Locating and isolating a gene, FISH, GISH, Chromosome walking and jumping, tetracycline expression system.

In situ hybridization
Fluorescent in situ hybridization
Genomic in situ hybridization
Positional cloning
Chromosome walking
Chromosome jumping
Tetracycline inducible expression system

1. LOCATING AND ISOLATING A GENE & TETRACYCLINE EXPRESSION SYSTEM 1

2. CONTENTS In situ hybridization Fluorescent in situ hybridization Genomic in situ hybridization Positional cloning Chromosome walking Chromosome jumping Tetracycline inducible expression system 2

3. Locating and isolating a gene In situ hybridization Positional cloning Chromosome walking Chromosome jumping 3

4. IN-SITU HYBRIDIZATION DNA in situ hybridization is used to identify the position of genes and localize and detect the specific DNA sequences in cells.
In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain information about gene expression and genetic loci.
In situ hybridization was invented by Joseph G. Gall.
It is a biological assay (like ELISA, PCR) for molecular diagnosis. 4

5. 1. Sample Preparation Tissue sections or cells are fixed onto a slide, preserving their morphology and integrity. 2. Probe Design A complementary DNA or RNA probe is designed to target the sequence of interest. This probe is often labeled with a detectable marker, such as a fluorescent dye, radioactive isotope, or enzyme, for visualization. 3. Denaturation The sample is treated to denature the DNA or RNA, separating the double helix into single strands, making them accessible for hybridization. STEPS 5

6. 7. Analysis The signal intensity and distribution of the probe within the tissue are analyzed to determine the location and abundance of the target gene. 6 . Detection The location of the hybridized probe is visualized using techniques appropriate to the labeling method, such as fluorescence microscopy, autoradiography, or enzyme-linked detection. 4. Hybridization A labeled nucleic acid probe, complementary to the target sequence, is added to the sample. The probe will bind specifically to its complementary sequence within the sample. 5. Washing Unbound probe is washed away to reduce background signal. 6

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8. PROBES Probes are labeled DNA or RNA oligonucleotides that are complementary to genes or genomic regions of interest. Strands will anneal with complementary nucleotides bonding back together with their homologous partners when cooled. Chances of a probe finding a homologous sequence other than the target sequence decreases as the number of nucleotides in the probe increases. TYPES Double Stranded DNA Probes Single Stranded DNA Probes RNA Probes Oligonucleotide Probes 8

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10. LABELLING OF PROBES A probe is a labeled fragment of DNA or RNA used to find its complementary sequence or locate a particular clone. The presence of the label should not interfere with the hybridization reaction. Two types; Radioactive labelling Radioactive labels are the isotopes which emit beta particles and are detected by autoradiography. It involves incorporating radioactive isotopes with the probe molecule. E.g., ³H, ³⁵S, ³²P Non- radioactive labelling It involves attaching non-radioactive markers to the probe molecule. Two types, Direct method Indirect method 10

11. Non-radioactive labelling (1) Direct labelling It involves, the label is directly attached to the probe molecule itself. This means the label is physically linked to the probe being used for detection. The labels are, Enzyme Radioisotope Fluorescent marker (2) Indirect labelling It involves attaching a secondary molecule, such as an antibody or another probe, which carries the label. This secondary molecule binds specifically to the primary probe, allowing for detection indirectly through the presence of the label attached to the secondary molecule. A hapten , Biotin Digoxigenin Fluorescein 11

12. ISH FISH GISH Types of in situ hybridization Fluorescent in situ hybridization (FISH) Genomic in situ hybridization (GISH) 12

13. 1. Fluorescence in situ hybridization (FISH) Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind only those parts of the chromosome with a high degree of sequence complementarily. 13

14. STEPS Basic elements are a DNA probe and a target sequence. B) Before hybridization, the DNA probe is labeled indirectly with a hapten or directly labeled via the incorporation of a fluorophore. 14

