The document provides information about a seminar on the application of polymerase chain reaction (PCR) in plant genome analysis. It discusses the basics of PCR including its history, the three main steps of PCR (denaturation, annealing and extension), and key enzymes involved such as DNA polymerase. It also summarizes some applications of PCR in genetic disease testing, crop improvement, and plant molecular biology research. PCR is a powerful technique for amplifying specific DNA sequences that has many uses in molecular biology and biotechnology.
Introduction
Enzyme involve of DNA repair
Types of DNA repair
direct DNA repair
excision repair system
mismatch repair system
Conclusion
Reference
DNA polymerase –a class of enzyme to all synthesize 5’ to 3’ direction of nucleotides.
DNA polymerase I – a class of enzyme 1st isolated by Escherichia coli, and function is removes of RNA primers ,during DNA replication.
Helicase- any of a group of enzyme that unwind the two strand of DNA to facilitate DNA replication.
Exonuclease – an enzyme capable of cutting phosphodiester bonds between nucleotides located at an end of a DNA strand .
Endonuclease – an enzyme capable of cleaving phosphodiester bonds between nucleotide located internally in a DNA strand .
DNA ligase – a enzyme that fill the gap of nucleotides.
Introduction
Enzyme involve of DNA repair
Types of DNA repair
direct DNA repair
excision repair system
mismatch repair system
Conclusion
Reference
DNA polymerase –a class of enzyme to all synthesize 5’ to 3’ direction of nucleotides.
DNA polymerase I – a class of enzyme 1st isolated by Escherichia coli, and function is removes of RNA primers ,during DNA replication.
Helicase- any of a group of enzyme that unwind the two strand of DNA to facilitate DNA replication.
Exonuclease – an enzyme capable of cutting phosphodiester bonds between nucleotides located at an end of a DNA strand .
Endonuclease – an enzyme capable of cleaving phosphodiester bonds between nucleotide located internally in a DNA strand .
DNA ligase – a enzyme that fill the gap of nucleotides.
Prediction of the three dimensional structure of a given protein sequence i.e. target protein from the amino acid sequence of a homologous (template) protein for which an X-ray or NMR structure is available based on an alignment to one or more known protein structures
GENOMICS
Genomics is the study of all genes in an organism, also known as its genome. Genomics includes identifying the specific building blocks of all the genes in a cell, mapping their location in relation to the rest of the DNA, and studying the function of those genes or combination of those genes.
Types of Genomics :
1. Structural Genomics
2. Comparative Genomics
3.Functional Genomics
4. Epigenomics
5. Metagenomics
6. Pharmacogenomics
7. Mutation Genomics.
PROTEOMICS : (PROTEin in complement to genOME)
Proteomics is the study of proteome [Proteome is a protein molecule that interacts to give the cell its individual character]. Proteomics is a subset of functional genomics.
The proteome of a cell is all the proteins expressed by its genome. The proteome is of intense interest to investigators because proteins are the major functional components of the cell.
Proteomics is the study of proteins in order to revolutionize the understanding of cell behaviour and disease.
1. It studies the translation of process of RNA into proteins as well as the overall process of DNA into proteins.
2. It studies the diseases through proteins because disease process manifest themselves at the level of protein activity.
3. Most drugs act by targeting proteins or protein receptors, so Proteomics is important in new generation of drugs.
4. Proteins are more complex than genes because they can be modified after formation.
5. Proteomics is the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes.
6. Proteomics can use analysis techniques to determine all of the post translational modifications that proteins undergo and therefore determine what makes a diseased or mutant protein different from a normal protein.
Proteins are fundamental components of all living cells. Proteins help us digest our food, fight infections, control body chemistry, keep our bodies function smoothly. Identifying a proteins’ shape or structure is key to understanding its biological function and its role in health and disease.
A protein needs to adopt a final and stable 3-dimensional shape in order to function properly.
• The Tertiary Structure of a protein is the arrangement of the secondary structures into this
final 3-dimensional shape.
Recognizable folding pattern of proteins involved two or more elements of its secondary structure.
In a motif two elements of secondary structure folded against each other.
Because it is falling between secondary and tertiary structure and describe small part of protein or entire polypeptide chain.
