212 A SIMPLE PROTOCOL TO EXTRACT DNA FROM SMALL NUMBERS OF CELLS

Abstract

Reproductive biotechnology frequently requires improvements in our ability to isolate DNA from fewer and fewer cells. It is now possible to find several commercially available kits to meet this demand, but they frequently come at a relatively high cost. The aim of this project was to evaluate the efficiency of a simple handmade protocol to extract DNA from a small number of cells. To evaluate this method we chose 3 different bovine cell lines, Mac T-cells, sperm, and fibroblasts, for the analysis. Five different quantities (30, 100, 150, 300, and 500 cells) were evaluated for each cell line. The endpoints examined were the purity of the extracted DNA by NanoDrop analysis and visualisation of bands on agarose gels after PCR. The samples were initially diluted in 20 µL of PBS. The cell lysis buffer contained 15 mM Tris-HCl (pH 8.9), 50 mM KCl, 2.5 mM MgCl₂, 0.1% Triton X-100, and 150 µg mL⁻¹ of proteinase K. Twenty microlitres of lysis buffer were added to each sample. The tubes were then incubated at 55°C for 1 h. Last, proteinase K was inactivated by incubation at 90°C for 10 min. After extraction, 3 samples from each cell quantity for each replicate were evaluated by NanoDrop. After NanoDrop analysis, a standard PCR procedure was carried out using the homologue gene, tRep-137, as the amplification target. As a control, the Gene Wizard DNA extraction kit was used for all cell treatments. The DNA fragments resulting from PCR were separated for visualisation on agarose gels. Five replicates of the experiment were performed with 3 individual samples for each cell quantity evaluated. There were 80 total samples evaluated, including 5 control samples (1 per replicate). The PCR results were analysed using chi-square analysis with Fisher correction. An α-level of 0.05 was selected a priori for significant differences. The agarose gel analysis showed that each cell line gave different results. The data presented in Table 1 shows that this protocol is less effective for Mac T-cells, but satisfactory for both sperm and fibroblast cells. When the quality of the DNA extracted was evaluated with the NanoDrop method, DNA was found in all samples, but the purity of the samples was very low. None of the samples had a 260/280 ratio over 1, the range being 0.6 to 0.8. This high impurity could explain why not all samples showed PCR product bands on the gel. The next step will be to purify the DNA by removing all of the protein from the samples after isolation from the cells. In conclusion, these data show that it is possible to extract DNA at a low cost from a small number of cells.

Table 1.  The mean (SD in parentheses) percentage of samples with PCR bands for each cell line and each cell quantity