It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
Polymerase chain reaction is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
Polymerase chain reaction is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
Real Time Polymerase Chain Reaction
Basics of Real Time PCR
Definition
Advantages
Principles
Instruments (Thermal Cyclers)
Useful terms
Real Time PCR Chemistry
Fluorescence Dyes
SYBR Green
EvaGreen
Melt Doctor
Fluorescence Probes
TaqMan Probe
Molecular Beacons
Scorpion Primers
SYBR Green In details
qPCR Set-Up
Assay Design
Data Analysis
Troubleshooting
In this ppt, the various types of PCR such as real time PCR, Reverse transcription PCR, multiplex PCR, ligation chain PCR, nested PCR which is applied in diagnosis of diseases, identification of genetic disorders, determination of polymorphism and also in DNA fingerprinting analysis are described.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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Real Time Polymerase Chain Reaction
Basics of Real Time PCR
Definition
Advantages
Principles
Instruments (Thermal Cyclers)
Useful terms
Real Time PCR Chemistry
Fluorescence Dyes
SYBR Green
EvaGreen
Melt Doctor
Fluorescence Probes
TaqMan Probe
Molecular Beacons
Scorpion Primers
SYBR Green In details
qPCR Set-Up
Assay Design
Data Analysis
Troubleshooting
In this ppt, the various types of PCR such as real time PCR, Reverse transcription PCR, multiplex PCR, ligation chain PCR, nested PCR which is applied in diagnosis of diseases, identification of genetic disorders, determination of polymorphism and also in DNA fingerprinting analysis are described.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
https://www.patreon.com/biotechlive
SUPPORT EDUCATION... SUPPORT US
Gene cloning and polymerase chain reaction Abhay jha
In these you are able to know about the gene cloning basic steps and Polymerase chain reaction process also there is an brief description about the ideal property shown by vectors which are lambda and M13 phases and there are lots of things in these slides
Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
PCR is a biochemical technology in molecular biology. This technique used in DNA cloning for sequencing, gene cloning and manipulating, diagnosis of hereditary diseases.
Polymerase chain reaction .... Very important topic which is explained in very simple and convenient way. Learning objectives and references are given in the presentation for the detailed learning. The presentation was Guided by Dr Shilpa Jain and made by Ms. Nidhi Argade.
Infection characterized by severe local inflammation, usually with pus formation, generally caused by one of the pyogenic bacteria.
Sepsis:The term sepsis covers numerous and diverse pyogenic infections which includes superficial skin infections,wound infections,infection of burns,infection of eyes,peritonitis and abscesses.
Pus is an exudate typically white yellow or yellow formed at the site of inflammation during infection.
Abscesses are localized collection of pus composed of living and dead WBC, components of tissue break down.
70% of tissue infection is mainly caused by
Staphylococcus aureus.
Etymology:
Greek word, pyon meaning pus, genein, meaning to produce
Pus is a fluid composed of : dead & dying WBC, dead & dying bacteria (in bacterial cause of pus),tissue debris, edema, fibrin, lipid and nucleic acid.
Pus cells : it is degranulated wbc, neutrophils.
The body responds to invasion by a wide variety of bacteria by an increased blood supply to the area and by an outpouring of serous fluid and white blood cells.
This is the typical inflammatory response.
The white cells which pass from the blood into the infected tissues attempt to ingest the bacteria (phagocytosis), many cells die and the resultant material consisting of both living and dead white cells (leucocytes or pus cells) and bacteria, together with damaged local tissues and blood proteins, constitutes PUS.
Infections in which pus is produced are known as pyogenic, i.e. pus-producing infections.
Pus may be present as a localised collection deep in the tissues—an ABSCESS, it may be produced on a surface, e.g. the mucosa of the pharynx, the mucosa of the bladder, the méninges, indeed any body surface, it is then known as a PURULENT EXUDATE.
Alternatively infection may spread evenly through the tissues causing a diffuse inflammation :CELLULITIS.
The type of pus production will depend on the organism causing the infection, on the tissue in which the infective process is taking place, and also on the body resistance to the infection.
