Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap

Kalendar, R.; Ivanov, K.I.; Samuilova, O.; Kairov, U.; Zamyatnin, A.A.

Статья опубликована в высокорейтинговом журнале

Информация о цитировании статьи получена из Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 15 февраля 2024 г.

Авторы: Kalendar Ruslan, Ivanov Konstantin I., Samuilova Olga, Kairov Ulykbek, Zamyatnin Andrey A.
Журнал: Analytical Chemistry
Том: 95
Номер: 48
Год издания: 2023
Издательство: American Chemical Society
Местоположение издательства: United States
Первая страница: 17818
Последняя страница: 17825
DOI: 10.1021/acs.analchem.3c03894
Аннотация: 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.
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Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trapстатья

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