SGER: Testing and Optimization of DNA-based Methods of Zooplankton Analysis

University Of Louisiana At Lafayette, Lafayette LA

Investigators

Abstract

The life histories of the large majority of marine species include planktonic stages, and the availability of planktonic larvae may regulate benthic as well as pelagic populations. Because the distributions of planktonic organisms are extremely variable in space and time, large numbers of samples are needed to adequately characterize them. Using conventional methods, organisms must be identified and counted in each sample by tedious microscopic examination by highly skilled biologists. The PI will attempt to resolve this sampling bottleneck by developing a novel approach for rapid, quantitative analysis of larval abundance using inexpensive molecular methods. This new approach is referred to as CADRE, for "competitive amplification of a diagnostic repetitive element. CADRE exploits the widespread occurrence of taxon-specific repetitive DNA sequence elements. Amplification of these elements by the polymerase chain reaction (PCR) will be used to detect and quantify the target species in mixed plankton samples. Quantification of larval biomass can be achieved by competitive PCR, in which a known amount of a DNA template standard is used as a control for variable PCR conditions. The standard has the same primer binding sequences as the targeted template DNA, and therefore it acts as an internal control for the efficiency of amplification. The products amplified from the standard and target can be distinguished by size, and the ratio of these products, the PCR product ratio, provides an estimate of the initial amount of the target sequence. Although other DNA sequences, including ribosomal RNA genes and mitochondrial DNA genes can be used to detect or quantify planktonic organisms, repetitive elements offer a unique combination of advantages. They are easily detected and quantified, taxon-specific, and relatively independent of the physiological state of the organism.