Site-directed mutagenesis: a two-step method using PCR and DpnI.

with impervious exteriors, such as shells or chitinous exoskeletons. Advantages to this method are high throughput, high success rate and enough template DNA for several different assays (FRE-PCR used only 5 μL of the 20-μL supernatant). Other extraction methods that were tried and discarded for low success rates included boiling in water or PCR buffer, grinding before boiling, grinding with commercial pestles and grinding without Chelex. Until now, PCR-based assays of small marine eukaryotes using probes and restriction enzymes have been applied to a pooled sample of 10 individual larvae (2) or to large, soft-bodied, individual larvae (6,7). Côrte-Real et al. (5) amplified single bivalve larvae with a 67% success rate, but larvae were cultured, and all were selected for extraction before the shell had developed. Now FRE-PCR can be applied to numerous microscopic individuals from marine environmental samples as well as to microscopic organisms in the same size range (e.g., nematodes, insects, polychaetes) from other environments.