15. C) The labeled probe and the target DNA are denatured to yield single-stranded DNA. D) They are then combined, which allows the annealing of complementary DNA sequences. 15

16. E) If the probe has been labelled indirectly, an extra step is required for visualization of the non-fluorescent hapten that uses an enzymatic or immunological detection system. F) Finally, the signals are evaluated by fluorescent microscope. 16

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19. PROBES USED IN FISH Chromosome painting probe They are fluorescently labeled DNA sequences that bind to complementary sequences on chromosome 19

20. 2. Genomic in situ hybridization (GISH) Genomic in situ hybridization (GISH), which is a modification of fluorescent in situ hybridization. It is a cytogenetic technique that allows the detection and localization of specific nucleic acid sequences on morphologically preserved chromosomes using genomic DNA of donor species as probe. It involves hybridizing labeled genomic DNA probes to the chromosomes of the target organism. This technique involves the extraction of labeling DNA of one organism and to use as a probe to target the genome of another organism.
The part of genome that are similar to the probe hybridize to from a probe target complex. 20

21. GISH FISH It is is primarily used for comparing and visualizing the distribution of entire genomes or large segments of genomes between different species or individuals. It is used for detecting and locating specific DNA sequences, genes, or chromosomal abnormalities within chromosomes or cells. It involves hybridizing labeled genomic DNA from one species to the chromosomes of another species. It involves hybridizing fluorescently labeled DNA probes to specific DNA sequences within chromosomes or cells. The target is the entire genome or large genomic segments of one species, which are hybridized with labeled DNA probes from a different species. The target is specific DNA sequences or genes within chromosomes or cells, which are hybridized with fluorescently labeled DNA probes complementary to those sequences. 21

22. POSITIONAL CLONING Positional cloning, also known as map-based cloning, is a technique for the positioning of a trait-associated gene in the genome and involves methods such as linkage analysis, association mapping, and bioinformatics. 22

23. STEPS Initial Mapping : Linkage Analysis : This method examines the co-segregation of genetic markers with the trait or disease within families. It helps identify regions of the genome that are likely to contain the gene of interest. Association Studies : These investigate the association between genetic markers and the trait or disease in a population. It can identify regions of interest without requiring family data. 23

24. 2. Fine Mapping Physical Mapping: Utilizes physical markers along the chromosome, such as restriction fragment length polymorphisms (RFLPs) or microsatellites, to narrow down the region of interest. High-Resolution Mapping: Involves the use of more densely spaced markers, like single nucleotide polymorphisms (SNPs), and larger sample sizes to further refine the region. 3. Candidate Gene Identification Bioinformatics Analysis: Involves computational methods to prioritize genes within the narrowed region based on their known functions, expression patterns, and relevance to the phenotype or disease.
Functional Studies: Candidates are assessed for their biological plausibility through expression analysis, protein-protein interaction studies, and functional assays in relevant cell lines or model organisms. 24

25. 5.Sequencing and Confirmation Sequencing Candidate Genes: Involves sequencing the coding and regulatory regions of candidate genes to identify mutations or variations associated with the trait or disease.
Confirmation Studies: Validates the association between identified mutations and the phenotype through segregation analysis in affected families or case-control studies in larger populations. 25

26. Example Recently map-based cloning was employed to clone the plant resistance gene that follows the gene-for-gene interaction.
The tomato gene that was cloned was Pto and it provides resistance against bacterial speck disease of tomato caused by Pseudomonas syringae pv . Tomato. 26

27. CHROMOSOME WALKING Chromosome walking is a technique used to clone a target gene in a genomic library by repeated isolation and cloning of adjacent clones of the genomic library. Chromosome walking is a tool which explores the unknown sequence regions of chromosomes by using overlapping restriction fragments. 27