Although the pyogenic infections have very similar appearances whatever the causative organism, different sites of the body have a tendency to be infected with particular species of bacteria.
Always submit two swabs so that Gram stain can be performed.
Limit swab sampling to wounds that are clinically infected or those that are chronic and are not healing.
To minimize contamination, it is important to cleanse the wound to remove superficial debris by thorough irrigation and cleansing with non-bacteriostatic sterile saline.
If the wound is relatively dry, collect the specimen with two cotton-tipped swabs moistened with sterile non-bacteriostatic saline. Gently roll the swab over the surface of the wound approximately five times, focusing on an area where there is evidence of pus or inflamed tissue.
casoni test is an immediate hypersensitivity skin test previously used in the diagnosis of hydatid disease.
Intradermal injection of 0.2ml of hydatid fluid collected from animal/human cyst which is sterilized by seitz filtration OR membrane filtration.
equal volume of saline(control) injected on the other forearm and observation made for next 30 min and after 1 to 2 days.
As a precaution anaphylactic tray must be kept ready before carrying out the test.(Type 1 hypersensitivity reaction)
Interpretation: Sensitive patients develop large wheal measuring 5 cm or more with formation of pseudopodia like projection within 30 minutes occuring at injection site, considered positive result.(immediate hypersensitivity) .
No reaction in the control arm.
Disadvantage: It has low sensitivity (60-80%)
and gives false positive results in cross reactive cestode infections.
It is no longer used nowadays and replaced largely by the serological tests.
Less reliable than imaging technique.
Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.
Introduction to calcium
Sources of calcium
Dietary requirement of calcium
Calcium absorption
Biochemical function of calcium
Calcium in blood
Calcium estimation
Factors regulating calcium level in blood
Disease states of calcium
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
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.
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
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
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
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.
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
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.
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!
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
2. • It was invented in
1983 by Dr. Kary Mullis,
for which he received the
Nobel Prize in
Chemistry in 1993.
• It is called “polymerase” because the only enzyme
used in this reaction is DNA polymerase.
• It is called “chain” because the products of the first
reaction become substrates of the following one, and
so on.
2
5. Principle
• The method relies on thermal cycling, consisting
of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic
replication of the DNA.
• Artificial process which imitates natural DNA
replication
5
7. Components of PCR
• DNA template- the sample DNA that contains the target sequence. At the
beginning of the reaction, high temperature is applied to the original
double-stranded DNA molecule to separate the strands from each other.
• Usually target sequence is 100bp-1500bp length
• .DNA polymerase- a type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly
used of these enzymes is TaqDNA polymerase (fromThermis aquaticus),
whereas PfuDNA polymerase (from Pyrococcus furiosus) is used widely
because of its higher accuracy when copying DNA. Although these
enzymes are subtly different, they both have two capabilities that make
them suitable for PCR:
1) they can generate new strands of DNA using a DNA template and primers,
and
2) they are heat resistant.
7
8. Taq polymerase
• A thermostable DNA polymerase named after
the thermophilic bacterium Thermus aquaticus
• Taq's optimum temperature for activity is 75–
80°C
• Can replicate a 1000 base pair strand of DNA in
less than 10 seconds at 72°C
8
9. • Primers- short pieces of single-stranded DNA that are
complementary to the target sequence. The polymerase
begins synthesizing new DNA from the end of the primer.
-Primers are oligonucleotides with 15-20 bases in length
-Two primers forward and reverse primer .
-Annealing temperature depends upon primer sequence (~
50% GC content)
-Avoid primer complementary (primer- dimer formation)
-The last 3 nucleotides at the 3` end is the substrate for DNA
polymerase - G or C
-The primers must not base pair with each other or with
themselves or form hairpins.
9
11. • Nucleotides (dNTPs or deoxynucleotide
triphosphates)- single units of the bases A, T, G, and
C, which are essentially "building blocks" for new
DNA strands.
