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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9031944 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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