its about the microscopes types and there significance in the world for diagnostic purposes .advantages and disadvantages of the types of different microscopes
its about the microscopes types and there significance in the world for diagnostic purposes .advantages and disadvantages of the types of different microscopes
Electrophoresis, Light Microscope, Transmission & Scanning electron microscop...Prajwal Gowda M.A
Steps involved in Electrophoresis, Light Microscope, Scanning and Transmission Electron Microscope.
Differences among them, Advantages and Limitations.
Proteomics Practical (NMR and Protein 3D softwareiqraakbar8
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
This presentation is an overview of Hemolytic anemia with respect to enzyme deficiencies. The inheritance pattern, clinical manifestations, symptoms complications, diagnosis and management of different types of enzyme deficiencies such as G6PD and Pyruvate Kinase have been discussed in detail.
Rheumatology MCQs Practice questions with explanationDr. Almas A
Topic: Rheumatology
Exam type: MCQs Practice questions
Q. A 26-year-old female presented to ER with dry cough and shortness of breath and often complains of chest pain. Chest x-ray shows bilateral hilar lymphadenopathy. Which of the following will indicate that the patient suffers from sarcoidosis?
Q: A 50-year-old female presents to ER with dyspnea on exertion and orthopnea, red painful eyes. She complains of chronic dull pain in the gluteal region for the last 5 years and stiffness in the lower back that wakes her up in the morning. X-ray spine reveals squaring of vertebrae with bone spur formation. On MRI sacroiliitis is seen. Which of the following is the most likely diagnosis?
Q: A 60-year-old female presents in OPD with knee joint stiffness in the morning and increases with activity and decreases on rest. She also complains about a crackling noise on joint movement. X-ray shows narrowing of the joint space and osteophytes. Which of the following treatments is recommended in this patient?
Q: A 70-year-old female presented to ER with swelling of knee joint and severe pain. Arthrocentesis revealed rhomboid-shaped crystals that stained deeply blue with H&E stain, and show weak positive birefringence on light microscopy. X-ray reveals chondrocalcinosis. Which of the following statements is true?
Q: A 40-year-old female comes to OPD with dry eyes and dyspareunia for the last 6 months. She also complains of cough and fatigue with joint pains. On examination, her parotid gland was enlarged and laboratory tests revealed anti-Ro antibodies are positive. Which of the following tests is recommended to this patient?
Electrophoresis, Light Microscope, Transmission & Scanning electron microscop...Prajwal Gowda M.A
Steps involved in Electrophoresis, Light Microscope, Scanning and Transmission Electron Microscope.
Differences among them, Advantages and Limitations.
Proteomics Practical (NMR and Protein 3D softwareiqraakbar8
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus.
FLOW CYTOMETRY, PRINCIPLE, APPLICATION, USE IN HAEMATOLOGY, COMPONENT OF FLOW CYTOMETRY, DATA INTERPRETATION, DATA ANALYSIS, CELL SHORTING ADVANTAGES AND DISADVANTAGES, IMMUNOLOGICAL CLASSIFICATION OF ACUTE
LEUKEMIA
This presentation is an overview of Hemolytic anemia with respect to enzyme deficiencies. The inheritance pattern, clinical manifestations, symptoms complications, diagnosis and management of different types of enzyme deficiencies such as G6PD and Pyruvate Kinase have been discussed in detail.
Rheumatology MCQs Practice questions with explanationDr. Almas A
Topic: Rheumatology
Exam type: MCQs Practice questions
Q. A 26-year-old female presented to ER with dry cough and shortness of breath and often complains of chest pain. Chest x-ray shows bilateral hilar lymphadenopathy. Which of the following will indicate that the patient suffers from sarcoidosis?
Q: A 50-year-old female presents to ER with dyspnea on exertion and orthopnea, red painful eyes. She complains of chronic dull pain in the gluteal region for the last 5 years and stiffness in the lower back that wakes her up in the morning. X-ray spine reveals squaring of vertebrae with bone spur formation. On MRI sacroiliitis is seen. Which of the following is the most likely diagnosis?
Q: A 60-year-old female presents in OPD with knee joint stiffness in the morning and increases with activity and decreases on rest. She also complains about a crackling noise on joint movement. X-ray shows narrowing of the joint space and osteophytes. Which of the following treatments is recommended in this patient?
