Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Principle: The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane.
It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts.
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.
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
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
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
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Principle: The method is characterized by transferring the protein which run on a gel by electrophoresis on to a nitro cellulose membrane.
It is widely used analytical technique in molecular biology to detect specific proteins in a sample of tissue homogenate or extracts.
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.
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
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
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.
Southern blotting is a laboratory technique used to detect specific DNA sequences in DNA samples. It involves several steps:
Restriction Digestion: DNA from a biological sample (such as blood or tissue) is broken into smaller fragments using restriction enzymes, which cut the DNA at specific sequences.
Electrophoresis: The DNA fragments are separated based on their molecular weights using gel electrophoresis. This process allows smaller fragments to move faster than larger fragments.
Transfer to Membrane: The DNA fragments are transferred from the gel onto a solid membrane, typically a positively charged nylon membrane, using capillary action.
Hybridization: The membrane is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The probe is designed to be complementary to the target DNA sequence, allowing it to bind to the specific DNA fragment on the membrane.
Detection: The bound probe is detected using methods such as X-ray film, phosphorimaging, or chemiluminescent substrates, depending on the type of label used.
Southern blotting is used in various applications, including:
Identifying specific DNA sequences in DNA samples
Studying gene rearrangements and mutations
Analyzing viral and bacterial infections
Forensic analysis and personal identification
Gene mapping and restriction enzyme mapping
Identifying methylated sites in genes.
This technique is named after its inventor, Dr. Edwin Southern, who first published it in 1975.
This is all about southern blotting
a brief on thyroid gland covering following titles:
Introduction
Anatomy and physiology of thyroid gland
Synthesis of thyroid hormones
Regulation
Mechanism of action
Biological function
MI is one of the CVS complication leading to mortality whose diagnosis is mainly dependent on clinical presentation and other supportive investigation. clinical laboratory plays crucial role in its diagnosis, prognosis and monitoring therapy.
introduction of Purine and Pyrimidine metabolism, biosynthesis and degradation of nucleotides, biological functions and metabolic disorders, chemical analogues and therapeutic drugs, uric acid metabolism
Triacylglycerol and compound lipid metabolismDipesh Tamrakar
Biosynthesis and metabolic regulation of triglyceride and other compound lipids: glycerophospholipids, sphingophospholipids, ether glycerolipids and glycolipids
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
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
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
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
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.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
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.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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
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.
2. Definition
Visualization of specific DNA, RNA & protein by BLOT transfer
Types:
1. Southern
blotting
( to detect
DNA )
1.2. Northern
blotting
2.( to detect
RNA )
1.3. Western
blotting
2.( to detect
protein )
1.4. Eastern
blotting
2.( to detect
protein of post
translational
modifications )
2
3. 1. Southern Blotting
The technique was developed by E.M. Southern in 1975
Used to detect the presence of a particular fragment of DNA in a
sample
This DNA can be:
• Single gene
• Part of a larger piece of DNA s/a viral genome
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 )
3
4. “Used to detect the DNA”
• This method Involves:
1. Separation
2. Transfer
3. Hybridization.
“The key to this method is Hybridization”
4
5. DNA
Each individuals unique genetic blueprint is stored in material known
as DNA.
DNA is found in all cells containing a nucleus.
DNA can be extracted for analysis from hair, bones, saliva, sperm, skin,
organs, all body tissues and blood.
Temperature Storage for DNA
Purified DNA may be refrigerated at 4°C for up to 3 years.
Samples kept over 3 years should be frozen at -70°C.
• Specimens used in DNA testing:
• Whole blood, Solid tissue, Serum and plasma
• Urine, Bone marrow and many others
5
6. 1. Separation
The DNA sample is digested by restriction endonucleases , producing
small fragments & that are amenable for analysis .
Fragments are separated 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 .
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 .
6
7. Separated nucleic acids are visualized by fluorescent dye ethidium
bromide
The agarose gel is soaked in a solution of dye & washed for remaining
excess dye
illumination of the rinsed slab with UV light reveals red orange stains
where nucleic acids are located
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.
7
8. 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
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
P32 is a strong β emitter
Bands of labeled DNA on electrophoresis gel can located by
autoradiography
8
9. • Labelling molecule before analysis with coenzyme biotin , biotin forms
a strong complex with enzyme linked streptavidin .
