Polymerase chain reaction (PCR) is a technique used to amplify a single copy of a DNA segment across orders of magnitude, generating thousands to millions of copies. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Two primers are used to target the specific segment that will be amplified. During each cycle, the DNA polymerase enzyme adds nucleotides to the primers, duplicating the targeted DNA segment. As the cycles repeat, the copy number increases exponentially. PCR is widely used in clinical diagnostics and research for applications such as disease diagnosis, genetic testing, and forensic analysis.
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
b pharmacy
pharmaceutical biotechnology
Polymerase chain reaction
History
Purpose
Components of PCR
Steps of PCR
Denaturation of DNA template
Annealing of primers
Extension of ds DNA molecules
Reaction Condition & Experimental Protocol
General PCR Protocol
Application
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
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
b pharmacy
pharmaceutical biotechnology
Polymerase chain reaction
History
Purpose
Components of PCR
Steps of PCR
Denaturation of DNA template
Annealing of primers
Extension of ds DNA molecules
Reaction Condition & Experimental Protocol
General PCR Protocol
Application
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
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
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
What is PCR ? What is Real Time PCR ? Polymerase Chain Reaction ? What is Reverse Transcriptase Enzyme ?
Presented By:
Bharat Bhushan Negi
M.Tech. Biotechnology
IIT Guwahati
Oxford Nanopore was founded in Oxford Nanolabs by Dr.Gordon Sanghera, Dr.Spike Willcocks and Professor Hagan Bayley. Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors.
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
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
What is PCR ? What is Real Time PCR ? Polymerase Chain Reaction ? What is Reverse Transcriptase Enzyme ?
Presented By:
Bharat Bhushan Negi
M.Tech. Biotechnology
IIT Guwahati
Oxford Nanopore was founded in Oxford Nanolabs by Dr.Gordon Sanghera, Dr.Spike Willcocks and Professor Hagan Bayley. Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors.
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
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
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
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
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
For more information, visit-www.vavaclasses.com
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
How to Create Map Views in the Odoo 17 ERPCeline George
The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptx
PCR
1.
2. Polymerase
technique
chain
used
reaction (PCR) is a
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.
3. Developed in 1983 by Kary Mullis, PCR is
now a common technique used in clinical
and research laboratories for a broad variety
of applications.
In 1993, Mullis was awarded the Nobel Prize
in Chemistry for his work on PCR.
4. DNA template is target DNA sequence. It is
the DNA molecule that contains the region
(segment) to be amplified, the segment we
are concerned which is the target sequence.
5. DNA polymerase:
DNA polymerase sequentially adds
nucleotides complimentary to template
strand at 3’-OH of the bound primers and
synthesizes new strands of DNA
complementary to the target sequence. The
most commonly used DNA polymerase is
Taq DNA polymerase (from Thermus
a thermophillic bacterium)aquaticus,
because of high temperature stability.
6. DNA polymerasePfu (fromPyrococcus
furiosus) is also used widely because of its
higher fidelity (accuracy of adding
complimentary nucleotide).
Mg2+ ions in the buffer act as co-factor for
DNA polymerase enzyme and hence are
required for the reaction.
7. Primers are synthetic DNA strands of about
18 to 25 nucleotides complementary to 3’end
of the template strand. DNA polymerase
starts synthesizing new DNA from the 3’ end
of the primer .
8. Two primers must be designed for PCR; the
forward primer and the reverse primer.
9. The forward primer attaches to the start codon of the template DNA (the anti-
sense strand), while the reverse primer attaches to the stop codon of the
complementary strand of DNA (the sense strand).
The 5' ends of both primers bind to the 3' end of each DNA strand.
10. Primers should bind to template with good
specificity and strength. If primers do not
bind to correct template, wrong sequence
sequences and appropriate
will get amplified. Optimal primer
primer
concentrations are essential for maximal
specificity and efficiency in PCR.
11. Complementary nucleotide sequences within
a primer and between primers should be
avoided. If there are complimentary
sequences in two primers used (one primer
for each will
hybridize
DNA strand), the primers
with each other thus forming
primer-dimmers and will not be available for
binding with template. If there are
complementary sequences within a primer,
it will make hairpin loop structures as shown
below.
12.
13.
14. The primers should preferably end on a
Guanine and Cytosine (GC) sequence so
that it can attach with sufficient strength
with template. This increases efficiency of
priming due to stronger bonding of G and C
bases.