Q: A 70-year-old female presented to ER with swelling of knee joint and severe pain. Arthrocentesis revealed rhomboid-shaped crystals that stained deeply blue with H&E stain, and show weak positive birefringence on light microscopy. X-ray reveals chondrocalcinosis. Which of the following statements is true?
Q: A 40-year-old female comes to OPD with dry eyes and dyspareunia for the last 6 months. She also complains of cough and fatigue with joint pains. On examination, her parotid gland was enlarged and laboratory tests revealed anti-Ro antibodies are positive. Which of the following tests is recommended to this patient?
Laboratory waste disposal steps are important to be followed. we cannot drain the laboratory waste directly into the drain, or it can cause corrosive damage to the drain system and the sewerage. Alkali waste requires proper neutralization for proper disposal
The correct disposal of acid waste includes the following steps. Pouring direct into the sink in the laboratory will cause corrosive damage. Thus these steps need to be followed when disposing acid waste.
what are the steps you need to follow when you get a needle stick injury in the lab, while handling needles or any sharp instruments. We should deal with each needle stick injury as it is contaminated by any infectious disease like hepatitis, AIDS or others.
Operative Dentistry Viva questions. To help you revise your syllabus for examination.
If you found it helpful, please leave a feedback.
Thank You,
Dr. Almas Muhammad Arshad
Dr. Muaaz Amjad
The periodontium and pulp are two most important entities of the tooth, infection from one can travel towards other by different pathways. Neglect of either one can lead to failure. This presentation will help you learn clear steps towards diagnosis and treatment planning of such lesions
Hepatitis B and C infection and it's clinical implication in Dental practice, how to management patients of hepatitis and what clinical features patients with hepatitis show in oral cavity.
CEPHALOSPORINS (First Genertaion)
Introduction:
First discovered in 1945
A class of Beta Lactam Antibiotics
Are derivatives of 7-aminocephalosporanic acid
They were first isolated from Cephalosporium acremonium (fungus)
Structure:
Are Beta-lactam compounds
In which the beta-lactam ring is fused to a 6-membered dihydrothiazine ring, thus forming the cephem nucleus.
Mechanism of action:
They are Bactericidal agents by cell lysis.
Bind to the Penicillin-binding proteins (PBPs) on the bacterial cell membrane and inhibit cell wall synthesis.
Inhibit Peptidoglycan synthesis by inhibiting the transpeptidation reaction – failure of cross-linking of peptidoglycan.
Mechanism of resistance
Acquired resitance to cephalosporins could be due to:
Alternation of the PBPs (target protiens)
Impermeability to the antibiotic thus preventing it to reach it’s site of action.
Production of Beta lactamases by many bacteria that inactivate the drug.
Resistance developed by penicilinase produced by staphylococci (less than penicillin)
Classification of cephalosporins:
Based on their spectrum of activity, Cephalosporins can be broadly categorized into four generations.
1st Generation (Cefazolin, Cephalexin)
2nd Generation (Cefotetan, Cefoxitin)
3rd Generation (Cefoperazone, Cefixime)
4th Genertaion (Cefepime)
First Generation drugs:
Also called Narrow spectrum Cephalosporins
Include;
ORAL:
CEPHALEXIN
CEFADROXIL
CEPHRADINE
PARENTERAL:
CEFAZOLIN (prototype)
CEPHAPIRIN
Anti-baterial spectrum:
First generation cephalosporins are very active against gram positive cocci which include:
Pneumococci
Streptococci
staphylococci.
Against gram negative bacilli
E. coli
Klebsiella
Proteus
Active against most penicillin-susceptible anaerobes found in the oral cavity,
except those belonging to the Bacteroides fragilis (that are Gram-negative bacillus bacterium species, and an obligate anaerobe of the gut ) group.
