A simple method for isolation of PCR fragments from silver-stained polyacrylamide gels by scratching with a fine needle

Abstract

▼PCR-based DNA and RNA fingerprinting techniques, including arbitrarily primed PCR (AP-PCR) and differential display reverse transcriptase PCR (ddRT-PCR), represent comprehensive genetic screening methods (Ref. 1). Recently, sensitive nonradioactive detection techniques have been introduced for visualization of fingerprints using silver staining of the PCR products after polyacrylamide gel electrophoresis (Ref. 2, 3). Characterization of polymorphic PCR fragments depends on prior isolation of the corresponding bands from stained and dried gels. In most protocols, PCR products of interest have been cut out of the dried gel complete with the paper support, and DNA eluted by boiling in elution buffer (Ref. 4). Reamplification of the eluted PCR fragments has been successful in up to 75% of the experiments. However, additional bands neighbouring the fragment of interest were frequently coamplified. To avoid coamplification of contaminating bands, the whole procedure of cutting, boiling and reamplification was repeated up to five times by some protocols (Ref. 5). Here we demonstrate simple and rapid reamplification of PCR products after isolation by gently scratching over the silver-stained band using a sterile disposable needle [Microlance 3 (Becton-Dickinson; 0.7 mm)] prewetted with PCR mastermix, without destroying the gel. After scratching the band from the gel, the needle is placed into 20 μl standard PCR reaction mix for 1 min and then discarded. The scratched PCR products are reamplified by 30 PCR cycles (Fig. 1). Using this scratching technique, we are able to reamplify PCR products of interest in more than 90% of the experiments. In addition to the simplicity and speed of