Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
Blotting technique including Southern , Northern and Western blotting Rohit Mondal
he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Concept: reannealing nucleic acids to identify sequence of interest.
Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.
Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.
Size bands and quantify abundance of molecule.
Similar to VAISHNAVI GHUGAL(SCREENING OF RECOMBINANTS).pptx (20)
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 .
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
3. INTRODUCTION
SCREENING: A population of viable cells is subjected to some sort of analysis that
enables the desired sequence to be identified.
SCREENING METHODS
DIRECT
METHOD
Use of
marker or
reporter
gene
Selection by
complement
ation or
nonsense
suppression
Marker
inactivatio
n
techniques
INDIRECT
METHOD
Restriction
enzyme cleavage
pattern
Hybridization
techniques
Colony or
plague
hybridization
Colony
lift
Blotting
technique
s
Southern
blotting
Northern
blotting
Western
blotting
Detection of specific
proteins
Protein
synthesis in
mini cells
Immunologica
l method
4. SCREENING BY COMPLEMENTATION
Functional complementation assay is an in vivo that is widely used to elucidate the
function/role of genes/enzymes.
This technique is very common in biochemistry, genetics and many other disciplines.
EXAMPLES:
Blue-white screening(α complementation of β-galactosidase)
5. BLUE-WHITE SCREENING(α complementation
of β galactosidase)
Scientist discovered that deleting a section from lacZ gene
(a mutation called lacZΔM15) creates a nonfunctional
β-galactosidase enzyme. Providing DNA encoding this
section of amino acid(called α-peptide) to a lacZΔM15
mutant bacterial cell in trans complements the mutation
allowing for a functional enzyme. This process
is called α -complementation.
BLUE COLONY= NON-RECOMBINANT
WHITE COLONY=RECOMBINANT
Foreign DNA in MCS, no α fragment
No α fragment, no β -gal
No β -gal, no blue color(white colonies)
http://www.sigmaaldrich.com/content/dam/sigma-
aldrich/articles/biology/blue-white-screening/typical-blue-white-screening-
procedure.jpg
6. COLONY LIFT
Colony hybridization is a screening method used in the selection of bacterial colonies
with a desired DNA sequence with high density.
The procedure of colony hybridization was first developed by Grunstein and Hogness
in 1975. It is also called colony blot hybridization, colony lift or replica plating.
https://tse2.mm.bing.net/th?id=OIP.ZXDTVTS7cX8JIXY-eQkMAgHaFj&pid=Api&P=0&w=220&h=165
7. SOUTHERN BLOTTING
Southern blotting technique is employed for the:
I. detection of specific DNA sequences/genes
II. detection of abnormalities in a given gene structure
The method was named in honor of the British biologist Edwin M. Southern,
University of Oxford who first published it in 1975, and hence the name Southern
Blotting.
Major steps involved may be summarized as follows:
Step 1: Extraction and Purification of DNA from cells.
Step 2: Restriction Digestion (Fragmentation of sample).
Step 3: Electrophoretic separation of DNA fragments in the sample.
Step 4: Partial depurination to promote higher efficiency transfer of DNA fragments.
Step 5: Denaturation with mild alkali and subsequent blotting.
Step 6: Probe-hybridization (binding of analytical probe) to target molecule.
Step 7: Visualization of the bound probe by Autoradiography
8. APPLICATIONS
It is a widely accepted analytical technique used in
molecular biology and immunogenetics to identify the
DNA of interest from a mixture of DNA samples or to
detect a specific base sequence within a strand of DNA.
Identification of methylated sites in particular genes.
Preparation of RFLP maps.
Detection of point-mutations, deletions or gene
rearrangements in DNA.
In forensic medicine, for criminal identification and DNA
fingerprinting (VNTR).
Detection and identification of transgene in transgenic
individuals.
For diagnosis of infectious diseases and prognosis of
cancer.
Useful in prenatal diagnosis of genetic diseases.
https://www.onlinebiologynotes.com/wp-content/uploads/2017/12/southern-blotting.gif
9. NORTHERN BLOTTING
Northern blotting is used in molecular biology to study gene expression by detection of
RNA (isolated mRNA) in a sample.
