Introduction to PCR and RT-PCR, RT-PCR
PCR, Contents of PCR, Steps in PCR, PCR VS RT-PCR, Applications
Presented by
N. Ramya
Department of Pharmacology
In this slide contains contents, steps, different and application of PCR and RT-PCR
Presented by: RAMYA NAGARAJU GARI (Department of pharmacology).
RIPER, anantapur
In this slide contains contents, steps, different and application of PCR and RT-PCR
Presented by: RAMYA NAGARAJU GARI (Department of pharmacology).
RIPER, anantapur
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
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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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.
Probes are used for hybridization purposes. different types of probes can be used on the basis of what we want to hybridize. May be Radioactive or Non-Radioactive.
Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts.
cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.
Target Validation
Introduction,Drug discovery, Target identification and validation, Target validation and techniques
By
Ms. B. Mary Vishali
Department of Pharmacology
In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis.
Presented by: A.Geethanjali (Department of pharmacology),
RIPER, anantapur.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
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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SUPPORT EDUCATION... SUPPORT US
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.
Probes are used for hybridization purposes. different types of probes can be used on the basis of what we want to hybridize. May be Radioactive or Non-Radioactive.
Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts.
cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.
Target Validation
Introduction,Drug discovery, Target identification and validation, Target validation and techniques
By
Ms. B. Mary Vishali
Department of Pharmacology
In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis.
Presented by: A.Geethanjali (Department of pharmacology),
RIPER, anantapur.
In this slide contains introduction, genomic materials of virus and testing method of covid 19 by using RT-PCR.
Presented by: R.Rekha (Department of pharmacology),
RIPER, anantapur.
Introduction to Analytical Techniques in Phaese III,
Spectrophotometry, Reflectance photometry, Nephelometry & Turbidimetry, Osmometry, Potentiometry, Flowcytometry, Densitometry, Electrophoresis, LC-MS, ICP-MS
Presented by
B. Kranthi Kumar
Department of Pharmacology
In this slide contains analytical techniques in phase-3 clinical trials.
Presented by: KRANTHI KUMAR BONALA (Department of pharmacology).
RIPER, anantapur
Introduction to Screening Models Of Anti Cancer Drugs
Need for novel anti cancer drugs, In - vitro methods, In - vivo methods, Advantages and disadvantages
Presented by
T. Niranjan Reddy
Department of Pharmacology
In this slide contains instrumentation of Fourier-Transform Nuclear Magnetic Resonance (FT-NMR).
Presented by: P. Venkatesh. (Department of pharmaceutical analysis).
RIPER, anantpur.
In this slide contains deep explanation about Ionization Techniques in LC-MS.
Presented by: G Chiranjeevi. (Department of pharmaceutical analysis)
RIPER, anantpur.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
In this slide contains principle, advantage, dis advantage and application of UPLC.
Presented by: P. Sudheer Kumar. (Department of pharmaceutical analysis)
RIPER, anantapur.
JOURNAL CLUB PRESENTATION (20L81S0402-PA & QA)
Presented by: K VENKATSAI PRASAD (Department of pharmaceutical analysis and quality assurance).RIPER, anantapur
In this slide contains Study of Quality of Raw Materials and General methods of analysis of Raw materials used in cosmetic manufacture as per BSI
Presented by: P.PAVAN KALYAN (Department of pharmaceutical analysis).RIPER, anantapur
In this slide contains Determination of Acid value, Saponification value and Ester value.
Presented by: P.NARESH (Department of pharmaceutical analysis).RIPER, anantapur
More from Raghavendra institute of pharmaceutical education and research . (20)
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
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
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
(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.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
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.
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.
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PCR and RT-PCR
A Seminar as a part of curricular requirement
for M. Pharmacy, I Year - I semester
Presented by
N. Ramya
(20L81S0110)
PHARMACOLOGY
Under the guidance of
A. Sudheer, M. Pharm
Associate Professor Dept. of pharmacology
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RT-PCR
PCR
Contents of PCR
Steps in PCR
PCR VS RT-PCR
Applications
References
contents
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• principle
• In RT- PCR, the RNA template is first converted into a
Complementary DNA (cDNA) using a reverse transcriptase (RT).
