Publicação
Microfluidic based on aqueous two-phase system for plasmidic DNA purification
| Resumo: | Through the last four decades, plasmids played a crucial role in the development of biotechnology. They are the workhorse of contemporary molecular biology being used for DNA manipulation, transfer, and gene expression in a variety of microorganisms and animal cells. As the next generation of biotechnology products (gene-based medicines and DNA vaccines) makes their way into clinical trials aspiring the pharmaceutical marketplace, the demand for high yield production and purification methods of pDNA, particularly supercoiled (sc) pDNA, has vastly increased. The approaches currently employed for pDNA purification are time consuming, laborintensive and expensive. Additionally, these are hampered by their low binding capacity. Aqueous two-phase system (ATPS) – a liquid-liquid extraction method that involves the transfer of solute between incompatible aqueous solutions – are an appealing alternative method for sc pDNA purification that reduces the number of unit operations of the expensive purification process flow presently employed, thus reducing its overall cost. Nevertheless, pDNA single-stage batch extractions in some cases does not meet sufficient purity requirements considering the product final application; and multi-stage extractions demands an increase in unit operations, time consumption, sample amount and handling, and a reduction on the amount of pDNA recovered. In this context, microfluidics-based platforms are an attractive alternative to standard batch processes currently used at the bench scale. These platforms demand smaller sample volumes, reduced reagent consumption, reduced production of potentially harmful byproducts, decreased analysis time, higher levels of throughput, automation and precise control, and have the potential of being low cost, disposable and portable. Hence, the present work aimed to perform the proof-of-concept concerning the purification of pDNA from E. coli lysates using a microfluidic based on PEG600– Ammonium Sulfate ATPS. The obtained results proved that pDNA purification using a microfluidic device was successfully achieved without evidence of RNA contamination. Also, the dimensional analysis of the relationships behind the physical phenomena explored in this study may be used to build a mathematical model and minimize the number of experiments for the microfluidic design parameters; ultimately leading to the optimization of pDNA microfluidic purification. |
|---|---|
| Autores principais: | Samy, Silvina Maria Ribeiro |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| _version_ | 1867439603143671808 |
|---|---|
| author | Samy, Silvina Maria Ribeiro |
| author_facet | Samy, Silvina Maria Ribeiro |
| author_role | author |
| contributor_name_str_mv | Marcos, João Carlos RepositóriUM - Universidade do Minho |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Samy, Silvina Maria Ribeiro\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Marcos, João Carlos RepositóriUM - Universidade do Minho |
| datacite.creators.creator.creatorName.fl_str_mv | Samy, Silvina Maria Ribeiro |
| datacite.date.Accepted.fl_str_mv | 2015-01-01T00:00:00Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| datacite.titles.title.fl_str_mv | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| dc.contributor.none.fl_str_mv | Marcos, João Carlos RepositóriUM - Universidade do Minho |
| dc.creator.none.fl_str_mv | Samy, Silvina Maria Ribeiro |
| dc.date.Accepted.fl_str_mv | 2015-01-01T00:00:00Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/1822/41108 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| dc.title.fl_str_mv | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_bdcc |
| description | Through the last four decades, plasmids played a crucial role in the development of biotechnology. They are the workhorse of contemporary molecular biology being used for DNA manipulation, transfer, and gene expression in a variety of microorganisms and animal cells. As the next generation of biotechnology products (gene-based medicines and DNA vaccines) makes their way into clinical trials aspiring the pharmaceutical marketplace, the demand for high yield production and purification methods of pDNA, particularly supercoiled (sc) pDNA, has vastly increased. The approaches currently employed for pDNA purification are time consuming, laborintensive and expensive. Additionally, these are hampered by their low binding capacity. Aqueous two-phase system (ATPS) – a liquid-liquid extraction method that involves the transfer of solute between incompatible aqueous solutions – are an appealing alternative method for sc pDNA purification that reduces the number of unit operations of the expensive purification process flow presently employed, thus reducing its overall cost. Nevertheless, pDNA single-stage batch extractions in some cases does not meet sufficient purity requirements considering the product final application; and multi-stage extractions demands an increase in unit operations, time consumption, sample amount and handling, and a reduction on the amount of pDNA recovered. In this context, microfluidics-based platforms are an attractive alternative to standard batch processes currently used at the bench scale. These platforms demand smaller sample volumes, reduced reagent consumption, reduced production of potentially harmful byproducts, decreased analysis time, higher levels of throughput, automation and precise control, and have the potential of being low cost, disposable and portable. Hence, the present work aimed to perform the proof-of-concept concerning the purification of pDNA from E. coli lysates using a microfluidic based on PEG600– Ammonium Sulfate ATPS. The obtained results proved that pDNA purification using a microfluidic device was successfully achieved without evidence of RNA contamination. Also, the dimensional analysis of the relationships behind the physical phenomena explored in this study may be used to build a mathematical model and minimize the number of experiments for the microfluidic design parameters; ultimately leading to the optimization of pDNA microfluidic purification. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | masterThesis |
