methods+provide+the+best+results+in+terms+of+DNA+yield+and+purity%2C+PCR+and+identification+success.+4+pairs+of+primers+were+used+for+the+PCR+(ITS1-+ITS4%2C+ITS5-ITS4%2C+trnL%2C+psbA-trnH)+and+their+efficiency+was+compared.+After+collection+of+the+root+samples%2C+the+material+is+prepares+distinguishing+two+tissues%3A+phloem+(bark)+and+xylem+(wood).+Then%2C+DNA+extractions+with+different+commercial+kits+and+traditional+CTAB+methods+are+performed%3B+different+modifications+are+conducted+searching+for+an+improvement+in+the+effectiveness.+After+the+extractions+results+of+DNA+yield+and+purity+are+recorded.+Subsequently%2C+PCR+is+carried+out+with+the+extracts+obtained+and+the+rate+of+success+is+determined+by+means+of+a+gel+electrophoresis+that+shows+the+size+of+the+DNA+fragments.+Finally%2C+the+DNA+samples+which+showed+enough+band+intensity+are+cleaned+up+and+sequenced.+Sequencing+results+are+compared+with+the+data+from+the+GenBank+online+dataset%2C+using+the+algorithm+BLAST+to+achieve+the+correct+identification.+At+the+same+time%2C+secondary+questions+such+as+the+effect+of+repeated+cycles+of+freezethawing+on+DNA+concentration+and+the+relation+between+amount+of+initial+sample+and+DNA+yield+are+analysed.&rft.publisher=E.T.S.I.+Montes+(UPM)&rft.rights=https%3A%2F%2Fcreativecommons.org%2Flicenses%2Fby-nc-nd%2F3.0%2Fes%2F&rft.date=2016-05&rft.type=info%3Aeu-repo%2Fsemantics%2FbachelorThesis&rft.type=Final+Project&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=eng&rft.rights=info%3Aeu-repo%2Fsemantics%2FopenAccess&rft.identifier=https%3A%2F%2Foa.upm.es%2F52985%2F