Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
In situ Hybridization (ISH) and Fluorescence in Situ Hybridization (FISH) Creative-Diagnostics
In situ Hybridization (ISH) and Fluorescence in Situ Hybridization (FISH) by Creative Diagnostics, learn more http://www.creative-diagnostics.com/in-situ-hybridization-and-fluorescence-in-situ-hybridization.htm
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
It is used to identify chromosomal rearrangements in cancer patients.
Chromosomal identification in cell.
Detect the specific nucleotide sequence within cell and tissues.
Unique point among the studies of cell, biology, cytogenetics and molecular genetics
It is possible to detect single copy sequence on chromosome with probes.
genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics
It will useful for investigating the origins of wild and cultivated polyploid plant species
Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
In situ Hybridization (ISH) and Fluorescence in Situ Hybridization (FISH) Creative-Diagnostics
In situ Hybridization (ISH) and Fluorescence in Situ Hybridization (FISH) by Creative Diagnostics, learn more http://www.creative-diagnostics.com/in-situ-hybridization-and-fluorescence-in-situ-hybridization.htm
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.
According to the ICSM (Intergovernmental Committee on Surveying and Mapping), there are five different types of maps: General Reference, Topographical, Thematic, Navigation Charts and Cadastral Maps and Plans.
It is used to identify chromosomal rearrangements in cancer patients.
Chromosomal identification in cell.
Detect the specific nucleotide sequence within cell and tissues.
Unique point among the studies of cell, biology, cytogenetics and molecular genetics
It is possible to detect single copy sequence on chromosome with probes.
genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics
It will useful for investigating the origins of wild and cultivated polyploid plant species
The Evolution of In Situ Genetic Technologyasclepiuspdfs
In situ genetic technology was historically developed and mainly focused on detection purpose, allowing specific nucleic acid sequences to be visualized in morphologically preserved tissue sections. With the synergy of genetics and immunohistochemistry, in situ detection can correlate microscopic topological information with gene activity at the transcriptional or post-transcriptional levels in specific tissues. Furthermore, its resolution allows spatial distribution of nucleic acid products to be revealed in a heterogeneous cell population. The newest member to the franchise of in situ genetic technology is a direct-on-specimen enrichment methodology specifically for cell-free DNA liquid biopsy. Contrary to in situ detection, this in-well in situ innovation tackles the very first sample preparation step to reduce material loss, thereby improving overall sensitivity. Genomic nucleic acids purified from specimens have been proven to be time consuming and suffered from damages and losses; the evolution of in situ genetic technology offers a powerful tool for precision functional genomics, enabling cross-check between in vitro and in vivo findings. It further opens the door to ultimate genetic engineering in situ.
MOLECULAR BIOLOGY TECHNIQUES USED IN ZOONOTIC DISEASE Nataraju S M
Zoonotic pathogens cause diseases and death both in human & animals which ultimately leads to man power and economic loss of the country. Traditional diagnostic methods identify a pathogen based on its phenotype.
The correct assessment of a clinical isolate takes more time. Faster and simpler methods of diagnosis is of great advantage. That is why molecular biology technique is the first and foremost choice .
What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
Creative Bioarray offers high quality, time efficient and cost effective kinase solution for assay development, high throughput screening (HTS) and selectivity profiling.
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Creative Bioarray provides an extensive range of high quality RNA samples which are ideal for Northern blotting, ribonuclease protection assay, SI nuclease assay, RT-PCR/Q-PCR analysis, rapid amplification of cDNA ends (RACE) and purification of mRNA for library construction.
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Immunohistochemistry (IHC) is the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
https://www.creative-bioarray.com/protocol/immunohistochemistry-protocol.htm
A fluorescent compound has the property of absorbing light energy at a range of specific wavelengths. This absorption of light causes electron to rise from the ground state to a higher energy level (excited state). The excited electron quickly decays to its ground state while releasing the excess energy in the form of photon of light. This transition of energy is called fluorescence.
https://www.creative-bioarray.com/support/fluorochromes-in-flow-cytometry.htm
Creative Bioarray is offering Caco-2 permeability assay to help determine the absorption and the bioavailability of drug candidates, facilitating the lead optimization process in drug discovery.
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Comparison of caco 2 with other cell-based models for intestinal permeability...Creative-Bioarray
The use of cell cultures provides a method to predict drug permeability by utilizing cell monolayers in a two-chamber diffusion system to simulate the passage of drugs from the intestinal lumen into the blood.
