Characterizing the abundance and activity of soil microbes using single-strand conformational polymorphism by capillary electrophoresis.

摘要

Bioremediation is the process by which microbes are used to break down soil contaminants. The traditional methods for assessing soil microbial communities are both time consuming and can underestimate the numbers due to the inability to culture many soil organisms. The increased speed and automation of capillary electrophoresis (CE) allows for more rapid and reliable results. The identification and assessment of diversity and activity of microbial soil communities can be accomplished by isolating DNA and RNA from soil samples. These DNA/RNA samples can then be characterized by a technique called single-strand conformational polymorphism (SSCP) analysis of the 16S gene and its variable regions. These variable regions have been amplified by polymerase chain reaction (PCR) giving species-dependent fragments of the gene sequence. SSCP, a method that is normally used to detect mutations, is the heat denaturing of these fragments resulting in single strand DNA (ssDNA). Snap cooling of the ssDNA causes it to refold into different secondary structures (conformations) depending on the nucleotide sequence. These different conformations can be separated due to their different electrophoretic mobility, even if they are the same size.;CE-SSCP has been used to identify mixed populations from DNA extracted from pure culture and soil samples and to begin preliminary evaluation of the activity of the microorganisms by examining the products of reverse transcription (RT)-PCR which relates to the sample's RNA. By isolating both DNA and RNA from the same soil sample, at the same time, a more accurate picture of the microbial community in those samples has been made available. This is an important development in the study of contaminated soils and the monitoring of the progress of bioremediation.