Molecular Identification of Microbes: III. Pseudomonas

Abstract

The molecular identification protocol for Pseudomonas introduces PCR cycle sequencing and some basic bioinformatics tools. First follow a simple protocol to isolate genomic DNA from the bacteria. This protocol involves breaking the cells open with a series of freeze/thaw cycles and then centrifuging to remove cellular debris. Many techniques exist for the identification of Pseudomonas. We present a detailed explanation for setup of 16S rRNA amplification and 16S rRNA RFLP. First after isolation of DNA a PCR reaction has to set up to amplify a region of the 16S rRNA gene. The PCR product should be cleaned up by using PCR purification kit, which cleaves excess primers and inactivates free nucleotides. The cleaned PCR product is then used as the template for a sequencing reaction. Sequencing should be done by using BigDye reagents and the reactions are run in a thermocycler (PCR machine). The completed samples are then sent to a core facility to obtain the sequence. Finally, view the electropherograms from the sequencing reaction, then use the sequence in a BLAST search limited to a bacterial data base. Students can identify their unknown bacteria by examining the top-scoring sequences from the BLAST search results. 16S rRNA-RFLP is an important tool to understand the phylogenic similarity between isolates so that one can avoid the sequencing of the isolates which are similar and it leads to the reduction of cost.