Clinical uses:
For dental surgical prophylaxis (Cephalexin and Cefazolin)
Skin and bone infections (Cefazolin)
Pharyngitis
Tonsilitis
Otitis
Pneumonia
UTI
Skin infections
Toxicity:
Diarrhea
Nausea
Vomiting
Abdominal discomfort
Headache
Fever
Rashes
Pruritis
Urticaria
Serum sickness like reaction
Disturbance in liver enzymes
Transient Hepatitis
Cholestatic jaundice
Eosinaphilia
Blood disorders
Antibiotic associated colitis (rare)
Medical Technology Tackles New Health Care Demand - Research Report - March 2...pchutichetpong
M Capital Group (“MCG”) predicts that with, against, despite, and even without the global pandemic, the medical technology (MedTech) industry shows signs of continuous healthy growth, driven by smaller, faster, and cheaper devices, growing demand for home-based applications, technological innovation, strategic acquisitions, investments, and SPAC listings. MCG predicts that this should reflects itself in annual growth of over 6%, well beyond 2028.
According to Chris Mouchabhani, Managing Partner at M Capital Group, “Despite all economic scenarios that one may consider, beyond overall economic shocks, medical technology should remain one of the most promising and robust sectors over the short to medium term and well beyond 2028.”
There is a movement towards home-based care for the elderly, next generation scanning and MRI devices, wearable technology, artificial intelligence incorporation, and online connectivity. Experts also see a focus on predictive, preventive, personalized, participatory, and precision medicine, with rising levels of integration of home care and technological innovation.
The average cost of treatment has been rising across the board, creating additional financial burdens to governments, healthcare providers and insurance companies. According to MCG, cost-per-inpatient-stay in the United States alone rose on average annually by over 13% between 2014 to 2021, leading MedTech to focus research efforts on optimized medical equipment at lower price points, whilst emphasizing portability and ease of use. Namely, 46% of the 1,008 medical technology companies in the 2021 MedTech Innovator (“MTI”) database are focusing on prevention, wellness, detection, or diagnosis, signaling a clear push for preventive care to also tackle costs.
In addition, there has also been a lasting impact on consumer and medical demand for home care, supported by the pandemic. Lockdowns, closure of care facilities, and healthcare systems subjected to capacity pressure, accelerated demand away from traditional inpatient care. Now, outpatient care solutions are driving industry production, with nearly 70% of recent diagnostics start-up companies producing products in areas such as ambulatory clinics, at-home care, and self-administered diagnostics.
Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctors’ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratory
to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
Navigating the Health Insurance Market_ Understanding Trends and Options.pdfEnterprise Wired
From navigating policy options to staying informed about industry trends, this comprehensive guide explores everything you need to know about the health insurance market.
Leading the Way in Nephrology: Dr. David Greene's Work with Stem Cells for Ki...Dr. David Greene Arizona
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Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
R3 Stem Cells and Kidney Repair A New Horizon in Nephrology.pptxR3 Stem Cell
R3 Stem Cells and Kidney Repair: A New Horizon in Nephrology" explores groundbreaking advancements in the use of R3 stem cells for kidney disease treatment. This insightful piece delves into the potential of these cells to regenerate damaged kidney tissue, offering new hope for patients and reshaping the future of nephrology.
Navigating Women's Health: Understanding Prenatal Care and Beyond
Techniques in Biochemistry Notes.docx
1. Microscope:
Optical Instrument.
To view tiny objects that are not visible to the naked eye.
Electron Microscope:
Uses electrons to illuminate the specimen.
High resolution.
Magnify images 2 million times (light = 2000x).
Magnification:
The increase in the size of an object when viewed through the microscope.
Resolution:
The level of detail that the microscope can reveal in a specimen.
Measured in nanometers (nm) or micrometers (μm).
Contrast:
The intensity between adjacent parts of an image or specimen helps to differentiate and
distinguish details.
degree of difference in brightness between the light and dark areas of the specimen
being viewed.
Methods of enhancing contrast:
Brightfield illumination: The most common method uses reflected light to provide
essential contrast by creating a bright background and dark features.
Darkfield illumination: This method uses scattered light to produce a dark background
and brightly illuminated features.
Phase contrast: This method uses interference of light waves to highlight differences in
refractive index within a specimen, making it possible to see otherwise invisible
features.
Fluorescence: Fluorescent dyes or stains - making them visible under UV light.
Differential Interference Contrast (DIC): Polarized light to produce contrast.
Nature of Image:
Close-up, highly magnified representation.
