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Covalent “Click Chemistry”-Based Attachment of DNA onto Solid Phase Enables Iterative Molecular Analysis

Author:
Lau, Billy T., Ji, Hanlee P.
Source:
Analytical chemistry 2019 v.91 no.3 pp. 1706-1710
ISSN:
1520-6882
Subject:
DNA, DNA libraries, agarose, exons, genetic analysis, mutation, neoplasms, reference standards
Abstract:
Molecular analysis of DNA samples with limited quantities can be challenging. Repeatedly sequencing the original DNA molecules from a given sample would overcome many issues related to accurate genetic analysis and mitigate issues with processing small amounts of DNA analyte. Moreover, an iterative, replicated analysis of the same DNA molecule has the potential to improve genetic characterization. Herein, we demonstrate that the use of “click”-based attachment of DNA sequencing libraries onto an agarose bead support enables repetitive primer extension assays for specific genomic DNA targets such as gene exons. We validated the performance of this assay for evaluating specific genetic alterations in both normal and cancer reference standard DNA samples. We demonstrate the stability of conjugated DNA libraries and related sequencing results over the course of independent serial assays spanning several months from the same set of samples. Finally, we finally applied this method to DNA derived from a tumor sample and demonstrated improved mutation detection accuracy.
Agid:
6300609