FLUORESCENT IN SITU HYBRIDIZATION (FISH)

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- BCT-600 SEMINAR ON FLUORESCENT IN SITU HYBRIDIZATION (FISH) & IT’S APPLICATION PRESENTED BY DR. DHIREN B. BHOI M. V. Sc. VETERINARY GYNAECOLOGY AND OBSTETRICS COLLEGE OF VETERINARY SCI. & ANIMAL HUSBANDRY ANAND

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OVERVIEW Introduction History of Development of FISH Advantage Over Isotopic Technique Types Of FISH Probes Types Of FISH Procedure Steps Advantages Limitations Applications In Identification Of Microbial Community Analysis

INTRODUCTION : 

Fluorescence in situ hybridization (FISH) uses fluorescent molecules to “paint” genes or chromosomes. This technique is for gene mapping, identification of chromosomal abnormalities and identification of cultured/uncultured microorganisms in environment. FISH involves the use of short sequences of single-stranded DNA (probes) which are labeled with fluorescent tags, to hybridize, or bind, to the complementary DNA to see the location of those sequences of DNA under the fluorescent microscope. INTRODUCTION

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HISTORY OF DEVELOPMENT OF FISH The abilities to detect specific molecular identities was first demonstrated using antigen-antibody interactions. In 1977, the first antibody dependent fluorescent detection of nucleic acid was achieved. The first application of fluorescent in situ detection come in 1980, when RNA was directly labeled on the 3 end with fluorophore.

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ADVANTAGE OVER ISOTOPIC TECHNIQES 1 Isotopic probes are unstable. Sensitivity is high but resolution is limited. 3 Long exposure are often required to produce measurable signal on radiography. 4 Radoilabled probe is costly and hazardous.

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Gene/locus specific probes used to detect the presence absence or location of a particular gene, both in metaphase and interphase cells Centromere probes designed to hybridize to alpha satellite repeat regions in centromeres, fluoresce brightly due to large number of repeats in centromeres useful for determining the number of copies of a particular chromosome Whole chromosome paint probes made from flow-sorted or microdissected chromosomes used to determine composition of marker chromosomes, confirm the presence of chromosome rearrangements FISH PROBES

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TYPES OF FISH PROBES

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METAPHASE FISH DISEASE INVOLVED DI-GEORGE SYNDROME WILLAMS SYNDROME STEROID SULFATASE DEFICIENCY KALLMAN SYNDROME

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INTERPHASE FISH ADVANTAGEOUS OVER METAPHASE ANEUPLOID SCREEN TEST

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Procedures Sample preparation and hybridization Prepare slides with metaphase chromosomes or interphase nuclei Dehydrate in ethanol Denature DNA at 70oC Denature labeled probe Incubate at 37oC for 4-16 hours for hybridization

ADVANTAGES OF FISH : 

ADVANTAGES OF FISH Rapid High efficiency of hybridization and detection Lots of cells can be analyzed Cells do not have to be replicating

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PROBLEMS WITH IN SITU HYBRIDIZATION Permeabilization problems Uneven cell penetration No signals High amount of background autofluorescene

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1 Identifying/quantifying uncultivated and/or cultivated organisms in natural environmental samples, within organisms like endosymbionts, or enrichments. 2 Monitoring population dynamics in environments and/or enrichments in communities like biofilms, sludge, etc. 3 Identifying morphology or relationships among types of organisms. APPLICATIONS IN IDENTIFICATION OF MICROBIAL COMMUNITY

Bacterial Taxonomy : 

Bacterial Taxonomy Bacterial species is the base unit for taxonomy Definition of any given species is subjective >70% sequence similarity of genome >98% sequence similarity of rRNA Each species is phenotypically distinct Difficult to classify Tools:FISH

Evolutionary Chronometers : 

Evolutionary Chronometers Phenotypic characteristics Mole percent Guanine + Cytosine DNA sequence similarity (gross sequence similarity) Small-subunit RNA (16S rRNA of prokaryotes)

Phenotypic Taxonomy : 

Phenotypic Taxonomy Phenotype determination is classic taxonomic method Today more reliance on molecular methods for taxonomy above the genus level Still, phenotypic differentiation is considered requirement for separation of species

Range of G+C contents : 

Range of G+C contents

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DNA-DNA hybridization Signature sequences identify phylogenetic groups 16S& 18S sequences identify Bacteria, Archaea, and EucaryaProbes can be developed for FISH (fluorescent in situ hybridization)

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r-RNA Features Well Conserved Single Strand Ancient Molecules Constant Functions Universally Distributed Phylogenitically

Community analysis by molecular methods : 

Community analysis by molecular methods

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Extraction of total community Preparation of a short gun DNA library in bacteriophage lambda Screening by the hybridization with a 16S RNA specific probe Sequence determination from clones containing 16S RNA genes Comparative analysis of the retrieved sequence THE PRINCIPLE STEPS OF THE PROPOSED PROCEDURE

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FISH using r-RNA targeted probes is the method of choice for all studies in which exact cell numbers and cellular locations need to be determined. Development & design of more r-RNA targeted probes for novel microorganisms in environment The methodology is being continuously improved so far however, microscopic analysis by FISH has not been automated sufficiently which would be desirable in many investigations Accurate quantification still remains a challenging task and study needs careful controls. Conclusion

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THANK YOU