| fulltext.url.fl_str_mv | https://repositorium.uminho.pt/bitstreams/fe2ef923-4ef9-4ebe-9631-9cbd477b6da1/download |
| id | rum_6bdf6f41104f059d56be0aa5d46b6c97 |
| identifier.url.fl_str_mv | https://hdl.handle.net/1822/41108 |
| instacron_str | repositorium |
| institution | Universidade do Minho |
| instname_str | Universidade do Minho |
| language | eng |
| network_acronym_str | rum |
| network_name_str | RepositóriUM - Universidade do Minho |
| oai_identifier_str | oai:repositorium.uminho.pt:1822/41108 |
| organization_str_mv | urn:organizationAcronym:repositorium |
| person_str_mv | Samy, Silvina Maria Ribeiro |
| publishDate | 2015 |
| reponame_str | RepositóriUM - Universidade do Minho |
| repository_id_str | urn:repositoryAcronym:rum |
| service_str_mv | urn:repositoryAcronym:rum |
| spelling | engporThrough the last four decades, plasmids played a crucial role in the development of biotechnology. They are the workhorse of contemporary molecular biology being used for DNA manipulation, transfer, and gene expression in a variety of microorganisms and animal cells. As the next generation of biotechnology products (gene-based medicines and DNA vaccines) makes their way into clinical trials aspiring the pharmaceutical marketplace, the demand for high yield production and purification methods of pDNA, particularly supercoiled (sc) pDNA, has vastly increased. The approaches currently employed for pDNA purification are time consuming, laborintensive and expensive. Additionally, these are hampered by their low binding capacity. Aqueous two-phase system (ATPS) – a liquid-liquid extraction method that involves the transfer of solute between incompatible aqueous solutions – are an appealing alternative method for sc pDNA purification that reduces the number of unit operations of the expensive purification process flow presently employed, thus reducing its overall cost. Nevertheless, pDNA single-stage batch extractions in some cases does not meet sufficient purity requirements considering the product final application; and multi-stage extractions demands an increase in unit operations, time consumption, sample amount and handling, and a reduction on the amount of pDNA recovered. In this context, microfluidics-based platforms are an attractive alternative to standard batch processes currently used at the bench scale. These platforms demand smaller sample volumes, reduced reagent consumption, reduced production of potentially harmful byproducts, decreased analysis time, higher levels of throughput, automation and precise control, and have the potential of being low cost, disposable and portable. Hence, the present work aimed to perform the proof-of-concept concerning the purification of pDNA from E. coli lysates using a microfluidic based on PEG600– Ammonium Sulfate ATPS. The obtained results proved that pDNA purification using a microfluidic device was successfully achieved without evidence of RNA contamination. Also, the dimensional analysis of the relationships behind the physical phenomena explored in this study may be used to build a mathematical model and minimize the number of experiments for the microfluidic design parameters; ultimately leading to the optimization of pDNA microfluidic purification.application/pdfporMicrofluidic based on aqueous two-phase system for plasmidic DNA purificationSamy, Silvina Maria RibeiroMarcos, João CarlosHostingInstitutionOrganizationalRepositóriUM - Universidade do Minhoe-mailmailto:repositorium@usdb.uminho.ptrepositorium@usdb.uminho.ptTID201258153201520152015-01-01T00:00:00ZHandlehttps://hdl.handle.net/1822/41108http://purl.org/coar/access_right/c_abf2open accesshttp://www.oecd.org/science/inno/38235147.pdfFields of Science and Technology (FOS)Ciências Naturais::Ciências Biológicas17587413 bytesliteraturehttp://purl.org/coar/resource_type/c_bdccmaster thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorium.uminho.pt/bitstreams/fe2ef923-4ef9-4ebe-9631-9cbd477b6da1/download |
| spellingShingle | Microfluidic based on aqueous two-phase system for plasmidic DNA purification Samy, Silvina Maria Ribeiro Ciências Naturais::Ciências Biológicas |
| status | SINGLETON |
| subject.other.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| title | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| title_full | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| title_fullStr | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| title_full_unstemmed | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| title_short | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| title_sort | Microfluidic based on aqueous two-phase system for plasmidic DNA purification |
| topic | Ciências Naturais::Ciências Biológicas |
| topic_facet | Ciências Naturais::Ciências Biológicas |
| url | https://hdl.handle.net/1822/41108 |
| visible | 1 |