PCR,polymerase chain reaction.Basic concept of PCR.naveed ul mushtaq
PCR.Basic concept of PCR. Steps in PCR.
Quantitative real time polymerase chain reaction.Fluorescent dyes and probes.
Advantages real-time PCR.
Real-time PCR primer
Primer design software
The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific detection and production of large amounts of DNA. PCR-based strategies have propelled huge scientific endeavors such as the Human Genome Project. The technique is currently widely used by clinicians and researchers to diagnose diseases, clone and sequence genes, and carry out sophisticated quantitative and genomic studies in a rapid and very sensitive manner. One of the most important medical applications of the classical PCR method is the detection of pathogens. In addition, the PCR assay is used in forensic medicine to identify criminals. Because of its widespread use, it is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome
PCR,polymerase chain reaction.Basic concept of PCR.naveed ul mushtaq
PCR.Basic concept of PCR. Steps in PCR.
Quantitative real time polymerase chain reaction.Fluorescent dyes and probes.
Advantages real-time PCR.
Real-time PCR primer
Primer design software
The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific detection and production of large amounts of DNA. PCR-based strategies have propelled huge scientific endeavors such as the Human Genome Project. The technique is currently widely used by clinicians and researchers to diagnose diseases, clone and sequence genes, and carry out sophisticated quantitative and genomic studies in a rapid and very sensitive manner. One of the most important medical applications of the classical PCR method is the detection of pathogens. In addition, the PCR assay is used in forensic medicine to identify criminals. Because of its widespread use, it is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
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.
A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting
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.
PCR- Steps;Applications and types of PCR (Exam point of view)Sijo A
The term PCR stands for Polymerase Chain Reaction.
It is an invitro amplification technique that allows synthesizing millions of copies of the DNA or gene of interest from a single copy.
It is called “Polymerase” because the only enzyme used in this reaction is DNA polymerase.
The PCR is invented by Kary Mullis in 1985.He received Nobel Prize in Chemistry in 1993.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
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.
A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting
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.
PCR- Steps;Applications and types of PCR (Exam point of view)Sijo A
The term PCR stands for Polymerase Chain Reaction.
It is an invitro amplification technique that allows synthesizing millions of copies of the DNA or gene of interest from a single copy.
It is called “Polymerase” because the only enzyme used in this reaction is DNA polymerase.
The PCR is invented by Kary Mullis in 1985.He received Nobel Prize in Chemistry in 1993.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
this ppt contain about pcr technique and its three process,primers in pcr,dna polymerase in pcr,melting temp of dna in pcr and applications of pcr technology
this doc is having basic information about PCR techmique. it contains history, principle, advantages, disadvantages, and applications.
it can give a brief idea about pcr technique.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
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Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
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2. Contents
What is PCR ?
History of PCR
Principle of PCR
PCR Machine:- Thermocycler
PCR Components
Basic requirementsfor PCR reaction
Mechanism of PCR
Advantage and Disadvantages of PCR
Application and it’s limitations
3. What is PCR ?
PCR is a technique that takes specific sequence of
DNA of small amount and amplifies it to be used
for further testing .
In-vitro technique.
Is a Molecular, Biochemical technology, Used to
amplify a single piece of DNA, By a series of
Heating and Cooling cycles, generating millions
of copies of a particular DNA sequence In Vitro.
4. DNA template:- contains the DNA region (target)
to be amplified.
DNA Primer:- Complementary to the 3’ (three prime) ends
of each of the sentences and anti-sence
strand of the DNA target.
5. History of PCR
In 1983, Dr.Karry Mullis described the technique of
in vitro gene amplification and named it as a Polymer
- ase Chain Reaction. Later on, he was awarded the
Noble prize for his findings.
1985:- First publication of PCR by Cetus Corporation
appears in science.
1986:- purified Taq polymerase is first used in PCR.
1988:- PerkinElmer introduces the automated thermal cycler.
1989:- Science declares Taq polymerase “molecules” of the
year.
6. 1990:- Amplification and detection of specific DNA
sequence using a fluorescent DNA- binding dye,
laying the foundation for future “real-time “ or
“kinetic” PCR.
1993:- Dr.Karry Mullis shares Noble prize in Chemistry for
conceiving PCR technology.
7. Principle of PCR
Purpose
To amplify a lot of double – stranded DNA molecules
(fragments) with same (identical) size and sequence
enzymatic method and cycling condition.
The principle of the PCR is based on the
temperature variations of heating and cooling –
thermocyclling reaction divided into three
steps :-
8. Denaturation:- The dsDNA becomes single stranded at a higher
temperature during denaturation. Here hydrogen
bonds betweentwo DNA strands break.
Annealing:- In the primer binds or anneals to its exact complem
-entry sequence on a DNA during the annealing steps.