Cell cycle refers to the set of events through which a cell grows, replicates its genome, and ultimately divides into two daughter cells through the process of mitosis.
https://www.creative-bioarray.com/cell-cycle-assays.htm
Creative Bioarray is offering Caco-2 permeability assay to help determine the absorption and the bioavailability of drug candidates, facilitating the lead optimization process in drug discovery.
https://www.creative-bioarray.com/Services/caco-2-permeability-assay.htm
A broad range of reprogramming systems is available including mRNA reprogramming, dox-inducible human 4F2A reprogramming and lentivirus reprogramming. These technologies have been adopted and developed in close collaboration with leading iPS cell pioneers. Our extensive range of products allows for customization of individual transcription factors, cell culture conditions, and delivery systems for the optimization of reprogramming parameters.
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Cell proliferation assays are used to monitor the dynamic growth of a cell population or to detect daughter cells in a growing population.
On the other hand, cell viability assays assess how healthy the cells are by measuring markers of cellular activity.
https://www.creative-bioarray.com/products/cell-viability,-proliferation-and-cytotoxicity-list-226.htm
By using flow cytometry, staining dyes are needed. Creative Bioarray can choose different dyes to perform the assays, including propidium iodide (PI), BrdU, 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI), based on the customer’s applications or requirements.
https://www.creative-bioarray.com/cell-cycle-assays.htm
By using flow cytometry, staining dyes are needed. Creative Bioarray can choose different dyes to perform the assays, including propidium iodide (PI), BrdU, 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI), based on the customer’s applications or requirements.
https://www.creative-bioarray.com/cell-cycle-assays.htm
Induced Pluripotent Stem Cells (iPSCs) are a type pf pluripotent stem cell artificially derived, and often referred to as programmed, from adult somatic cells using the expression of certain genes in culture.
https://www.creative-bioarray.com/products/ipsc-reprogramming-kit-list-239.htm
A fluorescent compound has the property of absorbing light energy at a range of specific wavelengths. This absorption of light causes electron to rise from the ground state to a higher energy level (excited state). The excited electron quickly decays to its ground state while releasing the excess energy in the form of photon of light. This transition of energy is called fluorescence.
https://www.creative-bioarray.com/support/fluorochromes-in-flow-cytometry.htm
Creative Bioarray provides our global clients the most comprehensive histology services. Drawing on many years of experience and in-depth knowledge, Creative Bioarray offers tissue processing, embedding, sectioning, and staining. Besides a histological examination of all major organs/tissues is provided, including immunohistochemistry (IHC), immunofluorescence (IF), in situ hybridization (ISH), fluorescent in situ hybridization (FISH), and transmission electron mircoscopy.
https://www.creative-bioarray.com/Services/Histology-Services.htm
Creative Bioarray offers 35 human cell systems with over 160 different cell types. Moreover, we also provide our customers with primary cells from over 13 types of other animals.
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Drug transporters mediate the uptake and efflux of a broad variety of drugs and drug metabolites. Most uptake transporters are members of the solute carrier (SLC) family, while most efflux transporters are members of the ATP-binding cassette (ABC) transporter family.
https://www.creative-bioarray.com/services/drug-transporters.htm
Acroscell provides ready-to-use beating human induced pluripotent stem cells (iPSC)-derived cardiomyocytes. Generated from mature cells that have been genetically reprogramed to a pluripotent stem cell state, induced pluripotent stem cells (iPSCs) can be readily expanded and induced to specialize or differentiate into cardiomyocytes in vitro.
https://www.creative-bioarray.com/acroscell/ipsc-derived-cardiomyocytes.html
PAMPA provides a simplified approach to permeability by addressing just a single transport mechanism. This avoids the complexities of active transport and metabolism, enabling ranking of the compounds on a single permeability factor.
https://dda.creative-bioarray.com/parallel-artificial-membrane-permeability-assay.html
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
3. In Situ Hybridization
In Situ Hybridization (ISH) is a
technique that allows for
precise localization of a specific
segment of nucleic acid within
a histologic section. The
underlying basis of ISH is that
nucleic acids, if preserved
adequately within a histologic
specimen, can be detected
through the application of a
complementary strand of
nucleic acid to which a reporter
molecule is attached.
Introduction
In Situ Hybridization
5. Probe types and their pros and cons
Probe Types Advantages Disadvantages
Double-stranded DNA (dsDNA)
probes
Stable, available, easier to obtain Self-hybridize, less sensitive, need
denaturation before hybridization
Single-stranded DNA (ssDNA)
probes
Stable, easier to work with, more
specific, resistant to RNases,
better tissue penetration, without
self-hybridize
Time consuming, expensive
RNA probes (riboprobes) Higher thermal stability, better
tissue penetration, more specific,
low background noise by RNase
Sensitive to RNases
Synthetic oligonucleotides Economical, stable, available,
easier to work with, more specific,
resistant to RNases, better tissue
penetration, better reproducibility
Know the information of
nucleotide sequence
Probes - ISH
6. Labeling Techniques - ISH
Probe Labeling and Signal Detection
32P
35S
3H
Biotin
Digoxigenin
Fluorescent dye
(FISH)
Radioactive isotopes Non-radioactive labels
7.
