CULTURE INDEPENDENT METHODS OF COMMUNITY ANALYSIS

CULTURE INDEPENDENT METHODS OF COMMUNITY ANALYSIS : 

CULTURE INDEPENDENT METHODS OF COMMUNITY ANALYSIS MICROBIOLOGY

CONTENTS : 

CONTENTS INTRODUCTION METHODS EMPLOYED FOR ANALYSIS FLUORESCENT IN SITU HYBRIDIZATION PHYLOGENETIC ANALYSIS PHOSPHOLIPID FATTY ACID ANALYSIS NUCLEIC ACID ANALYSIS

INTRODUCTION : 

INTRODUCTION The survey of biodiversity without culturing or otherwise observing the cells , is called ‘Culture Independent Method Of Community Analysis’ It is possible because it turns out that the most abundant members of the natural community are species that have so far defied laboratory culture Difference in lysis efficiency , effect of genome size , r RNA gene copy number of the target sequence , the composition of primers & concentration of DNA template are factors at work in culture independent approach

METHODS USED FOR ANALYSIS : 

METHODS USED FOR ANALYSIS 1. Fluorescent In Situ Hybridization 2. Phylogenetic Analysis 3. Phospholipid Fatty Acid Analysis 4. Nucleic Acid Analysis

Fluorescent In Situ Hybridization : 

Fluorescent In Situ Hybridization “ Fluorescent In Situ Hybridization is a cytogenetic technique which can be used to detect the presence or absence of specific DNA sequences on chromosomes.It uses fluorescent probes which bind only to those parts of the chromosomes with which they show a high degree of sequence similarity ”

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Fluorescent In Situ Hybridization has been nicknamed as FISH FISH can be applied directly to cells in culture or in a natural environment ( the term in situ means “in the environment” ). In essence FISH is a phylogenetic stain In the alongside figure is seen a metaphase cell positive for the bacterial arrangement using FISH.The chromosomes can be seen in blue.The chromosome labelled red & green spot is the one where wrong rearrangement is present.

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oligonucleotide probes can be made fluorescent with certain dyes, which are used to identify organisms containing a nucleic acid sequence complementary to the probe ; this technique is included in FISH Using FISH ,a particular organism or a group of related organism, depending on the specificity of the phylogenetic probe; can be identified or tracked in a natural sample

SCHEMATIC PRESENTATION OF PRINCIPLE OF FISH : 

SCHEMATIC PRESENTATION OF PRINCIPLE OF FISH

SCHEME OF PRINCIPLE OF FISH : 

Firstly a probe is constructed The probe has to be of such a length that it should be able to hybridize specifically exactly to its target It is directly tagged with fluorophores with targets for antibodies or biotin It is achieved by Nick translation and PCR using tagged nucleotides. (continued………. SCHEME OF PRINCIPLE OF FISH

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(continuation…………….. An Interphase or Metaphase chromosome preparation is produced, which is firmly attached to a Substrate; usually glass. The probe is then applied to the chromosome DNA and incubated for ~12 hrs. while hybridization. Several wash steps remove all unhybridized or partially hybridized probes The results are then visualized using a microscope that is capable of exciting the dye and recording the images. In the alongside structure shown are Interphase cells positive for a chromosomal rearrangement

VARIATION ON PROBES AND ANALYSIS : 

VARIATION ON PROBES AND ANALYSIS The differences between the various FISH techniques are due to the construction & content of the fluorescently labelled DNA probe The size,overlap,colour &mixture of the probe make possible cell FISH technique. FISH is a very general technique which is arbitarily divided into more specific categories based on application

FISH IN MICROBIAL ANALYSIS : 

FISH IN MICROBIAL ANALYSIS FISH can be used for the microbial identification and tracking of organisms directly in the environment. FISH offers a method for assessing the composition of microbial community directly by microscopy. FISH can be used to compare the genomes of two biological species, to deduce evolutionary relantionships.This is called ‘ZOO BLOT’

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Bacterial FISH probes are often primers for the 16S r RNA region. BIOFILMS are often composed of multispecies bacterial organizations.Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the dictribution of this specific species within the biofilm

KINDS OF FISH METHOD : 

KINDS OF FISH METHOD There are 2 kinds of FISH technologies :- a.) Chromosomal Painting b.) ISRT FISH a .) Chromosomal Painting is typically used to identify bacteria that contain specific , highly conserved genes; such as those that encode the enzyme nitrogenase enzyme ( used for nitrogen fixation ) The number of fluorescent cells indicate the number of nitrogen fixing bacteria respectively , in that natural sample. In the figure its shown Nitrifying bacteria in sewage sludge.Red , ammonia- oxidizing bacteria and Green ,nitrite-oxidizing bacteria.

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ISRT FISH is the abbreviated form of In Situ Reverse Transcription FISH. It is typically used for measuring the gene expression in microbial cells in a natural sample and understanding specific metabolic activities The two photos show a mixed culture of the bacteria Microbulbifer hydrolyticus(short fat rods) and Sagittula stellata(long thin rods) which led to the production of c DNA which is amplified by PCR & hybridized by fluorescently labelled probe.