• PAGE is useful for analyzing small fragments of DNA upto 500 bp
• Large molecules of DNA could be separated by pulsed field gel
electrophoresis.
9
10. • Ethidium bromide is an intercalating agent commonly used as a
fluorescent tag
• It is commonly abbreviated as "EtBr", which is also an abbreviation for
bromoethane
• When exposed to ultraviolet light (210 nm), it will fluoresce with an
orange colour, intensifying almost 20-fold after binding to DNA
• EtBr emits orange light with wavelength 605 nm.
Ethidium bromide
intercalated
between two
adenine-thymine
base pairs.
10
11. 2. Blotting
• 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 & neutralization ( hydrolyzes
phosphodiester back bone at depurinated sites )
• single strands bind to membranes more efficiently
• A buffer is used to facilitate the transfer .
11
12. a. Capillary blotting:
The gel is placed on a wet pad of buffer-
soaked filter paper & sheet of nitrocellulose
placed on the gel
Buffer is then drawn through the gel by
placing a pad of dry absorbent material
(usually filter paper) followed by a heavy
weight on top of the sheet
Passage of buffer by capillary action through
the gel carries the separated nucleic acids on
the nitrocellulose sheet to which they bind
irreversibly by hydrophobic interaction
12
13. b. Electro blotting:
Quicker & more efficient method of transfer
Sandwich of gel & nitrocellulose compressed in
a cassette & immersed in buffer between two
parallel electrodes
A current is passed at right angles to the gel
which causes the separated nucleic acids to
electrophorese out of the gel & into
nitrocellulose sheet
Obtained blot can be analyzed
13
14. 3. Hybridization
The key to this method is hybridization.
Hybridization-process of forming a double-stranded DNA molecule
between a single-stranded DNA probe and a single-stranded target
patient DNA.
There are 2 important features of hybridization:
The reactions are specific-the probes will only bind to targets with
a complementary sequence.
The probe can find one molecule of target in a mixture of millions
of related but non-complementary molecules.
14
15. • Steps for hybridization:
1. The mixture of molecules are separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds to the
location of the immobilized target molecule detected by
autoradiogarphy
15
16. Probe labelling & detection:
1. Radioactively labeled, often with 32P because of its high energy and
ease of incorporation into the phosphate groups of dNTPs &
detected using X-ray film.
2. Colorimetric detection (colored complex);
a. Alkaline Phosphatase that acts on substrates 5-bromo-4-chloro-
indoylphosphate (BCIP) to produce a dark purple product)
b. Horse Reddish Peroxidase (HRP) acts on H2O2 to produce insoluble brown
product
c. HRP acts on 4-chloro-1-napthol into insoluble blue product
3. Fluorescent detection
4. Chemiluminescence detection (luminol, isoluminol)
16
17. Applications
Southern blots are used in gene discovery, mapping , evolution &
development studies , diagnostics & forensics .
Allow for determination of molecular weights of restriction fragments
Presence of particular bit of DNA in the sample.
Identify: mutations, deletions, insertions and gene rearrangements
Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
Leukemias
Diagnosis of HIV-1 and infectious disease
Applications of DNA fingerprinting include:
• Paternity and Maternity Testing
• Criminal Identification and Forensics
• Personal Identification
17
18. Steps in southern blotting
The DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram
18
20. Northern 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
• Northern blotting was developed
20
21. • 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 .
Northern blotting
21
22. Applications
• A standard for the direct study of gene expression at the level of mRNA
(mRNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing - can detect alternatively spliced transcripts
• Study RNA half-life
• Study IRES (Internal Ribosomal Entry Site) – to remove possibility of
RNA digestion vs. 2nd cistron translation.
Northern blotting
22
23. Disadvantages
• Time consuming procedure
• RNA samples can be degraded by RNAses
• Use of radioactive probes
• Detection with multiple probes is a problem
Northern blotting
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24. Steps involved in Northern Blotting
RNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram
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25. Western blotting
• Dr. Douglas Lake of the University of Arizona School of Medicine's
Department of Microbiology and Immunology
• 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 .
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27. • 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
• Blot is incubated with primary and secondary antibodies on the
nitrocellulose
• Detection by autoradiography or fluorescence or colorimetric or
chemiluminescence detection
Western blotting
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28. Blocking
• Used to prevent the interactions between the membrane and the
antibody used for detection of the target protein.