A brief introfuction of label-free protein quantification methodsCreative Proteomics
If you want to know more about our services, please visit https://www.creative-proteomics.com/services/label-free-quantification.htm.
Label-free protein quantification is a mass spectrometry-based method for identifying and quantifying relative changes in two or more biological samples instead of using a stable isotope-containing compound to label proteins.
Protein docking is used to check the structure, position and orientation of a protein when it interacts with small molecules like ligands. Protein receptor-ligand motifs fit together tightly, and are often referred to as a lock and key mechanism. There are both high specificity and induced fit within these interfaces with specificity increasing with rigidity. The foremost thing that we need to start with a docking search is the sequence of our protein of interest. (Halperin et al., 2002).
Protein-protein interactions occur between two proteins that are similar in size. The interface between the two molecules tends to be flatter and smoother than those in interfaces of these interactions do not have the ability to alter protein-ligand interactions. Protein-protein interactions are usually more rigid, the conformation in order to improve binding and ease movement. (Smith and Sternberg, 2002).
The process of drug development has revolved around a screening approach, as nobody knows which compound or approach could serve as a drug or therapy. Such almost blind screening approach is very time-consuming and laborious. The goal of structure-based drug design is to find chemical structures fitting in the binding pocket of the receptor. Based on the three-dimensional structure of the target protein, it can automatically build ligand molecules within the binding pocket and subsequently screen them (Weil et al., 2004).
A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT, an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides. (O’Reilly et al., 2006).
A physical process by which a polypeptide chain (sequence of amino acids) folds into its characteristic & functional native structure from a random coil or a linear sequence.
Quantum Mechanics in Molecular modelingAkshay Kank
This slides gives you the information related to computer aided drug design and its application in drug discovery. Also you learn the Quantum mechanics related to the molecular mechanics. Theory related to molecular modeling and how the molecular modeling helps in drug discovery.
A Remarkable Conflict Between Biochemical Dogma And Radical Concept :
Traditional Concept :
By the 1950s it had been clearly established that oxidative phosphorylation involved the stepwise transfer of electrons through a series of carriers to molecular oxygen. But how the energy derived from these electron transfer reactions was converted to ATP remained a mystery. The general assumption was that ADP was converted to ATP by direct transfer of high-energy phosphate groups from some other intermediate. Thus it was postulated that high-energy intermediates were produced as a result of electron transfer reactions and that these intermediates drove ATP synthesis by phosphate group transfer.
Concept Proposed By Peter Mitchell :
The fundamental proposal of the chemiosmotic hypothesis was that the “intermediate” that coupled electron transport to ATP synthesis was a proton electrochemical gradient across the membrane. Mitchell postulated that such a gradient was produced by electron transport and that the flow of protons back across the membrane in the energetically favorable direction was then coupled to ATP synthesis
Prediction of the three dimensional structure of a given protein sequence i.e. target protein from the amino acid sequence of a homologous (template) protein for which an X-ray or NMR structure is available based on an alignment to one or more known protein structures
GENOMICS
Genomics is the study of all genes in an organism, also known as its genome. Genomics includes identifying the specific building blocks of all the genes in a cell, mapping their location in relation to the rest of the DNA, and studying the function of those genes or combination of those genes.
Types of Genomics :
1. Structural Genomics
2. Comparative Genomics
3.Functional Genomics
4. Epigenomics
5. Metagenomics
6. Pharmacogenomics
7. Mutation Genomics.
PROTEOMICS : (PROTEin in complement to genOME)
Proteomics is the study of proteome [Proteome is a protein molecule that interacts to give the cell its individual character]. Proteomics is a subset of functional genomics.
The proteome of a cell is all the proteins expressed by its genome. The proteome is of intense interest to investigators because proteins are the major functional components of the cell.
Proteomics is the study of proteins in order to revolutionize the understanding of cell behaviour and disease.
1. It studies the translation of process of RNA into proteins as well as the overall process of DNA into proteins.
2. It studies the diseases through proteins because disease process manifest themselves at the level of protein activity.
3. Most drugs act by targeting proteins or protein receptors, so Proteomics is important in new generation of drugs.
4. Proteins are more complex than genes because they can be modified after formation.
5. Proteomics is the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes.
6. Proteomics can use analysis techniques to determine all of the post translational modifications that proteins undergo and therefore determine what makes a diseased or mutant protein different from a normal protein.