• The cDNA is then used as a template for exponential
amplification using PCR.
• The two technique use the same process except that RT-PCR has
an added step of reverse transcription of RNA to DNA to allow
for amplification.
RT-PCR:
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• optimized one- step RT- PCR condition, supports the reverse
transcription of the RNA from unpurified or crude samples, such as
whole blood and serum.
• However, the starting RNA templates are prone to degradation in the
one- step approach, and the use of this approach is not recommended when
repeated assays from the same sample is required.
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Polymerase Chain Reaction
• Polymerase chain reaction (PCR) is a technique used in
molecular biology.
• To amplify a single copy or a few
copies of a segment of DNA generating
thousands to millions of copies of a
particular DNA sequence.
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• Components Of PCR constitutes the following:
1.DNA Template– The DNA of interest from the sample.
2.DNA Polymerase– Taq Polymerase is used. It is thermostable and does not
denature at very high temperatures.
3.Oligonucleotide Primers- These are the short stretches of single-stranded
DNA complementary to the 3’ ends of sense and anti-sense strands.
Components Of PCR
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4.Deoxyribonucleotide triphosphate– These provide energy for polymerization
and are the building blocks for the synthesis of DNA. These are single units
of bases.
5.Buffer System– Magnesium and Potassium provide optimum conditions for
DNA denaturation and renaturation. It is also important for fidelity,
polymerase activity, and stability.
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• 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.
1. Denaturation.
2. Annealing.
3. Extension.
Procedure:
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Denaturation at 94°C :
• During the denaturation, the reaction
mixture is heated
to 94°C for 1 min, which causes
separation of DNA double stranded.
• Now, each strand acts as template
for synthesis of complimentary strand.
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Annealing at 54°C :
• The reaction temperature is lowered to 54-60℃ for around 20-
40 seconds. Here, the primers bind to their complementary
sequences on the template DNA.
• Primers are single-strand sequences of DNA or RNA around 20
to 30 bases in length.
• They serve as the starting point for the synthesis of DNA.
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• The two separated strands run in the opposite direction
and consequently there are two primers- a forward primer
and a reverse primer.
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• At this step, the temperature is raised to 72-80℃.
• The bases are added to the 3’ end of the primer by the Taq polymerase
enzyme.
• This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds
about 1000bp/minute under optimum conditions.
• Taq Polymerase can tolerate very high temperatures. It attaches to the
primer and adds DNA bases to the single strand. As a result, a double-
stranded DNA molecule is obtained.
Extension:
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PCR RT-PCR
PCR is a technique to amplify a
segment of DNA, generating millions of
copies of a DNA Sequence.
RT-PCR is a variant of PCR used in
detection of gene expression in
molecular biology.
Denaturation, annealing, and
extension are the three steps.
RT-PCR is followed by PCR.
A double-standard DNA molecule serve
as the template.
A single standard RNA molecule is the
template for the reverse transcription.
DNA Polymerase is used as the
enzyme.
Reverse transcriptase and DNA
polymerase are used as enzyme.
Used in functional analysis of
genes, diagnosis, and
monitoring of heredity diseases,
DNA Cloning, DNA Sequencing.
Used in detection of gene
expression.
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• Gene Insertion: RT-PCR can also be very useful in the insertion of
eukaryotic genes into prokaryotes.
• It is useful in molecular identification, sequencing, genetic engineering.
• Cancer Detection: scientists are working on ways to use RT-PCR in cancer
detection to help improve prognosis and monitor response to therapy .
• Used as a tool in genetic fingerprinting.
• Compare the genome of two organisms in genomic studies.
• In the phylogenetic analysis of DNA from any source such as fossils.
Applications
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• Julia Bachman. Reverse Transcription PCR. Elsevier. 2013,
530. 67-74.
• Freeman WM, Walker SJ. Quantitative RT-PCR. Elsevier.
2012, 26(1): 124-125.
• Alexander A. Morley. Digital PCR. Elsevier. 2014, 272.
12-13.
References
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