Details about the structure, composition, or behavior of the specimen.
Purpose: Make clearer observations and analyses.
Parts of Microscope: The parts of a microscope typically include:
1. eyepiece lens (10x)
2. objective lenses (4x, 10x, 40x)
3. nosepiece (to hold the objective lenses)
4. coarse focus knob
5. fine focus knob
6. stage (to hold the specimen)
7. stage clips (to secure the specimen)
2. 8. light source (to illuminate the specimen)
9. diaphragm (to control the amount of light)
10. base (to support the microscope)
11. arm (to connect the head and the base)
12. microscope body (to hold all the components together)
Note: The number of lenses and components may vary depending on the type and model of the
microscope.
SEM vs TEM:
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
Both are electron microscopy techniques to study materials at the nanoscale.
Differences:
Image formation:
o SEM - scanning the sample surface with a focused electron beam and detecting
electrons that are emitted by the sample.
o TEM - transmitting electrons through a thin sample and detecting electrons that
pass through it.
Sample preparation:
o SEM – Bulkier sample required.
o TEM – Thin sample required / electron transparent.
Resolution:
o SEM – Lower Resolution than TEM.
o TEM - Higher resolution than SEM because it detects electrons that have passed
through the sample.
Information obtained:
o SEM - Surface morphology and elemental composition.
o TEM - Crystal structure, lattice spacings and defects.
Radiation damage:
o Higher risk of damage with TEM because of the transmission of electrons.
NMR:
Principle:
By changing the strength of a magnetic field, the nuclei of certain atoms will absorb and
then emit electromagnetiradiationns.
Change in magnetic field strength causes the spinning nuclei to precess and realign. This
produces an NMR signal.
The signal can be measured and then used to determine the chemical and structural
properties of the sample under study.
Clinical Application:
NMR - MRI.
MRI:
1. Brain and spinal cord imaging
2. Joint and skeletal imaging
3. Abdominal and pelvic imaging
3. 4. Cardiac and vascular imaging
5. Breast imaging
6. Neurological disorders diagnosis
7. Cancer diagnosis and treatment monitoring
8. Musculoskeletal injuries and disorders
9. Liver, pancreas and gallbladder imaging
10. Evaluation of pelvic floor disorders
11. Diagnosis of sports-related injuries
12. Diagnosis of multiple sclerosis
13. Detection of stroke and brain tumors
14. Evaluation of spinal cord and vertebral column disorders.
Hematology Analyzer:
Definition:
Laboratory instrument used to count and analyze the different types of cells in a blood
sample, including red blood cells, white blood cells, and platelets.
Used to diagnose and monitor various medical conditions, such as anemia, infection, and
bleeding disorders.
Types:
There are several types of hematology analyzers, including:
Three-part hematology analyzer:
o Separates three populations: RBC, WBC, Platelets.
o To study infections, anemia, and bleeding disorders.
Fiver part hematology analyzer:
o Separate five populations: RBC, WBC, Platelets, immature granulocytres and
lymphocytes.
Coulter Counter: An electrical impedance-based analyzer that measures cell size and
number.
Flow Cytometry: A laser-based technology that uses light scattering and fluorescent
labeling to analyze cells.
Coulter counter vs Flow Cytometry:
Principle:
o Coulter counter -
Blood cells are suspended in an electrolyte solution and passed through
a small aperture. Cells displace the electrolyte solution and cause a
change in electrical impedance.
The change in electrical impedance is proportional to the volume of the
cell.
Helps measure the cell size and number.
o Flow cytometry:
Blood cells are passed one by one through a laser beam.
The cells scatter the light.
The intensity and angle of the scattered light are measured.
Helps determine the size, shape and internal complexity of the cell.
Other parameters:
4. Fluorescence dye as cell marker – Cell characteristic and function,
surface antigen and intracellular proteins.
Technology:
o Coulter counter - electrical impedance-based analyzer
o Flow cytometry - laser-based technology that uses light scattering and
fluorescence.
Analysis:
o Coulter counter - Cell size and number.
o Flow cytometry - Detailed information on cell size, shape, and fluorescence
intensity, allowing for the simultaneous analysis of multiple cell parameters
(Cell characteristic and function, surface antigen and intracellular proteins).