It is a variant of Southern Blotting, developed in 1977 by James Alwine, David Kemp,
and George Stark at Stanford University, with significant contributions from Gerhard
Heinrich.
Major steps involved Northern Blotting :
• Step 1: Extraction of total RNA from a homogenized tissue sample or from cells.
• Step 2: Electrophoretic separation of RNA fragments in the sample.
• Step 3: Denaturation to limit the secondary structure followed by subsequent blotting.
• Step 4: Probe-hybridization (binding of analytical probe) to target molecule.
• Step 5: Visualization of the bound probe by Autoradiography.
10. APPLICATIONS
Used in diagnosis of environmental stress levels
and pathogen infection.
To detect the over-expression of oncogenes and
down-regulation of tumor-suppressor genes in
cancerous cells when compared to 'normal' tissue.
Used to identify gene expression in the rejection of
transplanted organs.
Helpful in studying mechanism of RNA
degradation and splicing.
Detection of molecular weight of a specific
mRNA.
Identification of mRNA produced by transgenes to
protect the recombinants.
https://s-media-cache-
ak0.pinimg.com/originals/4d/df/37/4ddf37f5a902dd879bee3c1bda2a7bd5.jpg
11. WESTERN BLOTTING
Western blotting is a widely applied analytical technique employed in molecular
biology and immunogenetics to identify and characterize specific proteins within a
sample of tissue homogenate or extract.
The method by itself originated in 1979 in the laboratory of Harry Towbin (Friedrich
Miescher Institute, Switzerland), but the term ‘western’ was later put forth by W.
Neal Burnette in 1981.
Major steps involved may be summarized as follows:
Step 1: Extraction and homogenization of the protein sample.
Step 2: Treatment with a suitable buffering solution.
Step 3: Electrophoretic separation of protein using SDS-PAGE.
Step 4: Electro-transfer into nitrocellulose/PVDF filter membrane.
Step 5: Preparation of membrane for antibody-staining.
Step 6: Incubation with primary antibodies, specific to detection of target protein.
Probe-hybridization (binding of analytical probe) to target molecule.
Step 7: Detection via (i). Colorimetry, (ii). CCD Camera, LED/Infrared Imaging.
12. APPLICATIONS
It is a routine method in molecular biology,
biochemistry, cell biology and
immunogenetics with a multitude of
applications.
Widely used method for detection of target
protein from complex samples, using
antibody-based probes.
Used to obtain information about quantity,
molecular weight and post-translational
modifications of proteins under study.
It is an essential tool for analyzing protein
moieties of complex systems.
Monitors changes in expression or post-
translational modifications in samples of
protein.
https://cdn.antibodies.com/image/resources/Western%20Blot%20-%20Figure%201.png
13. SOUTH-WESTERN SCREENING
South-western screening is a method for identifying a protein which binds to a protein-
binding site on the DNA .
cDNA is cloned into suitable expression vector to form
expression library.
Library is plated on nutrient agar.
Well separated phage plagues are formed.
Nitrocellulose filter is applied on agar & then peeled of to
give imprint of the phage.
Foreign cDNA sequence are expressed to give protein, and the
fusion proteins are adsorbed in nitrocellulose membrane.
Membrane is incubated in radiolabelled dsDNA representing
the known protein -binding sequence.
DNA probe binds to specific protein. This leads to
identification of specific binding protein.
14. IMMUNOPRECIPITATION
Immunoprecipitation is based on the use of antibodies to isolate the proteins against
which they are directed.
The process is as follows:
Cells are incubated
with radioactive
amino acids to
label the proteins.
Radiolabelled cell extract is
incubated with the antibody
against the target protein.
The antibody
binds to specific
protein forming
antigen-antibody
complex.
The antigen-antibody
complex is isolated by
incubating with beads
that bind the antibody.
Beads are
boiled to
dissociate
complex.
Proteins
recovered
are
analyzed by
gel
electrophor
esis.
Radioactive
proteins can be
detected by
autoradiography.