Southern, Northern, and Western blotting techniques allow for the detection of specific DNA, RNA, and protein fragments, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe for detection via hybridization. Northern blotting is similar but detects RNA. Western blotting separates proteins via gel electrophoresis, transfers them to a membrane, then uses primary and secondary antibodies for detection. These techniques are used for applications like gene mapping and the study of gene expression.
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.
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.
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
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
This presentation is about DNA fingerprinting, a brief description is given about its principle, working, technique and its application with a example.
WHAT IS BLOTTING?
Blotting is a technique for detecting any macromolecules that we deal with like DNA, RNA or proteins, which are initially present in a complex mixture.
TYPES OF BLOTTING:
Southern Blotting
Northern Blotting
Western Blotting
NORTHERN BLOTTING
A northern blotting is a laboratory method used to detect specific RNA molecules among a mixture of RNA (mRNA).
The technique was developed in 1979 by James Alwine and his colleagues.
Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the expression of particular genes.
Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence.
The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However the entire process is commonly referred to as northern blotting.
PROCEDURE
1.RNA isolation:
2.Separation of RNA using gel electrophoresis:
3.BLOTTING:
4.Hybridization with labelled probe:
5.WASHING OFF EXCESS PROBES
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites.
This presentation is about DNA fingerprinting, a brief description is given about its principle, working, technique and its application with a example.
Scintillation counter - instrumentation Principle, working, advantages and disadvantages and applications on various fields.
Reference : principles of biochemistry by wilson and walker.
Khaled El Masry, is an assistant Lecturer of Human Anatomy & Embryology, Mansoura University, Egypt. Great thanks to Prof. Dr Salwa Gawish, professor of Cytology & Histology, Mansoura University, for her great effort in explaining Genetics course.
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
What is blotting? Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
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2. DefinitionDefinition
Visualization of specific DNA , RNA &
protein among many thousands of
contaminating molecules requires the
convergence of number of techniques
which are collectively termed BLOT
transfer .
3. Types of blotting techniquesTypes of blotting techniques
1 ) Southern blotting ( to detect DNA )
2 ) Northern blotting ( to detect RNA )
3 ) Western blotting ( to detect
protein )
4. Southern BlottingSouthern Blotting
In 1975 Edward Southern developed this
technique that is widely used to detect
fragments of DNA .
This requires
1 ) Separation of DNA or DNA fragments by
agarose gel electrophoresis .
2 ) DNA fragments are blotted onto a strip of
nitrocellulose or a nylon membrane.
3 ) Identification by hybridization with a
labeled ,complementary nucleic acid probe.
5. DefinitionDefinition
Southern blot a method for transferring DNA
from an agarose gel to nitrocellulose filter ,
on which the DNA can be detected by
suitable probe ( eg : complementary DNA or
RNA ) .
6. ProcedureProcedure
The DNA sample is digested by restriction
endonucleases , producing small fragments &
that are amenable for analysis .
Fragments are seperated by agarose gel
electrophoresis or PAGE .
The mobility of nucleic acids in agarose gels is
influenced by agarose concentration ,
molecular size & molecular conformation of
the nucleic acid .
7. Agarose concentration of 0.3 – 2 %are
most effective for nucleic acid
separation .
Like proteins nucleic acids migrate at rate
that is inversely proportional to the
logarithm of their molecular weight .
8. contdcontd
Separated nucleic acids are visualized by
fluorescent dye ethidium bromide .
The agarose gel is soaked in a solution of dye
& washed for remain excess dye .
illumination of the rinsed slab with UV light
reveals red orange stains where nucleic acids
are located .
9. contdcontd
Ethidium bromide stains both single & double
stranded nucleic acids , the fluorescence is
much greater with double stranded molecules
.
The electrophoresis can be performed with
dye incorporated in the gel & buffer .
This has the advantage that the gel can be
illuminated with UV light during
electrophoresis to view the extent of
separation.
10. contdcontd
The mobility of DNA may be reduced by 10
-15 % in the presence of ethidium bromide .
Ethidium bromide must be used with great
care as it is a potent mutagen .
Gloves should be worn at all times while
using the dye solutions or handling gels .
11. contdcontd
Newer fluorescent SYBR dyes produced by
molecular probes offer several advantages ,
less toxic & 5 times more sensitive than
ethidium bromide.
Labeled DNA with radioisotope P32
at 5’ & 3’
ends .
P 32
is a strong β emitter .
Bands of labeled DNA on electrophoresis gel
can located by autoradiography .
12. contdcontd
Labelling molecule before analysis with
coenzyme biotin , biotin forms a strong
complex with enzyme linked streptavidin .
PAGE is useful for analysing small fragments
of DNA upto 3,50,00 daltons ( 500 bp ) in
molecular size .
