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Clinical Chemistry 55: 1686-1693, 2009. First published July 9, 2009; 10.1373/clinchem.2009.127266
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(Clinical Chemistry. 2009;55:1686-1693.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Aptamer-Based Regionally Protected PCR for Protein Detection

Jun Sheng Lin1,a and Kenneth P. McNatty2

1 Centre for Reproduction and Genomics, AgResearch, Invermay, Mosgiel, New Zealand; 2 School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

aAddress correspondence to this author at: Centre for Reproduction and Genomics, AgResearch, Invermay, Private Bag 50034, Mosgiel 9053, New Zealand. Fax +64-3-4893739; e-mail jun.lin{at}agresearch.co.nz.

Background: DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins.

Methods: Ovine follicle-stimulating hormone {alpha} subunit (oFSH{alpha}) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as "aptamer-based regionally protected PCR" (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR.

Results: Aptamers against oFSH{alpha} were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer’s secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSH{alpha} by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10–14 mol/L.

Conclusions: Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .






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Copyright © 2009 by the American Association for Clinical Chemistry.