PCR has revolutionized the scientist ’s ability to detect and quantify nucleic acid sequences for research and diagnostic purposes. However, the process is slowed in that PCR methods usually require a purification step prior to template amplification. This involves harvesting cells and/or solubilizing the nucleic acids, followed by separating the nucleic acids from lysis reagents using chromatography resins. Though these purification methods have evolved to be rapid, they still require time and materials that can be costly in a high-throughput environment. SPEX SamplePrep has applied its Geno/Grinder technology to a rapid and low-cost method for the preparation of cultured cells for PCR analysis of genomic DNA. This approach circumvents the need for purification methods using chromatography resins or columns by simply homogenizing the cells and performing the PCR analysis on the lysate. This approach has been developed for micro-well plates and is therefore amenable to high-throughput operations. The process is relatively simple and involves mixing cells, 200 µ zirconium beads (catalog #2180), and molecular biology grade water in a standard 96-well tissue culture plate, sealing the plate with film (catalog #2500 and 2501), and then grinding on a Geno/Grinder (catalog #2000). The entire process, which can be used on both adherent and non-adherent cells, can be performed in 10 to 15 minutes. The resulting lysate is used directly for PCR. Cultured cells mixed with water typically swell and rupture.This destroys the cell, but often the nucleus remains intact. The homogenization with the zirconium beads effectively destroys the nucleus, fragmenting the chromosomes in the process. These smaller DNA molecules are more readily amplified over the larger native molecules. Using this procedure, a DNA sequence from as few as 20 cells has been successfully detected.

Procedure for Adherent Cells

Cells are homogenized in standard 96-well cell culture plates at concentrations as low as 100 cells/well. Freshly plated cells are allowed to adhere before homogenization, while cultured cells can be lysed directly in the plate. For homogenization, the medium is removed and the cells are washed twice with 100 µl of phosphate buffered saline. The PBS is decanted and 150 mg of RNase-free, DNase-free treated Zirconium beads (200 µm, catalog #2180) are added to the wells using a commercial resin dispenser. Sterile molecular biology water (100 µ is added to the wells.The plate is sealed with either a press-on mat or, preferably, a heat-sealed film (catalog #2500 and 2501). Glue-based sealing tapes should not be used, as the beads will stick to the glue. The sealed plate is placed in the Geno/Grinder (catalog #2000) and run for 5 min. at 1200 rpm. The Geno/Grinder can run from one to eight plates in this procedure. Following homogenization, 1 to 20 µl of the lysate can be added directly to a PCR cocktail and amplified.

Procedure for Non-Adherent Cells

Homogenization of non-adherent cells is accomplished by trans- ferring cells to a 96-well plate,which already contains zirconium grinding beads and water. This procedure can also be used for adherent cells that are being sub-cultured and as a result are in suspension. Using either a sterile 96-well plate or tissue culture plate, dispense 150 mg of RNase-free, DNase-free treated zirconium beads (200 µ catalog #2180) into each well, using a resin dispenser. Then add, to each well,100 µ of sterile molecular biology grade water. Cultured cells are then added at concentrations as low as 100 cells/well; for best results, the volume of cells added should only be 1-2 µ per well. For dilute concentrations of cells,it is best to first concentrate the cells by centrifugation so that there are 100 to 1000 cells/µl. The plate is sealed using either press-on mats or, preferably, heat-sealed film (catalog #2500 and 2501). As noted above,tape with glue should not be used, as the beads will stick to the adhesive. The sealed plate is then placed in the Geno/Grinder (catalog #2000) and run for 5 min. at 1200 rpm, and 1 to 10 µl of the lysate can be added directly to a PCR cocktail and amplified.