Optimization of Emulsion PCR for Aptamer Selection

Cancers are known to produce unique biomolecules or biomarkers that can be detected using diagnostic tests. CA-125 and HE4 are two biomarkers used in clinical tests to detect ovarian cancer, which responds favorably to treatment when diagnosed at its earliest stage. These tests however, often produce both false positives, identifying cancer when it is not present, and false negatives, failing to detect biomarkers that are present. A diagnostic test based on recognition by nucleic acid aptamers may help improve detection of these biomarkers. SELEX is one method used to select aptamers, short oligonucleotides, (usually RNA or single stranded DNA) that bind to a target with high affinity and specificity. The selection process requires PCR to amplify the aptamers. At high cycle numbers, the use of PCR with aptamers is problematic due to byproduct formation and low yields of double stranded DNA (dsDNA). Emulsion PCR (ePCR) has been found to reduce byproducts at high cycle numbers through the use of an oil and water mixture. The emulsion reduces the aqueous phase to tiny droplets, creating individual reaction chambers which produce dsDNA with reduced byproduct. ePCR using a EurX kit has reliably produced dsDNA with less byproduct compared to conventional PCR. Successful optimization of ePCR will be used in the next SELEX selection of HE4