28. In chromosome walking, a part of a known gene is used as a probe and continued with characterizing the full length of the chromosome to be mapped or sequenced.
This goes from the marker to the target length.
In chromosome walking, the ends of each overlapping fragments are used for hybridization to identify the next sequence. 28

29. Steps Isolation of a DNA fragment which contains the known gene or marker near target gene
Preparation of the restriction map of the selected fragment and subcloning the end region of the fragment to use as a probe. Hybridization of the probe with the next overlapping fragment
Preparation of the restriction map of the fragment 1 and subcloning of the end region of the fragment 1 to use as a probe for the identification of the next overlapping fragment. Hybridization of the probe with the next overlapping fragment 2
Preparation of the restriction map of fragment 2 and subcloning of the end region of the fragment 2 to serve as a probe for the identification of the next overlapping fragment Above steps should be continued till the target gene or up to 3’ end of the total length of the sequence. 29

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31. Chromosome jumping Chromosomal jumping is a technique used in molecular biology for physical mapping of genomes of the organisms. This technique was introduced to overcome a barrier of the chromosomal walking which arose upon finding the repetitive DNA regions during the cloning process. Therefore, chromosome jumping technique can be considered as a special version of chromosomal walking. It is a rapid method compared to chromosomal walking and enables bypassing of the repetitive DNA sequences which are not prone to be cloned during chromosomal walking. Chromosomal jumping narrows the gap between the target gene and the available known markers for genome mapping. 31

32. Chromosome jumping tool starts with the cutting of a specific DNA with special restriction endonucleases and ligation of the fragments into circularized loops. Then a primer designed from a known sequence is used to sequence the circularized loops. This primer enables jumping and sequencing in an alternative manner. Hence, it can bypass the repetitive DNA sequences and rapidly walk through the chromosome for the search of the target gene. The discovery of the gene encodes for cystic fibrosis disease was done using the chromosomal jumping tool. 32

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34. INDUCIBLE EXPRESSION SYSTEM – TETRACYCLINE EXPRESSION SYSTEM The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. 34

35. Components Tet- Transactivator ( tTA )/Reverse Tet- Transactivator ( rtTA ): These are fusion proteins consisting of a Tet-repressor domain and a transcriptional activation domain. tTA activates gene expression in the presence of tetracycline, while rtTA activates gene expression in the absence of tetracycline. Tetracycline Response Element (TRE): This is a DNA sequence containing Tet-operator sites ( tetO ) to which the transactivator proteins bind. It is typically placed upstream of the gene of interest. Tetracycline analogs : Such as doxycycline (Dox) are used to regulate the system. These molecules bind to the transactivator proteins, altering their ability to bind to the TRE and thus modulating gene expression. 35

36. Tetracycline off system The Tet-Off system for controlling expression of genes of interest in mammalian cells was developed by Professors Hermann Bujard and Manfred Gossen (1992). The Tet-Off system makes use of the tetracycline transactivator ( tTA ) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the herpes simplex virus. In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. Tetracycline off is also known as the tTA -dependent system. 36

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38. Tetracycline On System The Tet-On system is based on a reverse tetracycline-controlled transactivator , rtTA . The tetracycline on system is also known as the rtTA -dependent system. Like tTA , rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding alters rtTA’s binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox. 38

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41. REFERENCE Das, A. T., Tenenbaum , L., & Berkhout , B. (2016). Tet-On Systems For Doxycycline-inducible Gene Expression. Current gene therapy, 16(3), 156–167. Jagodic , M., & Stridh , P. (2016). Positional Gene Cloning in Experimental Populations.  Methods in molecular biology (Clifton, N.J.) ,  1304 , 3–24. Shakoori A. R. (2017). Fluorescence In Situ Hybridization (FISH) and Its Applications. Chromosome Structure and Aberrations, 343–367. https://www.lifeasible.com/custom-solutions/plant/plant-breeding/positional-cloning/ https://www.differencebetween.com/difference-between-chromosome-walking-and-vs-jumping/amp/ 41