|Cook, Kimberly - Kim|
Submitted to: Kentucky Water Resources Research Institute Symposium
Publication Type: Proceedings
Publication Acceptance Date: 1/27/2012
Publication Date: 3/19/2012
Citation: Cook, K.L., Hill, L. 2012. Development of in-house methods for high-throughput DNA extraction. Kentucky Water Resources Research Institute Symposium. 25-26. Interpretive Summary:
Technical Abstract: Given the high-throughput nature of many current biological studies, in particular field-based or applied environmental studies, optimization of cost-effective, efficient methods for molecular analysis of large numbers of samples is a critical first step. Existing methods are either based on costly kits purchased from the biotech industry or are low-throughput, time consuming and/or not optimized for environmental analyses. Here we describe the development and optimization of a 96 well, high-throughput method for extraction of DNA from enrichment broths targeting pathogens and indicators. The DNA extraction method is derived from classic methods which involve microbial cell lysis, deprotenation, alcohol precipitation and washing of nucleic acid extracted and silica matrix-based DNA capture. The evaluation of extraction methods was carried out using overnight soil enrichments from samples collected from an on-going study established to evaluate survival of pathogens in conventional or no-till fescue plots with applied poultry litter or dairy manure. Samples (0.3 mL) from each broth type or from pooled broth samples were used for analyses. DNA concentration, DNA purity, PCR inhibition and amplification of targeted populations (particularly the Salmonella sp. ttr gene which is required for tetrathionate respiration) were measured. The final method used a HCl-based re-suspension solution for the production of silica matrix (AGM) by combining 4M-6M solution of a chaotropic solution (NaI or GITC) with the silica solution brought to pH 2 with 30% HCl. In this way, pH is maintained between pH 6.0 and pH 7.4 . This method is performed in a 96 well format using lab-based solutions for extraction and DNA binding. The high-throughput format permits preparation of up to 192 samples in a 4 hour time frame. By eliminating the use of purchased extraction solutions, extraction tubes and silica matrix, the cost per sample has been estimated to be as low at 10 cents.