Slide PCR: DNA amplification from cell samples on microscopic glass slides

The polymerase chain reaction (PCR) has been used to amplify sequences in polypropylene tubes from a wide range of template material. We have developed a method for amplifying DNA from cultured cells and smears directly on microscopic glass slides ('slide PCR'). Cells were either allowed to adhere as a monolayer or smeared onto silanised microscope slides (1), then fixed in methanol/acetic acid 3:1 (v/v), Carnoy's solution, absolute alcohol or 4% buffered paraformaldehyde for 5—15 min. These slides were rinsed in distilled water, dried, and then either used fresh or stored at -20°C until use. A 20x38 mm area on the slide was circumscribed using an immunohistochemistry barrier pen (Dako). 30 y\ of PCR mix comprising 10 mM Tris pH 8.3, 50 mM KC1, 1.5 mM MgCl2,200 ^M dNTP's, 100 nM primers, 0.01% (w/v) gelatin (Sigma), 0.2% (w/v) bovine serum albumin (BSA) and 2.5 U/100 /tl Taq polymerase (Cetus) was pipetted onto the slide. A 22 X40 mm glass cover slip was then placed over the rectangular area of the slide and its margins were sealed with mineral oil (Sigma). The slides, placed on the metal block of a thermal cycler (e.g. Koch-Light) such that there was maximum contact between the block and sample, were subjected to 30—40 rounds of amplification with parameters identical to those required for PCR in propylene tubes. Denaturation temperatures for later cycles could be lowered for short products (2). After the reaction, the cooled slides were then gently dipped into chloroform to remove most of the mineral oil, but without dislodging the cover slip. One corner of the rectangular cover slip was then carefully raised with a sharp pair of forceps and the PCR mix, which collected as a meniscus at the opposite corner, was retrieved with a pipette. Typically, at least 25 /xl could be collected; this solution was run on agarose gels or reamplified with nested primers,as for standard tube PCR reactions. 0.1 — 1% BSA is necessary for slide PCR. In its absence, amplification did not occur with cell samples on slides or with extracted DNA pipetted directly and dried on plain slides. Precoating slides with BSA allowed amplification though at lower efficiency. Gelatin (at least 0.001%) was also required for amplifying targets of about 1 kb, but did not affect the yield of smaller products. Slide PCR was successful with the different sample preparation and fixation methods discussed.