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

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.