Proteins are fundamental components of all living cells. Proteins help us digest our food, fight infections, control body chemistry, keep our bodies function smoothly. Identifying a proteins’ shape or structure is key to understanding its biological function and its role in health and disease.
A protein needs to adopt a final and stable 3-dimensional shape in order to function properly.
• The Tertiary Structure of a protein is the arrangement of the secondary structures into this
final 3-dimensional shape.
Recognizable folding pattern of proteins involved two or more elements of its secondary structure.
In a motif two elements of secondary structure folded against each other.
Because it is falling between secondary and tertiary structure and describe small part of protein or entire polypeptide chain.
A brief introfuction of label-free protein quantification methodsCreative Proteomics
If you want to know more about our services, please visit https://www.creative-proteomics.com/services/label-free-quantification.htm.
Label-free protein quantification is a mass spectrometry-based method for identifying and quantifying relative changes in two or more biological samples instead of using a stable isotope-containing compound to label proteins.
Protein docking is used to check the structure, position and orientation of a protein when it interacts with small molecules like ligands. Protein receptor-ligand motifs fit together tightly, and are often referred to as a lock and key mechanism. There are both high specificity and induced fit within these interfaces with specificity increasing with rigidity. The foremost thing that we need to start with a docking search is the sequence of our protein of interest. (Halperin et al., 2002).
Protein-protein interactions occur between two proteins that are similar in size. The interface between the two molecules tends to be flatter and smoother than those in interfaces of these interactions do not have the ability to alter protein-ligand interactions. Protein-protein interactions are usually more rigid, the conformation in order to improve binding and ease movement. (Smith and Sternberg, 2002).
The process of drug development has revolved around a screening approach, as nobody knows which compound or approach could serve as a drug or therapy. Such almost blind screening approach is very time-consuming and laborious. The goal of structure-based drug design is to find chemical structures fitting in the binding pocket of the receptor. Based on the three-dimensional structure of the target protein, it can automatically build ligand molecules within the binding pocket and subsequently screen them (Weil et al., 2004).
A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT, an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides. (O’Reilly et al., 2006).
A physical process by which a polypeptide chain (sequence of amino acids) folds into its characteristic & functional native structure from a random coil or a linear sequence.
Quantum Mechanics in Molecular modelingAkshay Kank
This slides gives you the information related to computer aided drug design and its application in drug discovery. Also you learn the Quantum mechanics related to the molecular mechanics. Theory related to molecular modeling and how the molecular modeling helps in drug discovery.
A Remarkable Conflict Between Biochemical Dogma And Radical Concept :
Traditional Concept :
By the 1950s it had been clearly established that oxidative phosphorylation involved the stepwise transfer of electrons through a series of carriers to molecular oxygen. But how the energy derived from these electron transfer reactions was converted to ATP remained a mystery. The general assumption was that ADP was converted to ATP by direct transfer of high-energy phosphate groups from some other intermediate. Thus it was postulated that high-energy intermediates were produced as a result of electron transfer reactions and that these intermediates drove ATP synthesis by phosphate group transfer.
Concept Proposed By Peter Mitchell :
The fundamental proposal of the chemiosmotic hypothesis was that the “intermediate” that coupled electron transport to ATP synthesis was a proton electrochemical gradient across the membrane. Mitchell postulated that such a gradient was produced by electron transport and that the flow of protons back across the membrane in the energetically favorable direction was then coupled to ATP synthesis
Polymerase chain reaction (PCR) is a technique in molecular biology used to
amplify (multiply) a single copy or a few copies of a piece of DNA, generating
thousands to millions of copies of that particular DNA sequence.
Lecture ON Polymerase Chain Reaction.
The polymerase chain reaction (PCR) is a powerful core molecular biology technique - Sometimes called "molecular photocopying. • Developed by Kary Mullis in 1985.
• It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from nucleic acids of various sources. •
It generates microgram (µg) quantities of DNA copies (up to billion copies) of the desired DNA (or RNA) segment.
