Microbial Diversity in Soil Assessed by Two Different Capillary Electrophoresis Methods

Abstract

Capillary electrophoresis can separate fragments of nucleic acids using single strand conformation polymorphism (SSCP) and terminal restriction fragment length polymorphism (T‐RFLP). These methods have been increasingly used in microbial ecology to develop fingerprints of the microbial community within soil. T‐RFLP is based on amplifying a region of DNA common to multiple species using conserved PCR primers whereas SSCP detects mutations in DNA fragments due to changes in the secondary structure of single‐stranded DNA fragments. The objective of this study is to use soil samples to compare molecular profiles generated by CE‐SSCP and T‐RFLP in detail. The soil samples will be collected from four locations representing four soil types. One set of soil subsamples will be analyzed immediately following collection and six subsamples will be placed in different storage treatments: cooling at −4C, freezing at −20C, freezing at −80C, freeze drying, air drying, and oven drying. DNA will be extracted from the samples, run through polymerase chain reaction (PCR) to amplify and fluorescently tag the genes of interest. T‐RFLP uses restriction enzyme MspI after PCR, while SSCP does not. Then, the microbial community fingerprints are analyzed. We hypothesize CE‐SSCP and T‐RFLP will generate similar fingerprints of the microbial community within soil. Supported by National Institute of Justice (25‐6228‐0159‐001).