Sample Preparation:
o Coulter counter requires a larger volume of the blood sample.
o Flow cytometry requires a smaller volume of a blood sample. And requires
additional processing steps, such as staining, to enhance the analysis.
Applications/Uses:
o Coulter counter - Routine complete blood cell count (CBC) analysis.
o Flow cytometry - Specialized applications; immunophenotyping and analysis of
rare cell populations.
Difference between oven and incubator:
Purpose:
o Oven - Heating, drying, and sterilizing samples.
o Incubator – Growing and maintaining living cells or microorganisms in a
controlled environment.
Temperature Control:
o Oven: ambient to 300°C
o Incubator: more precise and controlled temperature range, often between 20-
50°C.
Humidity:
o Oven: No control.
o Incubator: Controlled. Crucial for growing living cells or microorganisms.
Air Circulation:
o Oven: Poor.
o Incubator: Good to ensure a uniform temperature and prevent contamination.
CO2 Control:
o Oven: Does not control CO2 level.
o Incubator: Controlled CO2 levels.
Difference between Safety Cabinet and Laminar Airflow:
Lab equipment to protect samples and workers from contamination/ventilation.
Main difference: Type of airflow they use to maintain a clean environment.
Safety Cabinet:
Protects samples and workers from exposure to hazardous materials by an inward flow
of air to keep contaminants inside the cabinet while clean air is drawn from the room.
5. The air inside the cabinet is filtered to remove any contaminants before it is exhausted
back into the room.
Laminar Flow Cabinet:
Used to maintain a clean and contamination-free environment for delicate or sensitive
samples.
Uses continuous, unidirectional clean air filtered to remove contaminants.
The airflow is parallel and uniform, creating a "laminar" flow.
Helps prevent contamination of the samples inside the cabinet.
Use HEPA filter.
When is a Vertical Laminar Flow Cabinet the Best Choice?
Providing a contamination-free area for non-hazardous products that are safe for an operator to
inhale. Clean and particle free.
Uses:
Medical, Pharmaceutical, Scientific, and Electronics fields.
Tasks:
o Preparation, supply and testing of sterile pharmaceutical products
o To handle lab samples, products, and other specimens.
o Cell culture.
o Preparing and pouring bacterial growth media.
o Quality control.
o Electronic and optical assemblies and systems.
Sanger Sequencing vs NGS:
Sanger NGS
Throughput Low Throughput method,
1 Fragment at a time
High Throughput,
Millions of fragments at a time
Other name Di-deoxy chain termination Massive parallel sequencing
Generation First Second
Cost Higher cost More cost effective
Accuracy More accurate Higher error rate
Analogical Digital
Sensitivity Less sensitive High sensitive
Application Gold standard in research,
gene sequencing, mutation
analysis
Larger project, clinical lab
TaqMan and SYBR Green:
TaqMan and SYBR Green are different fluorescent dyes commonly used for real-time PCR
(polymerase chain reaction), a technique used to amplify DNA sequences.
Binding: SYBR Green is a generic dye that binds to double-stranded DNA. Taqman to single-
stranded DNA.
Principle: TaqMan is based on a probe-based system, while SYBR Green is a dye-based method.
6. Specificity: TaqMan is more specific (specific probes - target sequence). SYBR Green can detect
all amplified DNA.
Sensitivity: TaqMan is more sensitive than SYBR Green.
Cost: TaqMan is more expensive than SYBR Green.
Speed: TaqMan is generally slower than SYBR Green as it requires an additional step for the
binding and cleavage of the probe.
Interference: SYBR Green can be prone to interference from non-specific binding to other
sources of double-stranded DNA, while TaqMan is unaffected.
Detection limit: TaqMan has a lower detection limit than SYBR Green, as it can detect lower
levels of target DNA.
Relative vs absolute quantification:
SYBR Green and TaqMan are both commonly used for real-time PCR and can be used for both
relative and absolute quantification of target DNA.
Relative quantification: The goal is to determine the relative amount of target DNA in different
samples, often normalized to an internal control or reference gene. SYBR Green and TaqMan can
be used for relative quantification, where the change in fluorescence over the reaction
determines the relative amount of target DNA in each sample.
