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
INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
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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INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
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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Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
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.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
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.
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.
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
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.
Sanger sequencing is one of the DNA sequencing methods used to identify and determine the sequence (Nucleotide) of DNA .This is an enzymatic method of sequencing developed by Fred Sanger.
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.
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 power point presentation consists of 36 slides explaining about history, principle, different steps involved and applications of DNA fingerprinting. Recent Developments and the Future prospects of DNA profiling have also been mentioned
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
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
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
SLIDE CONTAIN BREIF NOTE ON PCR. IT CONTAINS 21 SLIDES INCLUDING, WHAT IS PCR? COMPONENTS, WORKING MECHANISM, APPLICATIONS, CONCLUSION, AND SOME REFRENCES, HISTORY ALSO
Basic Molecular Biology:
Molecular biology is the branch of biology that focuses on understanding the fundamental processes and mechanisms underlying life at the molecular level. It involves the study of biological molecules such as DNA, RNA, and proteins, and how they interact to regulate various cellular processes. Molecular biology techniques enable scientists to investigate genetic information, gene expression, and the structure and function of macromolecules.
Polymerase Chain Reaction (PCR):
Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify and replicate a specific segment of DNA in a laboratory setting. PCR allows scientists to make millions of copies of a target DNA sequence in a short period. It consists of repeated cycles of denaturation (separation of DNA strands), annealing (binding of short DNA primers to the target sequence), and extension (synthesis of new DNA strands using a heat-stable DNA polymerase enzyme). PCR has diverse applications, including DNA sequencing, genetic testing, forensics, and the study of gene expression.
Reverse Transcription Polymerase Chain Reaction (RT-PCR):
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a variation of the standard PCR technique that is specifically used to amplify RNA molecules. It involves a two-step process. First, the RNA is reverse transcribed into complementary DNA (cDNA) using the enzyme reverse transcriptase. Then, the cDNA is amplified using standard PCR. RT-PCR is essential for studying gene expression, viral RNA detection (e.g., for diagnosing diseases like COVID-19), and a range of other applications where RNA analysis is crucial.
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Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2. What is the PCR?
PCR is the technique that take specific sequence of DNA and
amplifies it to be used for further testing.
Purpose of the PCR is to amplify the lot of double stranded
DNA molecules(fragments) with same size and sequence by
enzymatic method and cycling condition.
3. PCR reaction :-
Essential components required:
DNA template
DNA polymerase
Primer
dNTPs
Buffer to maintain PH
Divalent cations (Mg+2)
4. principle :-
PCR contain three process
1. Denaturation
2. Annealing
3. Extension
cycle repeated according to requirement
5. Principle :-
Denaturation
Denaturation is the first step of PCR , in which the DNA strands are separated by
heating to 94–98 °C for 20–30 seconds.
It causes DNA melting of the DNA template by breaking the hydrogen bonds
between two strands and resulting single-stranded DNA molecules.
6. Principle :-
Annealing
The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing
annealing of the primers to the single-stranded DNA template.
This temperature must be low enough to allow for hybridization of the primer to the
strand, but high enough for the hybridization to be specific, i.e., the primer should
only bind to a perfectly complementary part of the template. If the temperature is
too low, the primer could bind imperfectly. If it is too high, the primer might not
bind.
7. Principle :-
Annealing
Typically the annealing temperature is about 3–5 °C below the melting
temperature of the primers used.
Stable DNA–DNA hydrogen bonds are only formed when the primer sequence
very closely matches the template sequence.
The polymerase binds to the primer-template hybrid and begins DNA formation.
8. Principle :-
Extension
The temperature at this step depends on the DNA polymerase used. Taq DNA
polymerase has its optimum activity temperature at 75–80 °C, and commonly a
temperature of 72 °C is used with this enzyme in this step.
The extension time depends both on the DNA polymerase used and on the length
of the DNA fragment to amplify.
9. Principle :-
Extension
At this step the DNA polymerase synthesizes a new DNA strand complementary
to the DNA template strand by adding dNTPs that are complementary to the
template in 5' to 3' direction.