OTHER APPLICATIONS OF FISH : 

OTHER APPLICATIONS OF FISH FISH has been used for the rapid identification of specific pathogens from parent specimens. By FISH diseased cells can be more easily detected than Cytogenic method .

PHYLOGENETIC ANALYSIS : 

PHYLOGENETIC ANALYSIS This is another method of Community Analysis. Phylogenetic Analysis method is widely used for analysis of several morphological and molecular data. For microbial community analysis, an estimation of evolutionary relantionship among organisms is done in case of Phylogenetic Analysis. Modelling mutation accurately is one of the challenges of Phylogenetic Analysis.

Properties of Analytical Methods : 

Properties of Analytical Methods CONSISTENCY:- A method is consistent if it is more likely to find the correct answer with more data. POWER:- A method is powerful if it can find the correct answer with very few data. ACCURACY:- A method is accurate if in multiple trials it produces answeres that follow a normal distribution centered on the correct answer. PRECISION:- A method is precise if in multiple trials it finds answers that are very close to each other i.e they have low variance.

BASIC MODEL OF PHYLOGENETIC ANALYSIS : 

BASIC MODEL OF PHYLOGENETIC ANALYSIS Nearly all Phylogenetic Analysis share a number of fundamental assumption:- Homologues sequences are in a multiple sequence alignment. The alignment is referred to as a character. The specific residue (nucleotide or amino acid) present in a given sequence is referred to as the character state. In most cases ancestral sequences are not known as so they are assumed to have undergone Mutation.

CATEGORIES OF PHYLOGENETIC METHODS : 

CATEGORIES OF PHYLOGENETIC METHODS Phylogenetic Methods are divided into 3 categories:- Parsimony:- It generates evolutionary trees based on the assumption that the minimal amount of sequence change necessary to diverge two lineages from a common ancestor usually occurred during their evolutionary divegence. Distance method:- Sequences are aligned and Evolutionary Distance (ED) Calculated by having the computer record every position in the database in which there is a difference in sequence. Likelihood:- The model of sequence evolution can be used to relate the data to a hypothesis(typically a tree topology )

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Transversion and Transition occur at different rates All nucleotides occur with equal frequency. In the evolution of real sequences the transitions are typically observed more often than transversion. TV indicates transversion & TS indicates transition and A,C,G,T are the nucleotides

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In Phylogenetic Analysis, an additional step is needed to resolve the different forms of the gene before sequencing can proceed There are 2 ways of achieving it:- A.) Molecular Cloning B.) DGGE Method

RESULTS OF PHYLOGENETIC ANALYSIS : 

RESULTS OF PHYLOGENETIC ANALYSIS Phylogenetic Analysis of microbial communities have been refined to allow for both qualitative as well as quantitative analysis It provides information about phylotypes present in the community that contain that specific gene. The number of band in DGGE gel is a clue to the boicomplexity of the habitat with respect to a given gene.

PHOSPHOLIPID FATTY ACID ANALYSIS : 

PHOSPHOLIPID FATTY ACID ANALYSIS Phospholipid fatty acid analysis is abbreviated as PLFA It’s a lipid based biochemical technique. Its used for characterizing microbial communities in aquifers and other deep surface habitat. It provides information on biomass Physiology,functional identity and Overall community composition structure of phospholipid fatty acid

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PLFA does not require culturing and can provide quantitative data on entire community. Phospholipid fatty acid are essentially the “skin” of microbe and provide a quantitative tool for assessing microbial responses to their environment

APPLICATION OF PLFA : 

APPLICATION OF PLFA It contributes to our understanding of ground water microbial ecology It has potential use in optimizing bioremediation of ground water pollutants It determines if biomass concentration are sufficient for bioremediation Monitoring the effect of electron donor addition in community composition

NUCLEIC ACID ANALYSIS : 

NUCLEIC ACID ANALYSIS Nucleic acid consists of DNA & RNA PCR amplified ribosomal RNA genes do not originate from a pure culture grown in the lab A nucleic acid probe is a DNA or RNA oligonucleotide complementary to a sequence in a target gene which hybridizes the target gene

STEPS IN NUCLEIC ACID ANALYSIS : 

STEPS IN NUCLEIC ACID ANALYSIS 1. Isolation of DNA 2. Heating up to separate strands 3. Addition of specific primers 4. Primer extension with DNA polymerase 5. Many copies of 16S r RNA is obtained 6. Agarose gel is run 7. The PCR product is purified & sequenced

SIGNIFICANCE OF NUCLEIC ACID ANALYSIS : 

SIGNIFICANCE OF NUCLEIC ACID ANALYSIS Signature Sequences can be highly diagnostic of a particular organism or a group of related organism Nucleic acid analysis can paint a phylogenetic picture of that community Signature defining a specific group within a domain or a particular genus or even a single species can be determined

CONCLUSION : 

CONCLUSION The recently expanded knowledge of prokaryotic diversity mainly due to culture independent approach has stimulated efforts to develop culturing methods that can recover its diversity as novel living organism The future study of prokaryotic diversity and the success in understanding its extent, clearly lie in the combination of culture and culture independent approach

REFERENCES : 

REFERENCES BROCK- ‘BIOLOGY OF MICROORGANISMS’ WEBSITES:- www.google.com www.wikipedia.com

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