Authors: Aman Jaiswal*, Ajay Kumar, Deepak Kumar Koli, Swati Sagar
Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi (India) 110 012
Introduction
Analyses of 16S rRNA gene sequences from environmental DNA have shown the extraordinary richness of phylogenetic types found in many microbial habitats. Culturing of microorganisms continues to be an important method for gaining better understanding of specific organisms. But this method is inadequate to represent the complex microbial communities often seen in the natural environments. The introduction of molecular microbial methods has brought out the profound influences on the studies of natural microbial communities. Signature molecules of the molecular DNA-based approaches that include 16S rDNA are prior-amplified by polymerase chain reaction (PCR) for further analyses. The pool of PCR products are either cloned for sequencing or are subjected to a variety of DNA fingerprinting methods. The fingerprinting methods include amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. These molecular DNA fingerprinting methods provide better resolution of microbial communities from the natural environments.
Genetic Fingerprinting
It is a method of identifying microbes on the basis of DNA. In genetic fingerprinting DNA is either digested by restriction enzyme or denatured by using temperature or chemical gradient.
Genetic fingerprinting techniques provide a pattern or profile of the community diversity based upon the physical separation of unique nucleic acid species.
Genetic finger printing is more important due to following reasons:--
- The methods are rapid and relatively easy to perform.
- It allow the simultaneous analysis of multiple samples, which makes it possible to compare the genetic diversity of microbial communities from different habitats.
- To study the behaviour of individual communities over time.
Molecular approaches to study microbial diversity
A number of approaches have been developed to study molecular microbial diversity. These include DNA reassociation, DNA–DNA and mRNA-DNA hybridisation , DNA cloning and sequencing, and other PCR-based methods such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel elec- trophoresis (TGGE), ribosomal intergenic spacer analysis (RISA) and automated ribosomal intergenic spacer analysis (ARISA).
- DGGE was invented by Dr. Leonard Lerman (1983).
- ARDRA was invented by Vaneechoutte et al. (1995).
- T-RFLP was introduced by Liu.et al. (1997).
Denaturing gradient gel electrophoresis (DGGE) is a molecular fingerprinting method that separates polymerase chain reaction (PCR)-generated DNA products. The polymerase chain reaction of environmental DNA can generate templates of differing DNA sequence that represent many of the dominant microbial organisms. However, since PCR products from a given reaction are of similar size (bp), conventional separation by agarose gel electrophoresis results only in a single DNA band that is largely non-descriptive. DGGE can overcome this limitation by separating PCR products based on sequence differences that results in differential denaturing characteristics of the DNA. During DGGE, PCR products encounter increasingly higher concentrations of chemical denaturant as they migrate through a polyacrylamide gel. Upon reaching a threshold denaturant concentration, the weaker melting domains of the double-stranded PCR product will begin to denature at which time migration slows dramatically. Differing sequences of DNA (from different bacteria) will denature at different denaturant concentrations resulting in a pattern of bands. Each band theoretically representing a different bacterial population present in the community. Once generated, fingerprints can be uploaded into databases in which fingerprint similarity can be assessed to determine microbial structural differences between environments or among treatments. Furthermore, with the breadth of PCR primers available, DGGE can also be used to investigate broad phylogenies or specific target organisms such as pathogens or xenobiotics degraders.
Principle
DGGE is a tool of molecular fingerprinting method which involves the separation of strands on the basis of GC content. Once the bands separate, they are sequenced for identification.
ARDRA
Restriction fragment length polymorphism (RFLP), also known as amplified ribosomal DNA restriction analysis (ARDRA) is another tool used to study microbial diversity that relies on DNA polymorphisms. In this method, PCR- amplified rDNA is digested with a 4-base pair cutting restriction enzyme. Different fragment lengths are detected using agarose or non-denaturing polyacrylamide gel electrophoresis in the case of community analysis. RFLP banding patterns can be used to screen clones or used to measure bacterial community structure.
Principle
ARDRA is a method which involves digestion of PCR amplified product by restriction enzymes and different fragment lengths are detected by PAGE. This method is useful for detecting the changes in microbial community.
T-RFLP
T-RFLP analysis is a technique used to study complex microbial communities based on variation in the 16S rRNA gene. T-RFLP analysis can be used to examine microbial community structure and community dynamics in response to changes in different environmental parameters or to study bacterial populations in natural habitats. It has been applied to the study of complex microbial communities in diverse environments such as soil, marine and activated sludge systems as well as in a study to characterize oral bacterial flora in saliva in healthy subjects versus patients with periodontitis. The T-RFLP technique is a culture independent, rapid, sensitive and reproducible method of assessing diversity of complex communities without the need for any genomic sequence information. The technique provides information on a collection of microorganisms that may be present in a given sample. After the completion of a T-RFLP project, if a researcher is interested in a finer level of analysis at a species level, the Applied Biosystems MicroSeq® identification kit may be used.
Principle
T-RFLP is a method which involves the powerful resolution of automated sequencing technology and avoids some of the limitations of RFLP. It involves the digestion of PCR products and analysis of labelled terminal fragments by using capillary gel electrophoresis.
Application of Genetic Fingerprinting
- Genetic diversity of microorganisms.
- Genetic relationship among organisms.
- Origin and evolution of species.
- Whole genome and comparative mapping.
- Unlocking valuable gene from wild species.
- Genetic drift and selection.
- Construction of exotic library.
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