Item Details

abstract

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content.

subject

Separations

Instrumentation

Capillary electrophoresis

Electrophoresis

Next-generation sequencing

Bioanalytical methods

contributor

Riley, Kathryn R (author)

Gagliano, Jason (author)

Xiao, Jiajie (author)

Libby, Kara (author)

Saito, Shingo (author)

Yu, Guo (author)

Cubiccioti, Roger (author)

date

2020-02-03T20:05:58Z (accessioned)

2020-02-03T20:05:58Z (available)

1/13/15 (issued)

identifier

Riley, Kathryn R., Jason Gagliano, Jiajie Xiao, Kara Libby, Shingo Saito, Guo Yu, Roger Cubicciotti et al. "Combining capillary electrophoresis and next-generation sequencing for aptamer selection." Analytical and bioanalytical chemistry 407, no. 6 (2015): 1527-1532. (citation)

https://doi.org/10.1007/s00216-014-8427-y (doi)

http://hdl.handle.net/10339/95965 (uri)

language

en_US (iso)

publisher

Springer

rights

https://creativecommons.org/licenses/by/4.0/ (uri)

source

Analytical and Bioanalytical Chemistry

title

Combining capillary electrophoresis and next-generation sequencing for aptamer selection

type

Article