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DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 μL of KSOM mouse embryo culture medium. Our group extrac...

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Autores principales: Alias, Anand Baby, Huang, Hong-Yuan, Wang, Yi-Wen, Lin, Kai-Ti, Lu, Pei-Jhen, Wu, Tzu-Hui, Jiang, Pei-Shin, Chen, Chien-An, Yao, Da-Jeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9031944/
https://www.ncbi.nlm.nih.gov/pubmed/35457812
http://dx.doi.org/10.3390/mi13040507
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author Alias, Anand Baby
Huang, Hong-Yuan
Wang, Yi-Wen
Lin, Kai-Ti
Lu, Pei-Jhen
Wu, Tzu-Hui
Jiang, Pei-Shin
Chen, Chien-An
Yao, Da-Jeng
author_facet Alias, Anand Baby
Huang, Hong-Yuan
Wang, Yi-Wen
Lin, Kai-Ti
Lu, Pei-Jhen
Wu, Tzu-Hui
Jiang, Pei-Shin
Chen, Chien-An
Yao, Da-Jeng
author_sort Alias, Anand Baby
collection PubMed
description Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 μL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 μL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept.
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spelling pubmed-90319442022-04-23 DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform Alias, Anand Baby Huang, Hong-Yuan Wang, Yi-Wen Lin, Kai-Ti Lu, Pei-Jhen Wu, Tzu-Hui Jiang, Pei-Shin Chen, Chien-An Yao, Da-Jeng Micromachines (Basel) Article Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 μL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 μL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept. MDPI 2022-03-25 /pmc/articles/PMC9031944/ /pubmed/35457812 http://dx.doi.org/10.3390/mi13040507 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Alias, Anand Baby
Huang, Hong-Yuan
Wang, Yi-Wen
Lin, Kai-Ti
Lu, Pei-Jhen
Wu, Tzu-Hui
Jiang, Pei-Shin
Chen, Chien-An
Yao, Da-Jeng
DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title_full DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title_fullStr DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title_full_unstemmed DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title_short DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform
title_sort dna sequencing from subcritical concentration of cell-free dna extracted from electrowetting-on-dielectric platform
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9031944/
https://www.ncbi.nlm.nih.gov/pubmed/35457812
http://dx.doi.org/10.3390/mi13040507
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