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

Detalhes bibliográficos
Autor(a) principal: Brinckmann Oliveira Netto, Alice
Data de Publicação: 2023
Outros Autores: Rojas Málaga, Diana, Kubaski, Francyne, Brusius Facchin, Ana Carolina, Giugliani, Roberto
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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spelling 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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