Large molecules of DNA could be separated
by pulsed field gel electrophoresis.
13. BlottingBlotting
Transfer of DNA from gel to nitrocellulose
membrane done by
1 ) Weak acid treatment to depurinate &fragment
the DNA , thus make it smaller & easier to elute
from the gel .
followed by
2) Denaturate with strong base& neutralisation
( hydrolyzes phosphodiester back bone at
depurinated sites )
single strands bind to membranes more efficiently )
A buffer is used to facilitate the transfer .
14. contdcontd
Original methods of transfer relied on
capillary action .
Vaccum or preassure systems can be used to
speed the transfer .
Faster & more efficient transfer is afforded by
the use of an electroblotter .
Electroblotting process is usually completes in
1-4 hours .
15. Hybridization assaysHybridization assays
All hybridization assays are based on the
ability of nucleic acids to form specific double
stranded hybrids .
The process requires
1 ) A probe that can target nucleic acids &
allow for specific complemenatary base
pairing .
2) A method to detect any resulting double
strands nucleic acids .
16. contdcontd
Conditions of high stringency in
hybridization assay are
1) Low salt concentration ,
2) High formamide levels ,
3) High temparature .
As the stringency of the assay is lowered
increasing number of base mismatches are
tolerated .
conditions of high stringency require exact base
pairing .
17. The time required to hybridize the probe to a
given fraction of the target remains
proportional to the probe concentration .
The rate of hybridization reaction is
influenced by temperature & ionic strength.
Above the Tm no stable hybrids are present .
Divalent cations like Mg+2
have stronger effect
on hybridization .
18. contdcontd
Unbound probes are removed by washing
Probe bound to the membrane is detected by
autoradiogarphy , which reveals the DNA
fragments to which the probe hybridized .
19. ApplicationsApplications
Southern blots are used in gene discovery, mapping ,
evolution & development studies , diagnostics &
forensics .
Deletions / insertions .
pointmutations / polymorphisms .
Structural rearrangements .
Allow for determination of molecular weights of
restriction fragments .
Presence of particular bit of DNA in the sample.
20. Northern blottingNorthern blotting
Northern blotting is a technique for detection of
specific RNA sequences .
Developed by James alwine & George stark.
RNA molecules have defined length & much shorter
than genomic DNA it is not necessary to cleave
RNA before electrophoresis .
RNA is more susceptible to degradation than DNA .
RNA sample are separated based on size by gel
electrophoresis .
21. contdcontd
RNA is blotted on to a nylon positively
charged membrane .
The membrane is placed in a hybridization
buffer with a labeled probe ( usually DNA )
Labeled probe is detected by autoradiography
Expression patterns of sequences of interest
in different samples can be compared .
22. ApplicationsApplications
A standard for direct study of the gene
expression at the level of mRNA .
Detection of mRNA transcript size .
Study of RNA splicing – can detect
alternatively spliced transcripts .
Study RNA half life
24. Western blottingWestern blotting
Western blotting is an immunoblotting
technique which rely on the specificity of
binding between the molecule of interest & a
probe to allow detection of molecule of
interest in a mixture of many other similar
molecules .
In western blotting the molecule of interest is
a protein & the probe is typically an antibody
raised against that particular protein .
25. ContdContd
SDS PAGE technique is a prerequisite for
western blotting .
Protein sample is subjected to
electrophoresis on SDS polyacrylamide gel .
Electroblotting transfers the separated
proteins from the gel to the surface of
nitrocellulose membrane .
26. contdcontd
Blot is incubated with generic protein
( such as milk protein )which binds to any
remaining sticky places on the nitrocellulose .
An antibody which is specific for the protein
of interest ( the primary antibody Ab 1 ) is
added to the nitrocellulose sheet & reacts
with the antigen . Only the band containing
protein of interest binds the antibody forming
a layer of antibody molecules .
27. contdcontd
Following several rinses for removal of
nonspecifically bound Ab1 , the Ab1 – antigen
complex on the nitrocellulose sheet is
incubated with second antibody Ab2 , which
specifically recognizes the Fc domain of the
primary antibody & binds it . Ab 2 is
radioactive labeled or is covalently linked to
reporter enzyme which allows to visualize
protein – Ab1 – Ab2 complex .
28. ApplicationsApplications
The confirmatory HIV test employs a western
blot to detect anti HIV antibody in a human
sample .
Proteins from known HIV infected cells are
separated & blotted on a membrane then the
serum to be tested is applied in the primary
antibody incubation step.
Free antibody is washed away & a second anti
human antibody linked to an enzyme signal
can be added .
29. contdcontd
The stained bands then indicate the proteins
to which the patient serum contains
antibody .
Western blot is also used as definitive test for
bovine spongiform encephalopathy . ( mad
cow disease )
Some forms of Lyme disease testing employs
western blotting .