Publication
Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. leaves
| dc.contributor.author | Sá, Olga | |
| dc.contributor.author | Pereira, J.A. | |
| dc.contributor.author | Baptista, Paula | |
| dc.date.accessioned | 2014-09-22T09:53:07Z | |
| dc.date.available | 2014-09-22T09:53:07Z | |
| dc.date.issued | 2011 | |
| dc.description.abstract | Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR. | por |
| dc.identifier.citation | Sá, Olga; Pereira, J.A.; Baptista, Paula (2011). Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. leaves. International Journal of Molecular Sciences. ISSN 1422-0067. 12:6, p. 4156-4164 | por |
| dc.identifier.doi | 10.3390/ijms12064156 | |
| dc.identifier.issn | 1422-0067 | |
| dc.identifier.uri | http://hdl.handle.net/10198/10515 | |
| dc.language.iso | eng | por |
| dc.peerreviewed | yes | por |
| dc.subject | Arbutus unedo L. | por |
| dc.subject | Strawberry tree | por |
| dc.subject | DNA isolation | por |
| dc.subject | RAPD | por |
| dc.subject | ISSR | por |
| dc.title | Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. leaves | por |
| dc.type | journal article | |
| dspace.entity.type | Publication | |
| oaire.citation.endPage | 4164 | por |
| oaire.citation.issue | 6 | por |
| oaire.citation.startPage | 4156 | por |
| oaire.citation.title | International Journal of Molecular Sciences | por |
| oaire.citation.volume | 12 | por |
| person.familyName | Pereira | |
| person.familyName | Baptista | |
| person.givenName | José Alberto | |
| person.givenName | Paula | |
| person.identifier.ciencia-id | 611F-80B2-A7C1 | |
| person.identifier.ciencia-id | 7D11-FE1E-CD0F | |
| person.identifier.orcid | 0000-0002-2260-0600 | |
| person.identifier.orcid | 0000-0001-6331-3731 | |
| person.identifier.rid | L-6798-2014 | |
| person.identifier.scopus-author-id | 57204366348 | |
| person.identifier.scopus-author-id | 14051688000 | |
| rcaap.rights | restrictedAccess | por |
| rcaap.type | article | por |
| relation.isAuthorOfPublication | 7932162e-a2da-4913-b00d-17babbe51857 | |
| relation.isAuthorOfPublication | 3f35226a-b17a-4f7d-8da1-3297105cbfe9 | |
| relation.isAuthorOfPublication.latestForDiscovery | 7932162e-a2da-4913-b00d-17babbe51857 |