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Old 11-23-2011, 05:27 AM   #1
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Default PubMed: A Highly Parallel Single Molecule Amplification Approach Based on Agarose Dro

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A Highly Parallel Single Molecule Amplification Approach Based on Agarose Droplet PCR for Efficient and Cost-effective Aptamer Selection.

Anal Chem. 2011 Nov 21;

Authors: Zhang W, Zhang W, Liu Z, Li C, Zhu Z, Yang CJ

Abstract
We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single molecule emulsion PCR based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library against cancer biomarker Shp2 protein which evolved by conventional SELEX was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high throughput fluorescence cytometry. DNA clones with high binding capacity and low Kd were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a Kd of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing, expensive and time consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient, cost-effective for molecular evolution approaches and will find its wide application in molecular evolution technologies including mRNA display, phage display and so on.


PMID: 22103644 [PubMed - as supplied by publisher]



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