Primer are extended by adding nucleotides (dNTPs) complementary to the DNA
template strand.
Now the first cycle over and second cycle continued, as PCR machine is
automated thermocycler the same cycle repeated up to 30-40 times.
12. PCR (contd…)
Primer in PCR
Primer are usually short, chemically synthesized oligonucleotides, with a length of
about 18-30 bases, Ideal G-C content 40-60% but play around the length.
Melting temperature should be between 42-65°C
Primers serve as a starting point for DNA synthesis and they are hybridized to the
target DNA, which is then copied by DNA polymerase.
Avoid primer-primer hybridization and pick the primer that do not form hairpins.
13. PCR (contd…)
Taq DNA polymerase in PCR
Taq polymerase is a thermostable DNA polymerase named after thermophilic
bacterium Thermus Aquaticus from which it was isolated.
Taq’s optimum activity between 75-80°C and in extension step applied
temperature is 72°C in PCR.
Drawbacks of Taq is relatively low replication fidelity, it is because lacks of
exonuclease proofreading activity.
Extension rate - 50 nucleotides/second.It has error rate measured at about 1 in
9,000 nucleotides.
14. PCR (contd…)
Pfu DNA polymerase in PCR
Pfu polymerase is a thermostable DNA polymerase named after hyperthermophilic
archaeon pyrococcus furiosus from which it was isolated.
Optimum activity of Pfu is between 72°C .
It has 3’ to 5’ exonuclease proofreading activity, this means Pfu polymerase
generate few errors than Taq polymerase.
Extension rate - 1000 nucleotides/minute.It has error rate measured at about 1 in
1.3 million nucleotides.
15. PCR (contd…)
DNA melting
The melting temperature defined as the temperature at which half of the DNA
strands are in the single-stranded (ssDNA) state.
A=T less energy required to break AT bonds
G=C more energy required to break GC bonds
16. APPLICATIONS OF PCR
1. Diagnosis of genetic diseases
The use of PCR in diagnosing genetic disease, whether inherited genetic changes or as a
result of a spontaneous genetic mutations, is becoming more common. Diseases can be
diagnosed even before birth. Examples include:
Genetic counseling-screening the parents for genetic diseases before deciding on having
children.
17. APPLICATIONS OF PCR (contd…)
2. Genetic fingerprints
One of the most famous uses for PCR is in the creation of a genetic fingerprint (also known
as DNA profiling) from a sample of blood or semen, or from a hair root. Much beloved by
writers of detective fiction, genetic fingerprints are profiles of specific stretches of DNA that
vary from person to person. PCR also plays a role in mitochondrial DNA analysis, used for
samples from hair shafts and bones when other samples are not available.
Genetic analysis based on PCR is also used in paternity testing, and in tissue typing for
organ transplantation.
18. APPLICATIONS OF PCR (contd…)
3. Detection and diagnosis of infectious diseases
PCR can detect infectious disease before standard serological laboratory tests (tests to
detect the presence of antibodies), so allowing treatment to start much earlier. Because of
this, PCR is also useful for screening donated blood for infections, and is especially useful for
infections that are difficult to culture in the laboratory, such as tuberculosis.
19. APPLICATIONS OF PCR (contd…)
4. Detection of infection in the environment
PCR is used to monitor and track the spread of infectious disease within an animal or human
population. PCR can also be used to detect bacterial and viral DNA in the environment, for
example looking at pathogens in water supplies.
5. Personalised medicine
PCR is used in personalised medicine to select patients for certain treatments, for example
in cancer when patients have a genetic change that makes a patient more or less likely to
respond to a certain treatment.
20. APPLICATIONS OF PCR (contd…)
6. PCR in research
Escherichia coli in genetic engineering, and to amplify stretches of genetic material for
Sanger sequencing – the Human Genome Project used PCR.
PCR can be used in analysis of gene expression, for example looking at levels of expression
and when genes are switched on and off in physiological processes, including in health and
disease.
