Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Clinical and Biomedical Research |
Texto Completo: | https://seer.ufrgs.br/index.php/hcpa/article/view/114246 |
Resumo: | Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting.Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS).Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample. |
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Clinical and Biomedical Research |
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Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disordersStandardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disordersDNA extractiondried blood spotsmolecular genetics diagnosisnewborn screeningtarget next-generation sequencingMolecular BiologyDNA extractiondried blood spotsmolecular genetic testingnewborn screeningtargeted next-generation sequencingIntroduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting.Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS).Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample.Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting. Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS). Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood. Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample.HCPA/FAMED/UFRGS2023-01-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionPeer-reviewed ArticleAvaliado por Paresapplication/pdfhttps://seer.ufrgs.br/index.php/hcpa/article/view/114246Clinical & Biomedical Research; Vol. 42 No. 3 (2022): Clinical & Biomedical ResearchClinical and Biomedical Research; v. 42 n. 3 (2022): Clinical and Biomedical Research2357-9730reponame:Clinical and Biomedical Researchinstname:Universidade Federal do Rio Grande do Sul (UFRGS)instacron:UFRGSporhttps://seer.ufrgs.br/index.php/hcpa/article/view/114246/87693Copyright (c) 2022 Clinical and Biomedical Researchhttp://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessBrinckmann Oliveira Netto, AliceRojas Málaga, DianaKubaski, FrancyneBrusius Facchin, Ana CarolinaGiugliani, Roberto2024-01-19T14:12:21Zoai:seer.ufrgs.br:article/114246Revistahttps://www.seer.ufrgs.br/index.php/hcpaPUBhttps://seer.ufrgs.br/index.php/hcpa/oai||cbr@hcpa.edu.br2357-97302357-9730opendoar:2024-01-19T14:12:21Clinical and Biomedical Research - Universidade Federal do Rio Grande do Sul (UFRGS)false |
dc.title.none.fl_str_mv |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
title |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
spellingShingle |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders Brinckmann Oliveira Netto, Alice DNA extraction dried blood spots molecular genetics diagnosis newborn screening target next-generation sequencing Molecular Biology DNA extraction dried blood spots molecular genetic testing newborn screening targeted next-generation sequencing |
title_short |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
title_full |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
title_fullStr |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
title_full_unstemmed |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
title_sort |
Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders |
author |
Brinckmann Oliveira Netto, Alice |
author_facet |
Brinckmann Oliveira Netto, Alice Rojas Málaga, Diana Kubaski, Francyne Brusius Facchin, Ana Carolina Giugliani, Roberto |
author_role |
author |
author2 |
Rojas Málaga, Diana Kubaski, Francyne Brusius Facchin, Ana Carolina Giugliani, Roberto |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Brinckmann Oliveira Netto, Alice Rojas Málaga, Diana Kubaski, Francyne Brusius Facchin, Ana Carolina Giugliani, Roberto |
dc.subject.por.fl_str_mv |
DNA extraction dried blood spots molecular genetics diagnosis newborn screening target next-generation sequencing Molecular Biology DNA extraction dried blood spots molecular genetic testing newborn screening targeted next-generation sequencing |
topic |
DNA extraction dried blood spots molecular genetics diagnosis newborn screening target next-generation sequencing Molecular Biology DNA extraction dried blood spots molecular genetic testing newborn screening targeted next-generation sequencing |
description |
Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting.Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS).Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-01-06 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Peer-reviewed Article Avaliado por Pares |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://seer.ufrgs.br/index.php/hcpa/article/view/114246 |
url |
https://seer.ufrgs.br/index.php/hcpa/article/view/114246 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
https://seer.ufrgs.br/index.php/hcpa/article/view/114246/87693 |
dc.rights.driver.fl_str_mv |
Copyright (c) 2022 Clinical and Biomedical Research http://creativecommons.org/licenses/by/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2022 Clinical and Biomedical Research http://creativecommons.org/licenses/by/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
HCPA/FAMED/UFRGS |
publisher.none.fl_str_mv |
HCPA/FAMED/UFRGS |
dc.source.none.fl_str_mv |
Clinical & Biomedical Research; Vol. 42 No. 3 (2022): Clinical & Biomedical Research Clinical and Biomedical Research; v. 42 n. 3 (2022): Clinical and Biomedical Research 2357-9730 reponame:Clinical and Biomedical Research instname:Universidade Federal do Rio Grande do Sul (UFRGS) instacron:UFRGS |
instname_str |
Universidade Federal do Rio Grande do Sul (UFRGS) |
instacron_str |
UFRGS |
institution |
UFRGS |
reponame_str |
Clinical and Biomedical Research |
collection |
Clinical and Biomedical Research |
repository.name.fl_str_mv |
Clinical and Biomedical Research - Universidade Federal do Rio Grande do Sul (UFRGS) |
repository.mail.fl_str_mv |
||cbr@hcpa.edu.br |
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1799767056279666688 |