8. Chromogenic In Situ Hybridization (CISH)
CISH enables examination of gene amplification,
gene deletion, chromosomal translocations, and
chromosomal number. This approach uses
conventional peroxidase or alkaline phosphatase
reactions using bright-field microscopy on
tissues fixed by formalin and embedded in
paraffin. These peroxidase or alkaline
phosphatase-labeled reporter antibodies
interact with a hybridized DNA probe, and are
then observed with an enzymatic reaction. Tissue
morphology and genetic abnormalities can be
viewed at the same time with CISH.
Introduction of CISH
9. Variations - CISH
SISH uses a similar method as
CISH, but a silver precipitate is
the end product rather than a
brown product.
Silver-enhanced in situ
hybridization (SISH)
10. 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0
DuoCISH is a variation of CISH
that addresses the need for
two different probes on the
same slide. It is a well-
established technique for HER-
2/neu amplification detection
even though it is sometimes
reported to be less effective
than FISH.
DuoCISH
Variations - CISH
11. Medical Applications
Gene amplification
CISH is frequently applied to assess gene
amplification, such as HER-2/neu status in
breast cancer samples.
Chromosomal rearrangements
CISH is also used for detection of chromosomal
rearrangements and fusions, such as the fusion
of ALK tyrosine kinase domain with the
promoter and 5’ region of EML4 in lung cancer.
Papillomavirus infections
Apart from cancers, CISH has also been shown
to be useful in detecting human papillomavirus
infections.
Chromogenic In Situ Hybridization (CISH)
12. Fluorescence In Situ Hybridization (FISH)
Fluorescence in
situ hybridization (FISH) is
a molecular
cytogenetic technique that
uses fluorescent
probes that bind to only
those parts of a nucleic
acid sequence with a high
degree of
sequence complementarity.
13. Single-molecule RNA FISH is a method of detecting
and quantifying mRNA and other long RNA
molecules in a thin layer of tissue sample.
Single-molecule RNA FISH assays can be performed
in simplex or multiplex, and can be used as a follow-
up experiment to quantitative PCR, or imaged
simultaneously with a fluorescent antibody assay.
The technology has potential applications in cancer
diagnosis, neuroscience, gene expression analysis,
and companion diagnostics.
Single-molecule RNA FISH
Variations - FISH
14. Variations - FISH
In an alternative technique
to interphase or metaphase
preparations, fiber FISH, interphase
chromosomes are attached to a slide in
such a way that they are stretched out
in a straight line, rather than being
tightly coiled, as in conventional FISH,
or adopting a chromosome
territory conformation, as in interphase
FISH. This is accomplished by applying
mechanical shear along the length of
the slide, either to cells that have been
fixed to the slide and then lysed, or to a
solution of purified DNA.
Fiber FISH
15. Q-FISH
MA-FISH
Variations - FISH
Microfluidics-assisted FISH (MA-FISH) uses a
microfluidic flow to increase DNA hybridization
efficiency, decreasing expensive FISH probe
consumption and reduce the hybridization time.
MA-FISH is applied for detecting
the HER2 gene in breast cancer tissues.
Q-FISH combines FISH with PNAs and
computer software to quantify
fluorescence intensity. This technique is
used routinely in telomere length research.
16. Hybrid Fusion FISH (HF-FISH) uses
primary additive excitation/emission
combination of fluorophores to
generate additional spectra through a
labeling process known as dynamic
optical transmission (DOT). Three
primary fluorophores are able to
generate a total of 7 readily detectable
emission spectra as a result of
combinatorial labeling using DOT.
Hybrid Fusion FISH enables highly
multiplexed FISH applications that are
targeted within clinical oncology panels.
Hybrid Fusion-FISH
Variations - FISH
18. General Procedure
- Select appropriate BAC for target of interest, extract, and amplify DNA
- Label probe with biotin or digoxigenin using random priming or nick translation
Probe
Design
- Fix tissue to a glass slide using paraffin
- Wash and heat slide several times to remove paraffin from surface
- Pepsin digestion to ensure access to target DNA sequence
Tissue
Prep
- Add 10-20 uL probe to tissue, cover slide, and seal coverslip
- Heat slide to 97℃ for 5-10 min to denature DNA
- Place slide in 37℃ oven overnight for probe to hybridize
Hybridization
- Add a blocker to bind nonspecific binding sites
- Add hydrogen peroxide to suppress endogenous peroxidase activity
Blocking
- - If digoxigenin is the label: add anti-digoxigenin fluorescein primary antibody
followed by a HRP-conjugated anti-fluorescein secondary antibody
- - If biotin is the label: HRP-conjugated streptavidin is used for detection
- - Add DAB which is converted to a brown precipitate by HRP
Probe
Detection