A simple PCR reaction consists of
i. A DNA preparation containing the desired segment to be amplified.
ii. A set of synthetic oligonucleotide primers that flank the target DNA
sequence, of about 20 bases long, specific, i.e., complementary.
iii. A thermostable DNA polymerase e.g., Taq isolated from the
bacterium Thermus acquaticus, Pfu – Pyrococcus furiosus and Vent
from Thermococcus litoralis. Pfu and Vent are more efficient than
Taq polymerase.
iv. Four deoxynucleoside triphosphate (dNTPs): TTP – thymidine
triphosphate, dCTP – deoxycyctidine triphosphate, dATP –
deoxyadenosine triphosphate and dGTP – deoxyguanosine
triphosphate
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.
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
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Chapter 3 - Islamic Banking Products and Services.pptx
APPLICATION OF PCR(SEMINAR).ppt
1. A SEMINAR
ON
APPLICATION OF PCR IN PLANT GENOME ANALYSIS
Submitted by- Miss. Trupti A. Raut
Roll No.: 15 (M. pharmacy First Year)
[Pharmacognosy & Phytochemistry]
Guided by : Archana Mohod Mam
1
GOVERNMENT COLLEGE OF PHARMACY, AMRAVATI
2. CONTENTS OF SLIDES
Sr. no. Contents Slide no.
1 Introduction 3
2 Short history of PCR 5
3 DNA Replication Vs PCR 6
4 Key Enzymes Involved in DNA Replication 8
5 Three Main Steps of PCR 16
6 Primers 24
7 Application of PCR – Genetic Diseases 25
8 Application of PCR in Crop Improvement 29
9 Application of PCR in short 30
10 Reference 31
2
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
3. INTRODUCTION
• 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.
3
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
4. POLYMERASE CHAIN REACTIONS (PCR)
• PCR is a means to amplify a particular piece of DNA.
• Amplify = making numerous copies of a segment of DNA.
• PCR can make billions of copies of a target sequence of DNA in a
few hours.
• PCR was invented by Kary Mullis (Cetus corporation, USA) in the
1983-1984 as a way to make numerous copies of DNA fragments in
the laboratory.
• Kary Mullis awarded Nobel Prize in chemistry in 1993.
• 1985, Saiki publishes the first application of PCR (beta – Globin)
• Its application are vast and PCR is now an integral part of Molecular
Biology/ Biotechnology
• 1976, Thomas Brock discovers Thermus aquaticus, a thermostable
bacteria in the hot springs of Yellowstone National Park.
• 1985, Cetus Corp. Scientists isolate Thermostable Taq Polymerase
(from T. aquaticus), which revolutionized PCR.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY) 4
5. SHORT HISTORY OF PCR
• 1983 : Dr. Kary Mullis developed PCR.
• 1985 : First publication of PCR by Cetus Corporation appears
in science.
• 1976 : Purified Taq polymerase is first used in PCR.
• 1988 : PerkinElmer introduces the automated thermal cycler.
• 1989 : Science declares Taq polymerase “molecule of the
year.”
5
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
6. DNA REPLICATION Vs PCR
• PCR is a laboratory version of DNA Replication in cells – In
vitro amplification of DNA.
• The laboratory version is commonly called “in vitro” since it
occurs in a test tube while “in vivo” signifies occurring in a
living cell.
6
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
7. DNA REPLICATION IN CELLS (in vivo)
• DNA replication is the copying of DNA.
• It typically takes a cell just a few hours to copy all of its DNA.
• DNA replication is semi- conservative (i.e. one strand of the DNA is
used as the template for the growth of a new DNA strand)
• This process occurs with very few errors ( on average there is one
error per 1 billion nucleotides copied)
• More than a dozen enzymes and proteins participate in DNA
replication.
7
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
8. KEY ENZYMES INVOLVED IN DNA
REPLICATION
• DNA Polymerase
• Helicase
• Primase
• Topoisomerase
• Single strand binding protein
• DNA Ligase
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
8
9. DNA REPLICATION ENZYMES: DNA POLYMERASE
Catalyzes the elongation of DNA by adding nucleoside
triphosphate to the 3’ end of the growing strand.
A nucleotide triphosphate is a 1sugar + 1 base + 3 phosphates
When a nucleoside triphosphate joins the DNA strand , two
phosphates are removed.
