Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap
Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA's electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA's negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2023 |
|---|---|
| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:95 |
|---|---|
| Enthalten in: |
Analytical chemistry - 95(2023), 48 vom: 05. Dez., Seite 17818-17825 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Kalendar, Ruslan [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
451W47IQ8X |
|---|
| Anmerkungen: |
Date Completed 06.12.2023 Date Revised 15.12.2023 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1021/acs.analchem.3c03894 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM364852372 |
|---|
| LEADER | 01000caa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM364852372 | ||
| 003 | DE-627 | ||
| 005 | 20231227132043.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231226s2023 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1021/acs.analchem.3c03894 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1226.xml |
| 035 | |a (DE-627)NLM364852372 | ||
| 035 | |a (NLM)37993972 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Kalendar, Ruslan |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap |
| 264 | 1 | |c 2023 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 06.12.2023 | ||
| 500 | |a Date Revised 15.12.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA's electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA's negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a DNA |2 NLM | |
| 650 | 7 | |a 9007-49-2 |2 NLM | |
| 650 | 7 | |a Sodium Chloride |2 NLM | |
| 650 | 7 | |a 451W47IQ8X |2 NLM | |
| 700 | 1 | |a Ivanov, Konstantin I |e verfasserin |4 aut | |
| 700 | 1 | |a Samuilova, Olga |e verfasserin |4 aut | |
| 700 | 1 | |a Kairov, Ulykbek |e verfasserin |4 aut | |
| 700 | 1 | |a Zamyatnin, Andrey A |c Jr |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Analytical chemistry |d 1965 |g 95(2023), 48 vom: 05. Dez., Seite 17818-17825 |w (DE-627)NLM000025771 |x 1520-6882 |7 nnns |
| 773 | 1 | 8 | |g volume:95 |g year:2023 |g number:48 |g day:05 |g month:12 |g pages:17818-17825 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1021/acs.analchem.3c03894 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 95 |j 2023 |e 48 |b 05 |c 12 |h 17818-17825 | ||