The primer provides a site for the initiation of synthesis.
Extension:- Taq DNA polymerase uses the 3’end of the primer and
starts DNA synthesis by adding nucleotides to the growing
DNA strand.
All three steps are repeated for 25 to 40 cycles and in each
cycle the DNA becomes double.
9. PCR Machine:- Thermocycler
The machine thermocycler provides
various temperature for each step to
complete Denaturation, annealing and
extension of DNA occur at different
temperature thus the machine is known
as Thermocycler.
10. PCR Components
Chemical:- dNTPs, distill water, PCR reaction buffer, enzyme Taq
DNA polymerase, primers and template DNA.
Instruments:- Thermocycler, spinner and agarose gel electrophoresis
unit.
Other utilities:- PCR tubes, stands, pipettes , tips.
PCR reagents:- Template DNA, PCR primers, dNTPs, Taq DNA polyme
rase and PCR buffer are the major reagents of PCR
reaction. The composition and quantity of each reagent
are very important. A single ųL variation in any
of the reagents leads to reaction failure.
11. Basic Requirements for PCR
reaction
DNA sequence of target region must be known.
Primer:- typically 20 – 30 bases in size. These can be readily
produced by commercial companies.
Thermo – stable DNA polymerase:- Taq polymerase
which is not inactivatedby heating to 95°C.
DNA thermocycler:- machine which can be programmed
to carry out heating and cooling of sample
overa number of cycles.
12. Mechanism of PCR
Various temperature zone governs each PCR steps, viz
Denaturation, annealing and extension followed by a single
initial denaturation and final extension steps.
In each step, different reactions occurs.
Denaturation:- Tem: 90°C to 95°C.
Time: 30 seconds to 90 sec.
13. In a denaturation two single – stranded DNA forms from the double –
stranded one. At 94°C temperature,the double – stranded DNA opens up
by breaking hydrogen bonds. The process of denaturationis followed by
the initial denaturation for 5 to 7 minutes at the same temperature.
Annealing:- Temperature:- 55°C to 65°C.
Time:- 30 to 60 sec.
Primer bind to their complimentary sequences.
14. After the denaturationprimer anneals to ssDNA at its exact
annealing temperature. Base on the GC content of primer, every primer
has its own annealing temperature.
The annealing temperature is usually raging from 55°C to 65°C .
Annealing temperature lower than that leads to non-specific binding
while highertemperature leads to amplification failure. 45 seconds to 1
minute are enough for the second step, annealing for more than 1minute
causes non-specific amplification.
Extension:- Temperature: 70°C to 72°C
Time: 45 sec
15. After the binding of the primer, it’s time to expand the DNA
strand. Here in extension step the Taq DNA polymerase comes in action
and add dNTPs to the DNA strand. The temperaturefor the extensionis
72°C for 45 seconds.
After completingall steps one more time the final extension is
performed for 7 minutes. The graphical representation of each PCR steps is
explained in the figure below:
16.
17. Advantage of PCR
Small amount of DNA is required for test.
Result obtained more quickly- usually within 1day for PCR.
Usually not necessary to use radioactivematerial (32P) for PCR.
PCR is much more precise in determining the size of alleles – essential for same disorders.
PCR can be used to detect point mutation.
Robust , making it possible to amplify DNA from degraded samples.
Disadvantages of PCR
Prior sequence knowledge.
Short size range of amplification products 100bp – 5000 bp.
Chance of Contamination.
Novel mutation can not be found using PCR, we have to do sequencing for that.
18. Application of PCR
The PCR has numerous applications in biological research as well as
diagnostics.
Diagnosis of inherited disease: the PCR is most routinelyused in the
diagnosis of some inheriteddisease such as
sickle cell anemia, thalassemia, gene mutation, etc.
This technique is appropriate for single – gene disorders.
Microbial identification: the microbial culture technique is traditional and
time consumingand the chance of infection is also high
in the case of culturing. In modern days, PCR is used in the
identificationof microbes.
19. DNA Fingerprinting and genetic imprinting:The PCR is first choice
for DNA Fingerprinting. Criminal
verification, identification of a person, and material
cell contaminationcan be detected using DNA
Fingerprinting.
The PCR is one of the best techniques for marker assistant selection. RFLP,
AFLP, RAPD ,VNTR and STR are some of the PCR based techniques.
PCR helps in detecting cancer genes and infections.
Further PCR is applicable to sex determination and sex identification.
The PCR is used in:
Gene editing.
Geneticengineering.
DNA and RNA quantification.
20. cDNA and gDNA library preparation.
Developingnew assays.
Limitations of PCR
Also Multigenic disorders cannot be
detected using PCR.
We cannot identify structural and numerical
chromosomal anomalies through PCR.
