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ARS Home » Pacific West Area » Corvallis, Oregon » Forage Seed and Cereal Research Unit » Research » Publications at this Location » Publication #58625
Title: A RAPID ONE-TUBE GENOMIC DNA EXTRACTION PROCESS FOR PCR AND RAPD ANALYSES

Author
item Steiner, Jeffrey
item POKLEMBA, CHRIS - 5358-05-00
item Fjellstrom, Robert
item Elliott, Lloyd

Submitted to: Nucleic Acids Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The polymerase chain reaction (PCR) is a powerful biochemical analysis tool that is used to look for genetic differences between different plants, animals, and microbes. However, PCR is limited when analyzing populations by the labor- intensive extraction of genomic DNA from large numbers of samples. We have developed a novel DNA extraction process that can be performed in a single tube from the time a tissue is collected until diluted aliquots of the extractant are taken for PCR reactions. The process does not require centrifugation, can prepare up to 6000 samples per day, results in enough DNA to perform 4000 PCR amplifications per sample, can be used for plant, microbial, and animal sources of DNA, including human blood. Additionally, none of the chemicals used are classified as hazardous waste, so this technique should not negatively impact the environment. The method can be used with primers for both random amplified polymorphic DNA and specific gene sequences. The cost per sample for DNA extraction is only $0.24 compared to $0.64 per sample for the commonly used CTAB procedure.

Technical Abstract: The polymerase chain reaction (PCR) is a powerful genetic tool, but the analysis of populations is limited by the labor-intensive extraction of genomic DNA from large numbers of samples. A novel DNA extraction process has been developed that can be performed in a single tube from the time a tissue is collected until diluted aliquots of the extractant are taken for PCR reactions. The process consists of two parts: sample preparation for extraction and DNA chemical extraction using a one-step buffer (ROSE). Using the ROSE buffer, 0.8 - 1.2 micro g of high molecular weight DNA (>40 Kb) can be extracted from 5.0 mg of lyophilized leaf tissue. The DNA extracts produce identical RAPD and single gene PCR amplification products as the standard CTAB method. DNA samples incubated at 37 deg C for 64 hours produce the same RAPD PCR products as samples stored at 4 deg C which demonstrates the stability of ROSE-extracted DNA. Due to the inhibitory effects of buffer components at their initial strength, and perhaps other natural compounds in the extract, the ROSE buffer extractant must be diluted 170-fold before the DNA can be amplified by PCR. The process does not require centrifugation; can prepare up to 6000 samples per day; results in enough DNA to perform 4000 PCR amplifications per sample; can be used for plant, microbial, and animal sources of DNA, including human blood; and does not generate hazardous waste.