Absolute quantification: In absolute quantification, the goal is to determine the exact number of
copies of target DNA in a sample. Absolute quantification typically requires a standard curve,
where a series of known amounts of target DNA is amplified alongside the unknown sample,
allowing the amount of target DNA in the sample to be determined. TaqMan is typically used for
absolute quantification, as the specificity of the probe system allows for a more accurate
determination of the number of target DNA copies in the sample.
It is worth noting that both methods have their limitations, and the choice of method will
depend on the specific requirements and goals of the experiment.
Taqman probe working:
The TaqMan probe is a fluorescent probe used in real-time PCR (polymerase chain reaction) to
detect and quantify specific DNA sequences. The probe consists of a fluorescent dye, a quencher,
and a probe-specific DNA sequence. Here's how the TaqMan probe works:
The TaqMan probe is added to the PCR reaction mixture and the template DNA, primers, and
polymerase.
During the PCR reaction, the primers anneal to the target DNA and the polymerase extends the
primers, creating new DNA strands. If the target sequence is present in the template DNA, the
TaqMan probe will bind to it.
The fluorescence from the dye is quenched by the quencher when the probe is intact.
As the polymerase cleaves the probe during amplification, the fluorescent signal is released and
can be measured. The fluorescence intensity increases proportionally with the amount of
amplified target DNA.
7. The fluorescence intensity can be monitored in real-time during the PCR reaction by measuring
the fluorescence intensity of the reaction mixture.
After the PCR reaction, the fluorescence intensity is plotted against the cycle number to
determine the amount of target DNA present in the sample.
The TaqMan probe is considered a more specific and sensitive method than other real-time PCR
methods, as it relies on a probe-specific sequence to detect only the target DNA and not non-
specific amplified DNA. However, designing TaqMan probes for each target gene is also more
expensive and time-consuming.
SYBR Green technology uses a fluorescent dye that binds to double-stranded DNA and
fluoresces in proportion to the amount of DNA present. The fluorescence intensity can then be
monitored in real-time during the PCR reaction.
Here's how the SYBR Green technology works:
The SYBR Green dye is added to the PCR reaction mixture and the template DNA, primers, and
polymerase.
During the PCR reaction, the primers anneal to the target DNA, and the polymerase extends the
primers, creating new DNA strands. As the DNA strands are amplified, the double-stranded DNA
increases and more SYBR Green dye binds to it.
The fluorescence emitted by the SYBR Green dye can be monitored in real-time during the PCR
reaction by measuring the fluorescence intensity of the reaction mixture. The fluorescence
intensity increases proportionally with the amount of amplified DNA.
After the PCR reaction, the fluorescence intensity is plotted against the cycle number to
generate a melting curve, which can be used to confirm the specificity of the amplification and
the identity of the amplified product.
SYBR Green technology is widely used in real-time PCR due to its simplicity, ease of use, and
relatively low cost compared to probe-based methods such as TaqMan. However, SYBR Green
can also produce non-specific signals and can be affected by interference from other sources of
double-stranded DNA, so it is important to confirm the specificity of the amplification using
methods such as melting curve analysis.
Principle of Real-Time PCR:
The principle of real-time PCR is based on the amplification of target DNA through repeated
cycles of heating and cooling and the measurement of the amplified DNA using fluorescent dyes.
The procedure of Real-Time PCR:
In real-time PCR, the sample DNA is mixed with specific primers, a polymerase enzyme, and
fluorescent dyes (such as SYBR Green or TaqMan probes).
The reaction mixture is then subjected to a series of temperature cycles in a
thermocycler.
During each cycle, the temperature is raised to denature the double-stranded DNA into
single strands, then lowered to allow the primers to anneal to the target DNA, and finally
raised again to allow the polymerase to extend the primers and amplify the target DNA.
As the target DNA is amplified, the fluorescent dye binds to the double-stranded DNA
and emits fluorescence in proportion to the amount of DNA present.
8. The fluorescence is monitored in real-time during the reaction, allowing for the rapid
and quantitative determination of the target DNA.
The final result is expressed as the number of copies of target DNA per reaction or per
unit of sample, which can be compared to a standard curve generated from known
amounts of target DNA.