Microorganisms - ENVIS Centre

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Methodologies/Protocol

Molecular Methods for monitoring complex microbial systems in the environment.

Traditional methods of microbial examination by microscopes and biochemical reactions are often insufficient for monitoring the specific microbes in mixed microbial communities in the environment. Molecular methods can detect on specific DNA sequences and relevant structural genes without culturing the microbes for the discovery if unique and previously unrecognized microbes as well as complex microbial diversity.

This page has described the recent molecular biology based techniques have led to rapid and accurate strategies for the identifications and characterization of novel microbial communities in the environment especially in contaminated soil and water.

I. Morphological tests for Bacteria

1. Gram Stain

II. Biochemical characterization

III. Molecular Techniques

I. Nucleic acid isolation

A. DNA Isolation

B. RNA Isolation

2. Agarose Gel Electrophoresis

3. Hybridization

4. DNA microarray

5. BAM: Bacillus cereus

6. qPCR

       Quantitative polymerase chain reaction (qPCR) or real-time PCR is used to simultaneously detect a specific DNA sequence in a sample and determine the actual copy number of this sequence relative to a standard. In real-time PCR, the DNA copy number can be established after each cycle of amplification.

       Apart from DNA, RNA can also be used as a template (e.g. in case of gene expression studies or detection of RNA viruses). In this case the RNA needs to be reverse transcribed into DNA (also termed complementary DNA or cDNA) before it is amplified with real-time PCR. There is a term for this combined method: real-time reverse transcription PCR or qRT-PCR (sometimes RT-qPCR) for short. More about qPCR...

        Designing qPCR Protocol