• Mg or Mn ions- Mg preferred, provides stable
conditions
• Buffer solution-suitable chemical environment for
optimum activity and stability of DNA polymerase
11
12. Very simple Lab procedure
• To perform PCR, the extracted sample (which
contains target DNA template) is added to a
tube containing primers, free nucleotides
(dNTPs), and Taq polymerase. The PCR
mixture is placed in a PCR machine. PCR
machine increases and decreases the
temperature of the PCR mixture in automatic,
programmed steps which generates copies of
the target sequence exponentially.
12
17. Initial Denaturation
• Heating separates the double
stranded DNA
– Denaturation
– heating the reaction to a
temperature of 94–96 °C for 5
mins depending on the GC
content of the template
• performed only once at the
beginning of the reaction
Heat Cool
17
18. Denaturation
• First regular cycling event and consists of
heating the reaction to 94–98 °C for 30–60
seconds.
• Causes melting of the DNA template by
disrupting the hydrogen bonds between
complementary bases, yielding single-
stranded DNA molecules.
18
19. Annealing (primer binding)
• Annealing of the primers to the
single-stranded DNA template.
• Typical annealing temperature is
between 50 and 55°C, for 30-60
seconds
• optimal temperature depends on
the primer sequence and length
• Typically the annealing temperature
is about 3-5 degrees Celsius below
the Tm of the primers used.
• The polymerase binds to the
primer-template hybrid and begins
DNA formation.
19
20. Extension(synthesis of new DNA)
o DNA polymerase duplicates DNA by synthesizing a new DNA
strand complementary to the DNA template strand (72C)-optimal
temperature for Taq DNA polymerase
o During this step dNTPs that are complementary to the template in
5' to 3' direction are added
20
22. Final extension
• This single step is occasionally performed at a
temperature of 70–74 °C for 5–15 minutes
after the last PCR cycle
• Its objective is to ensure that any remaining
single-stranded DNA is fully extended.
22
23. Final Hold
• This step at 4°C for an indefinite time may be
employed for short-term storage of the
reaction.
23
25. Detection of PCR products
Agarose Gel electrophoresis
easiest and commonest way to separate and analyze DNA on the
basis of molecular weight
have lower resolving power for DNA than acrylamide gels, but they
have greater range of separation, and are therefore usually used for
DNA fragments of 50-20,000 bp in size
the lower the concentration of the gel, the larger the pore size, and
the larger the DNA that can be sieved
-technique consist of 3 basic steps
• preparation of agarose
• Electrophoresis of DNA fragments
• Visualization of DNA fragments
25
26. Preparation of Agarose Gel
• Insoluble in water and buffer at RT but
dissolves on boiling
• On cooling undergoes polymerization (sugar
polymers crosslink with each other causing
solution to gel)
• Density or pore size determined by the
concentration of agarose
• 0.5 -2% agarose use, usually 1% preferred
• Buffer- 1x TBE(tris-borate-EDTA) or TAE
26
27. Electrophoresis of DNA
• Technique used to separate charged molecules
• DNA is negatively charged at neutral PH
• DNA migrates to anode when electric field is
applied across the gel
• Migration of DNA depends upon
-molecular size of DNA
-agarose concentration
-applied current
-conformation of DNA
27
28. • Progress of gel is monitored by observing the
migration of visible dye-tracking dye
• Commonly used are Xylene cynol and
Bromophenol blue
• 50 volts for 1-2 hrs
28
29. Visualization of DNA
• DNA is not visible in gel
• Stained with specific dye such as ethidium
bromide
-1 µl of 10 mg/ml
• Visualize under UV light-DNA floresces
*ethidium bromide must be handled carefully as it
is mutagen and carcinogen
29
31. PCR phases
Exponential
◦ If 100% efficiency – exact doubling of products. Specific and
precise
Linear
◦ High variability. Reaction components are being consumed
and PCR products are starting to degrade.
Plateau
◦ End-point analysis. The reaction has stopped and if left for
long – degradation of PCR products.
31
34. Lets see a short animation
video on Lab procedure of RT-
PCR for diagnosis of COVID-19.
34
35.
36. Real time PCR
• In order to amplify small amounts of DNA, the
same methodology is used as in conventional
PCR using a DNA template, at least one pair of
specific primers, deoxyribonucleotides, a
suitable buffer solution and a thermo-stable
DNA polymerase.
