Preparation of DNA from Numerous Individual Microscopic Organisms for PCR-Based Assays of Environmental Samples

Polymerase chain reaction (PCR)based assays have been developed to identify microscopic organisms that cause disease (8) or have an environmental impact (1). In both cases, DNA is extracted from pooled individuals. The pooled sample has typically been created for bacteria by culturing and for disease vectors by taking advantage of their reproduction in the host. PCRbased assays are being developed for a slightly larger class (50–500 μm) of microorganisms for which it is important to identify individuals rather than pooled samples (2,4). The usual extraction methods require many steps of centrifugation, vortex mixing, incubation and precipitation (4). Microorganisms with impervious exteriors (shells or chitinous exoskeletons) are especially resistant to lysis methods of DNA extraction. Rapid and easy methods for DNA extraction are needed so that large numbers of individuals can be screened in PCR-based assays of environmental samples. We describe a technique called fast resin extraction (FRE)-PCR to amplify the DNA from large numbers of individual microscopic organisms rapidly. To test the technique, we used early larval stages of marine bivalves (Phylum Mollusca) and naupliar stages of copepods (Phylum Arthropoda) from marine plankton samples. In both cases, the small size and lack of morphological differentiation at the magnifications available by light microscopy make them difficult to identify to species. Higher magnifications, available with scanning and transmission electron microscopy, are time-consuming and costly, limiting the number of individuals that can be examined. Larvae of the surfclam, Spisula solidissima, were cultured in the laboratory for use in testing the extraction methods. Plankton tows were taken with a plankton net on the continental shelf near Tuckerton, NJ. Individual bivalve larvae and copepod nauplii were sorted live with the aid of a dissecting microscope, transferred with <8 μL of seawater into individual tubes (200 or 500 μL or into wells in a 96-well V-bottom microplate) and frozen at -80°C until used. Plastic pipet tips were used to make pestles that fit snugly in the bottom of the microcentrifuge tubes, leaving no sample unground. The insufficient grinding of commercial pestles was discovered by examining the results with the aid of a compound microscope. Snugly fitting pestles were made by softening the pointed end of a pipet tip (200 μL) in a flame for a few seconds and pushing it to the bottom of a microcentrifuge tube. Tubes used to mold pestles were the same size as the sample tubes. Pestles for the 96-well plates were molded in 200-μL tubes because the 96-well V-bottom plates melted when exposed to hot pipet tips. Pestles were reused after soaking overnight in 3 N HCl and thorough rinsing in deionized water before autoclaving. Tubes and plates with individual larvae and nauplii were transferred directly from the freezer to a heating block and heated for 4 min at 100°C to denature DNases. The individual larva in each tube was ground with a plastic pestle. Twenty microliters of a 5% solution of Chelex-100 resin (Bio-Rad, Hercules, CA, USA) in sterile water were added to each tube before heating for 10 min at 100°C. After heating, the Chelex resin settled quickly to the bottom of the tube without centrifugation,