Polymerase Chain Reaction as an Amplification Method in Aptamer-Based Assays

5 POLYMERASE CHAIN REACTION AS AN AMPLIFICATION METHOD IN APTAMER-BASED ASSAYS 

Methods for the detection of specific nucleic acid sequences are much more sensitive with respect to the detection of proteins, since polymerase chain reaction (PCR) represents a very powerful amplification tool for nucleic acids. The fact that aptamers are made of DNA or RNA offers the opportunity to combine the detection of proteins with the amplification potentialities that until some years ago were restricted to the detection of nucleic acids. PCR has been used alone or combined with other molecular biology techniques for nucleic acid manipulation (proximity ligation or exonuclease protection) to develop extremely sensitive assays for the detection of a variety of proteins. 

A very simple and sensitive method has recently been published based on direct amplification of an aptamer specific for the reverse transcriptase of the human immunodeficiency virus type 1 (HlV-1 RTase) (Zhang et ah, 2006). After incubation of the protein with the aptamer, the complex formed is separated from the unbound aptamer by capillary electrophoresis. After collection of the aptamer-target complex, the aptamer was dissociated from the target and directly amplified by PCR. Approximately 10 molecules of target were detected using this method, improving the sensitivity of protein detection by several orders of magnitude. 

APTAMER-BASED BIOANALYTICAL ASSAYS AMPLIFICATION STRATEGIES 

In solid-phase proximity ligation assay, low-femtomolar concentrations of the protein were detected using the same pair of proximity probes as those used for the homogeneous assay. This demonstrated the high sensitivity of the detection method compared with the low-picomolar detection threshold of the ELISA for the same protein. 

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