37. Principal:
• Quantitative PCR is carried out in a thermal
cycler with the capacity to illuminate each
sample with a beam of light of a specified
wavelength and detect the fluorescence
emitted by the excited fluorophore.
• The thermal cycler is also able to rapidly heat
and chill samples, thereby taking advantage of
the physicochemical properties of the nucleic
acids and DNA polymerase.
38. • A substance marked with a fluorophore is
added to this mixture in a thermal cycler that
contains sensors for measuring the
fluorescence of the flurophore after it has been
excited at the required wavelength allowing
the generation rate to be measured for one or
more specific products.
38
39. • This allows the rate of generation of the amplified product
to be measured at each PCR cycle. The data thus generated
can be analysed by computer software to calculate relative
gene expression (or mRNA copy number) in several
samples.
• This measurement is made after each amplification cycle,
and this is the reason why this method is called real time
PCR (that is, immediate or simultaneous PCR) not at its
end, as in conventional PCR. In the case of RNA
quantitation, the template is complementary DNA (cDNA),
which is obtained by reverse transcription of ribonucleic
acid(RNA). In this instance the technique used is
quantitative RT-PCR or Q-RT-PCR.
40. • Two common methods for the detection of
products in quantitative PCR are:
- Non-specific fluorecent dyes that intercalate with
any double-stranded DNA
- Sequence-specific DNA probes consisting of
oligonucleotides that are labelled with a fluorescent
reporter which permits detection only after
hybridization of the probe with its complementary
sequence to quantify messenger RNA (mRNA) and
non-coding RNA in cells or tissues.
42. Advantages
• Real-Time chemistries allow for the detection of PCR
amplification during the early phases of the reaction.
• Measuring the kinetics of the reaction in the early phases of
PCR provides a distinct advantage over traditional PCR
detection. Traditional methods use Agarose gels for
detection of PCR amplification at the final phase or end-
point of the PCR reaction
• Sensitive assay, highly quantitative, and highly reproducible
• Can detect as few as 5 molecules
• Good Excellent dynamic range, linear over several orders of
magnitude
• Useful for diagnostic purposes
43. Disadvantages of Real time PCR:
• Expensive
• Can pick up RNA carryover or contaminating RNA leading to
false positives
• Need a skillful person
44.
45. Medical applications OF PCR
• genetic testing, where a sample of DNA is
analyzed for the presence of genetic
disease mutations. Prospective parents can be
tested for being genetic carriers, or their
children might be tested for actually being
affected by a disease.
• Prenatal testing can be obtained
by amniocentesis,chorionic villus sampling, or even
by the analysis of rare fetal cells circulating in
the mother's bloodstream. PCR analysis is also
essential to preimplantation genetic diagnosis,
where individual cells of a developing embryo are
tested for mutations.
45
46. Medical applications:
• PCR can also be used as part of a sensitive test
for tissue typing, vital to organ transplantation.
• blood type
• Oncogenes.
46
47. • Characterization and detection of infectious disease
organisms have been revolutionized by PCR:
• The human immunodeficiency virus
• tuberculosis,
• The spread of a disease organism through populations
• The sub-types of an organism that were responsible
for earlier epidemics can also be determined by PCR
analysis.
47
48. Forensic applications
• In its most discriminating form, genetic
fingerprinting can uniquely discriminate any one
person from the entire population of the world.
• DNA database of earlier evidence or convicts.
Simpler versions of these tests are often used
to rapidly rule out suspects during a criminal
investigation. Evidence from decades-old
crimes can be tested, confirming the people
originally convicted.
• parental testing, where an individual is
matched with their close relatives.
48
49. In Molecular diagnosis
• Viral load and identification of virus
• Bacterial infection: early detection specific targeting and better
treatment
detect Mycobacterium tuberculosis
Legionella pneumophila,
Listeria monocytogenes,
Neisseria gonorrhoea.
• Antibiotic resistance :
Staphylococcus aureus,
Staphylococcus epidermidis,
Helicobacter pylori,
Enterococcus faecalis
Enterococcus faecium