Slide 1: - 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 Slide 2: OVERVIEW
History of Development of FISH
Advantage Over Isotopic Technique
Types Of FISH Probes
Types Of FISH
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 Slide 5: 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. Slide 6: 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. Slide 7: Gene/locus specific probes
used to detect the presence absence or location of a particular gene, both in metaphase and interphase cells
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 Slide 8: TYPES OF FISH PROBES Slide 9: METAPHASE FISH DISEASE INVOLVED DI-GEORGE SYNDROME WILLAMS SYNDROME STEROID SULFATASE DEFICIENCY KALLMAN SYNDROME Slide 10: INTERPHASE FISH ADVANTAGEOUS OVER METAPHASE ANEUPLOID SCREEN TEST Slide 11: 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 Slide 14: PROBLEMS WITH IN SITU HYBRIDIZATION Permeabilization problems
Uneven cell penetration
High amount of background
autofluorescene Slide 15: 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 Slide 20: 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) Slide 21: r-RNA Features Well Conserved Single Strand
Phylogenitically Community analysis by molecular methods : Community analysis by molecular methods Slide 23: 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 Slide 25: 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 Slide 26: THANK YOU