Selecting DNA aptamers for 17β-estradiol

Akki, Spurti Umesh

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https://hdl.handle.net/2142/42165 Copy

Description

Title

Selecting DNA aptamers for 17β-estradiol

Author(s)

Akki, Spurti Umesh

Issue Date

2013-02-03T19:18:02Z

Director of Research (if dissertation) or Advisor (if thesis)

Werth, Charles J.

Department of Study

Civil and Environmental Engineering

Discipline

Environmental Engineering in Civil Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• DNA aptamer
• estradiol

Abstract

17β-estradiol (E2) is a potent estrogen that has been widely documented in water resources. It falls under the category of endocrine disrupting compounds (EDCs), and hence has been included in the EPA’s Unregulated Contaminant Monitoring Rule (UCM3) that will require the monitoring of this compound. Thus, there is an increasing demand to sensitively and selectively detect E2. Conventional detection methods are time consuming and/or expensive. We explore a promising approach which employs the use of DNA aptamers for the detection of E2. DNA aptamers are single stranded DNA molecules which are capable of binding target molecules with high affinity and selectivity. The aim of this project is to develop DNA aptamers that would function as sensors for the detection of this endocrine disrupting steroid hormone. The first step performed was iterative in vitro selections to identify aptamers from a pool of ~ 1014 DNA molecules with the application of appropriate selection pressures. This was achieved by passing the pool over a selection column containing the target immobilized on sepharose beads, followed by eluting the sequences bound to the column with the free target. The resulting pool with the desired sequences was enriched by carrying out polymerase chain reaction (PCR). The potential aptamers obtained were cloned and sequenced, following which they were screened for binding activity using a bead binding assay. The dissociation constant for each aptamer was determined by a DMS chemical probing assay. These aptamers can be incorporated into various real-time in-line sensing platforms to generally and selectively monitor the presence of E2 in environmental samples. They represent an improvement over the one existing aptamer for E2, because some are more selective and marginally more sensitive.

Graduation Semester

2012-12

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http://hdl.handle.net/2142/42165

Copyright and License Information

Copyright 2012 Spurti Umesh Akki

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