DNA footprinting is a technique to study how proteins interact with DNA. It involves treating DNA with nucleases or chemicals that cut DNA, but are blocked from cutting where a protein is bound. This leaves a "footprint" of protected DNA that reveals the protein's binding site. The technique was developed in 1978 and can detect even transient protein-DNA interactions to help understand transcriptional control within cells.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
DNA Protein interaction occur when a protein binds a molecule of DNA, often to regulate the biological function of DNA, usually the expression of a gene. DNA Protein interactions play very vital roles in any living cell. It controls various cellular processes which are very essential for living beings, viz. replication, transcription, recombination, DNA repair etc. There are several types of proteins found in a cell.Direct recognition occurs when the amino acid side chains of a protein interact with specific DNA bases.
Most protein-DNA interactions are mediated by direct physical interaction (hydrogen bonding or hydrophobic interactions) between the protein and the DNA base pairs.
DNA-binding proteins can be identified by many experimental techniques such as chromatin immunoprecipitation on microarrays, X-ray crystallography and nuclear magnetic resonance (NMR).
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
DNA Protein interaction occur when a protein binds a molecule of DNA, often to regulate the biological function of DNA, usually the expression of a gene. DNA Protein interactions play very vital roles in any living cell. It controls various cellular processes which are very essential for living beings, viz. replication, transcription, recombination, DNA repair etc. There are several types of proteins found in a cell.Direct recognition occurs when the amino acid side chains of a protein interact with specific DNA bases.
Most protein-DNA interactions are mediated by direct physical interaction (hydrogen bonding or hydrophobic interactions) between the protein and the DNA base pairs.
DNA-binding proteins can be identified by many experimental techniques such as chromatin immunoprecipitation on microarrays, X-ray crystallography and nuclear magnetic resonance (NMR).
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when two proteins or polypeptides of interest interact. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
Recombinant dna technology and DNA sequencinganiqaatta1
title: recombinant DNA technology and DNA sequencing
this lect will cover the pcr, isolation of DNA, detection of DNA and DNA manipulation joining DNA together. this is very important and it is required in research of every field especially medical related field.
The interface in a complex involves two structurally matched protein subunits, and the binding sites can be predicted by identifying structural matches at protein surfaces.
Identification of Protein–Ligand Binding Sites by Sequence & Identifying protein–ligand binding sites is an important process in drug discovery and structure-based drug design. Detecting protein–ligand binding sites is expensive and time-consuming by traditional experimental methods. Hence, computational approaches provide many effective strategies to deal with this issue. Recently, lots of computational methods are based on structure information on proteins. However, these methods are limited in the common scenario, where both the sequence of protein target is known and sufficient 3D structure information is available. Studies indicate that sequence-based computational approaches for predicting protein–ligand binding sites are more practical. Different methods were used to determine protein binding sites fir instance, chromatin immuno preciptitation assay ( ChIP),
Electrophoretic mobility shift assay (EMSA), Dnase footprinting assay etc.
Blotting
A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.
Types of blotting techniques
Southern Blotting
Northern Blotting
Western Blotting
A Southern blot is a method used
in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
The method is named after its inventor, the British biologist Edwin Mellor Southern.
- Methods in Southern blotting
- Advantages and disadvantages
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.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
4. (TCO 9) Provide a detailed description of the techniques used to .pdfarrowit1
4. (TCO 9) Provide a detailed description of the techniques used to make a DNA fingerprint.
What are some of the uses and applications of DNA fingerprinting?
Solution
DNA fingerprinting is a technique used to determine the nucleotides sequences of DNA which
are unique to each individual.
Technique
1. Extraction of the DNA from the source the DNA is extracted from blood sample, hair follicles
etc.available sample.
2.DNA is cut into fragments the DNA molecules broken with the help of restriction
endonuclease. Here the cleaning is double strand cut producing DNA fragments of different
lengths this fragment are also called restricted fragment length polymorphism Manyi of this
fragment contain vntr
3. Separation of the fragments using gel electrophoresis. As the DNA molecule is negatively
charged hence it will move towards positive or not in the setup the gel based matrix provides tiny
pores through which DNA molecules travel the larger molecules travel slowly where is the
smallest mens travel quickly from the loading point at the end of the experiment DNA pieces of
equal length obtained.
4. The DNA fragments or now treated with alkaline chemicals to facilitate denaturation into
single stranded DNA this is very important step.
5. Southern blotting technique in this technique nitrocellulose membrane is used the DNA is
bloated on suitable membrane like nitrocellulose or nylon membranes as they have good binding
capacity the membrane is subjected to gentle pressure due to this single stranded DNA fragments
are pulled and transfer onto the membrane . the membrane contains replica of the DNA.