• Blocking of non-specific binding is achieved by placing the membrane in a
dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or
non-fat dry milk in tris-buffered saline (TBS), with a minute percentage
(0.1%) of detergent such as Tween 20 or Triton X-100.
• The protein in the dilute solution attaches to the membrane in all places
where the target proteins have not attached.
• Thus, when the antibody is added, there is no room on the membrane for
it to attach other than on the binding sites of the specific target protein.
• This reduces background in the final product of the western blot, leading
to clearer results, and eliminates false positives.
Western blotting
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29. Tissue preparation
• Samples can be taken from whole tissue or from cell culture.
• Solid tissues are first broken down mechanically using a blender using a
homogenizer or by sonication.
• Assorted detergents, salts, and buffers may be employed to encourage
lysis of cells and to solubilize proteins.
• Protease and phosphatase inhibitors are often added to prevent the
digestion of the sample by its own enzymes.
• A combination of biochemical and mechanical techniques – comprising
various types of filtration and centrifugation – can be used to separate
different cell compartments and organelles.
Western blotting
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30. • 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
• 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 .
Western blotting
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31. Applications
• 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 .
Western blotting
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32. • 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
• Confirmatory test for Hepatitis B infection and HSV-2 infection
Western blotting
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33. Western blotting in Biochemistry
study different properties of protein based on molecular weight
Helps in confirm effective inhibition of myocardial mTOR kinase
activities by analyzing protein molecular wt. Vs expected values
investigations of mitochondrial uniporter selective calcium channels
in the organelle's inner membrane: Using the western blot technique,
researchers were able to determine through phylogenetic analysis
that the uniporter must have been a feature of the earliest
mitochondria.
Also used in Blood Doping to detect drug abuse in sports
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35. Eastern Blotting
• developed by Towbin in 1979
• The eastern blot is a biochemical technique used to analyze protein
post translational modifications (PTM) such as lipids, phosphomoieties
and glycoconjugates.
• It is most often used to detect carbohydrate epitopes.
• Thus, eastern blotting can be considered an extension of the
biochemical technique of western blotting.
• Multiple techniques have been described by the term eastern blotting,
most use proteins blotted from SDS-PAGE gel on to a nitrocellulose
membrane.
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36. • Transferred proteins are analyzed for post-translational modifications
using probes that may detect lipids, carbohydrate, phosphorylation or
any other protein modification.
• Eastern blotting should be used to refer to methods that detect their
targets through specific interaction of the PTM and the probe,
distinguishing them from a standard far-western blot.
• In principle, eastern blotting is similar to lectin blotting (i.e. detection
of carbohydrate epitopes on proteins or lipids).
Eastern Blotting
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37. Application
• detection of protein modifications in bacterial species Ehrlichia- E.
muris and IOE.
• The technique showed that the antigenic proteins of the non-virulent
E. muris is more post-translationally modified than the highly virulent
IOE
• PMT play an important role in translocation across biological
membranes.
• Expression of post-translated proteins is important in several diseases
Eastern Blotting
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38. • Far-eastern blotting is a technique developed in 1994 by Taki and
colleagues at the Tokyo Medical and Dental University, Japan for the
analysis of lipids separated by high-performance thin layer
chromatography (HPTLC).
• The lipids are transferred from the HPTLC plate to a polyvinyledene
difluoride (PVDF) membrane for further analysis, for example by
enzymatic or ligand binding assays or mass spectrometry.
Far-Eastern blotting
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39. Far-Western blotting
• Far-western blotting is a molecular biological method which is based
on the technique of western blotting to detect protein-protein
interaction in vitro.
• While usual western blotting uses an antibody to detect a protein of
interest, far-western blotting uses a non-antibody protein, which can
bind the protein of interest.
• Thus, whereas western blotting is used for the detection of certain
proteins, far-western blotting is rather employed to detect
protein:protein interactions.
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40. Northwestern blot
• The Northwestern blot, also known as the Northwestern assay, is a
hybrid analytical technique of the Western blot and the Northern
blot,
• used in molecular biology to detect interactions between RNA and
proteins.
• The Northwestern blot combines the two techniques, and specifically
involves the identification of labeled RNA that interact with proteins
that are immobilized on a similar nitrocellulose membrane.
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