15. All types of nucleotides are "building
blocks" fornew DNA strands and essential
for reaction. It includes Adenine(A),
Guanine(G), Cytosine(C), Thymine(T) or
Uracil(U).
16. Magnesium affects primer annealing and
template denaturation, as well as enzyme
activity.
An excess of magnesium gives non-specific
amplification products, while low magnesium
yields lesser amount of desired product.
17. There are three major steps in a PCR, which are
repeated for 30 or 40 cycles. This is done on an
automated cycler, which can heat and cool the
tubes with the reaction mixture in a very short
time.
18. • During the heating step (denaturation), the
reaction mixture is heated to 94°C for1min,
which causes separation of DNA double
stranded. Now, each strand acts as template for
synthesis of complimentary strand.
19.
20. This step consist of cooling of reaction mixture
after denaturation step to 54°C, which causes
hybridization (annealing) of primers to separated
strand of DNA (template). The length and GC-
content (guanine-cytosine content) of the primer
should be sufficient for stable binding with
template.
21. Guanine pairs
hydrogen bonding adenine binds
with cytosine with three
with
thymine with two hydrogen bonds. Thus,
higher GC content results in stronger
binding.
22.
23. • The reaction mixture is heated to 72°C which is
the ideal working temperature for the Taq
polymerase. The polymerase adds nucleotide
(dNTP's) complimentary to template on 3’–OH
of primers thereby extending the new strand.
24.
25. First three steps are repeated 35-40 times to
produce millions of exact copies of the
target DNA. Once several cycles are
completed, during the hold step, 4–15 °C
temperature is maintained for short-term
storage of the amplified DNA sample.
26. • As both strands are copied during PCR, there is
an exponential increase of the number of copies
of the gene. Suppose there is only one copy of
the desired gene before the PCR starts, after one
cycle of PCR, there will be 2 copies, after two
cycles of PCR, there will be 4 copies. Afterthree
cycles there will be 8 copies and so on.
27.
28.
29.
30.
31. Polymerase Chain Reaction
1. Diagnosis of bacterial and viral diseases
In early phases of tuberculosis, the sputum may contain only
very few tubercle bacilli, so that usual acid fast staining may be
negative.
But PCR could detect even one bacillus present in the specimen. Any
other bacterial infection could also be detected.
The specific nucleotide sequences of the bacilli are amplified by PCR
and then detected by Southern blot analysis.
32. 2. Medicolegal cases
PCR allows the DNA in a single cell or in a hair follicle to be
analysed.
The restriction analysis of DNA from the hair follicle from the
crime scene is studied after PCR amplification.
This pattern is then compared with the restriction analysis of DNA
samples obtained from various suspects.
The culprit's sample will perfectly match with that of PCR
amplified sample.
33. 3. Diagnosis of genetic disorders
The PCR technology has been widely used to amplify the
gene segments that contain known mutations for diagnosis of
inherited diseases such as sickle cell anemia, thalassemia, cystic
fibrosis, etc.
4. Prenatal diagnosis of inherited diseases
5. Cancer detection: PCR is widely used to monitor residual
abnormal cells present in treated patients. Similarly, identification of
mutations in oncosuppressor genes such as p53, or retinoblastoma
gene can help to identify individuals at high risk of cancer.
34.
35. Reverse Transcriptase PCR (RTPCR)
Instead of Taq polymerase, Tth polymerase from Thermus
Thermophilus may be used. This enzyme has both DNA
polymerase and reverse transcriptase activities at 95°C. So
mRNA is copied to cDNA synthesis followed by PCR
amplification. In ordinary PCR, DNA is detected; that
DNA could be from a living or non-living organism. But in
reverse PCR, mRNA is detected; that means, it is derived
from a living organism.
36. Reverse Transcriptase PCR (RTPCR)
By this method, quantitation of the number of virus
present in a sample can be calculated., e.g.,viral load in
HIV (human Immuno deficiency virus or HBV (hepatitis
B virus).
37. A real-time polymerase chain reaction (real-time PCR),
also known as quantitative polymerase chain
reaction (qPCR), 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), not at its end, as in
conventional PCR.
38. Real-time PCR is carried out in a thermal cycler with the capacity to detect the
fluorescence emitted by the dye applied.
The PCR process generally consists of a series of temperature changes that are
repeated 25–50 times.
These cycles normally consist of three stages:
the first, at around 95 °C, allows the separation of the nucleic acid's double
chain
the second, at a temperature of around 50–60 °C, allows the binding of the
primers with the DNA template
the third, at between 68–72 °C, facilitates the polymerization carried out by the
DNA polymerase.