Hybridisation with suitable DNA probe which is single stranded DNA having complementary
sequence to the desired DNA. Before using the probe the DNA of tagged with fluorescent dyes
to help in detection of the desired DNA excess probea are washed away.
5. the DNA sample is visualised using autoradiography the hybridisation pattern is called DNA
fingerprint having a sequence complementary to the probe.
6. PCR technique is a technique is useful to synthesise millions of copies of the DNA sequence
when low amount of DNA is available for the study this technique is used modifications of PCR
technique like r a p d PCR rflp PCR helps in giving accurate results.
Applications of DNA fingerprinting :
1.This test is used in the case of disputes regarding paternity testing .
2 it is useful tool in forensic applications
3.It is used to assess migration pattern of ancient population
4.it is used to determine Genetic diversity is in the evolutionary biology.
4. It is used to diagnose inherited disorders in both prenatal and newborn babies examples
huntington\'s disease Alzheimer\'s Sickle Cell anaemia Thalassemia haemophilia.
5. DNA fingerprinting is used to come from confirm cell line identity in a cell line collection.
6. It also helps in developing cures for inherited disorders..
The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...
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.
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.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
2. INTRODUCTION
• DNA footprinting is a method of investigating the sequence specific of
DNA – binding protein in vitro.
• This technique can be used to study protein – DNA interactions both
outside and within cell.
• Techniques like DNA footprinting help elucidate which protein bind to
these associated region of DNA and unravel the complexities of
transcriptional control.
3. HISTORY
• In 1978,David galas and Albert Schmitz developed the DNA
footprinting technique to study the binding specificity of the lac
repressor protein .
• It was originally a modification of the maxam – gilbert chemical
sequencing technique .
4. PRINCIPLE
• In this technique, nucleases like DNase I is used which will degrade
DNA molecule. Nucleases cannot degrade DNA if it is bounded by a
protein. Thus that region is protected from degradation by nucleases.
This protected DNA region is called the foot print.
5. METHOD
The simplest application of this technique is to assess whether a given
protein binds to a region of interest within a DNA molecule
• Polymerase chain reaction(PCR) amplify and label region if interest
that contain a potential protein- binding site.
• Add protein of interest to a portion of the labelled template DNA ; a
portion should remain separate without protein ,for later comparison
• Now, add a cleavage agent to both the portion of DNA template
(cleavage agent is a chemical or enzyme that will cut at random
locations in a sequence independent manner).
6. • Run both the samples side by side on a polyacrylamide gel
electrophoresis .
• The portion of DNA template without protein bill will be cut at
random locations , and thus when it is run on a gel ,will produce a
ladder- like distributions.
• The DNA template with a protein will result in ladder distribution with
a break in it , the “footprint” , where the DNA has been protected
from the cleavage agent .
7. Labeling
• The DNA template can be labeled at the 3’ or 5’ end , depending on
the location of binding sites .
• Labels that can be used are : radioactivity and fluorescence .
• Radioactivity has been traditionally used to label DNA fragments for
footprinting analysis.
• Radioactive labelling is very sensitive and is optimal for visualising
small amount of DNA .
• Fluorescence is a desirable advancement due to the hazards of using
radio-chemicals .
8. • However, it has been difficult to optimize because it is not always
sensitive enough to detect the low concentrations of the target DNA
strands used in a DNA footprintings experiments.
• Electrophoretic sequencing gels or capillary electrophoresis have
been successfully in analysing foot printing of fluorescent tagged
fragments .
9. Cleavage agent
• A variety of cleavage agent can be chosen .
• Ideally a desirable agent is one that is sequence neutral , easy to use ,
and is easy to control .
• The following cleavage agent are described in detail : DNase I is a
large protein that function as a double – strand endonuclease .
• It binds the minor group of DNA and cleaves the phospodiester
backbone .it is good cleavage agent for footprinting because its size
makes it easily physically hindered .thus is more likely to have to its
action blocked by a bound protein on a DNA sequence In addition ,
the DNase I enzyme is easily controlled by adding EDTA to stop the
reaction .
10. • Hydroxyl radicals are created from the Fenton reaction , which
involves reducing Fe2+ with H2o2 to form free hydroxyl molecules
.These hydroxyl molecules react with the DNA backbone resulting in a
break. Due to their small size , the resulting DNA footprint has a high
resolution .
• Ultraviolet irradiation can be used to excite nucleic acid and create
photoreactions , which result in damaged bases in a DNA strand.
Advantages of uv are that it reacts with very quickly and therefore
capture the interactions that are only momentary .
11. Applications
• DNA footprinting is often used to identify the binding sites of
proteins in a DNA molecule. Researchers often use this technique to
identify whether a particular protein can activate or inhibit
transcription. In addition, scientists also use this method to detect
where proteins bind to DNA in a living cell.