DNA polymerase can only add nucleotides to 3’ end of
growing strand.
9
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
10. COMPLEMENTARY BASE – PAIRING IN DNA
• DNA is a double helix, made up of nucleotides, with a sugar –
phosphate backbone on the outside of the helix.
Note : a nucleotide is a sugar + phosphate + nitrogenous base.
• The two strands of DNA are held together by pairs of nitrogenous
bases that are attached to each other via hydrogen bonds.
• The nitrogenous base adenine will only pair with thymine.
• During replication, once the DNA strands are separated, DNA
polymerase uses each strand as a template to synthesize new
strands of DNA with the precise, complementary order of
nucleotides.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
10
11. DNA REPLICATION ENZYMES: DNA LIGASE
• The two strands of DNA in a double helix are anti-parallel (i.e.
they are oriented in opposite directions with one strand oriented
from 5’ to 3’ and the other strand oriented from 3’to 5’
• Note : 5’ to 3’ refer to the numbers assigned to the carbons in the 5
carbon sugar.
• Given the anti-parallel nature of DNA and the fact that DNA
polymerases can only add nucleotides to the 3’ end, one strand
(referred to as the leading strand) of DNA is synthesized
continuously and the other strand (referred to as the lagging strand)
in synthesized in fragments (called Okazaki fragments).
• Okazaki fragments are joined together by DNA ligase.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
11
12. DNA REPLICATION ENZYMES: PRIMASE
DNA Polymerase cannot initiate the synthesis of DNA.
Remember that DNA polymerase can only add nucleotides to
3’ end of an already existing strand of DNA.
In humans, primase is the enzyme that can start an RNA chain
from scratch and it creates a primer ( a short stretch RNA with
an available 3’ end) that DNA polymerase can add nucleotides
to during replication.
Note that the RNA primer is subsequently replaced with DNA.
12
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
13. DNA REPLICATION ENZYMES
(Helicase, Topoisimerase and Single - strand binding
protein)
• Helicase untwists the two parallel DNA strands.
• Topoisomerase relieves the stress of this twisting.
• Single- strand binding protein binds to and stabilizes the
unpaired DNA strands.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
13
14. PCR : The in vitro version of DNA Replication
• The following components are needed to perform PCR in the
laboratory:
1) DNA (your DNA of interest that contains the target sequence you
wish to copy)
2) A heat-stable DNA polymerase (like Taq Polymerase)
3) All four nucleotide triphosphates
4) Buffers
5) Two short, single stranded DNA molecules that serve as primers
6) Thin walled tubes
7) Thermal cycler (a device that can change temperatures dramatically
in a very short period of time).
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
14
15. PCR
• The DNA , DNA polymerase,
buffer, nucleoside triphosphates,
and primers placed in a thin –
walled tube and then these tubes
are placed in the PCR thermal
cycler.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
15
16. THE THREE MAIN STEPS OF PCR
• The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.
• In a PCR reaction, the following series of steps is repeated
20-40 times.
Note – 30 cycles usually takes about 2-3 hours and amplifies the DNA
fragment of interest 1,000,000,000 fold.
Step 1: Denature DNA
At 950C, the DNA is denatured (i.e. the two strands are separated)
Step 2 : Annealing (of Primers)
At 400C-650C, the primers anneal (or bind to) their complementary
sequences on the single strands of DNA.
Step 3: Extension ( of the DNA chain by DNA polymerase)
At 720C, DNA Polymerase extends the DNA chain by adding
nucleotides to the 3’ ends of the primers.
16
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
17. 17
HEAT STABLE DNA POLYMERASE
• Given that PCR involves very high temperatures it is
imperative that a heat – stable DNA polymerase be used in
the reaction.
• Most DNA polymerases would denature ( and thus not
function properly ) at the high temperatures of PCR.
• Taq DNA polymerase was purified from the hot springs
bacterium Thermus aquaticus in 1976.
• Taq has maximal enzymatic activity at 750C to 800C , and
substantially reduced activities at lower temperatures.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
18. Step 1 : Denaturation of DNA
This occurs at 950C mimicking the function of helicase in the
cell.
18
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
19. Step 2: Annealing or Primers Binding
Reverse Primer
Forward Primer
Primers bind to the complimentary sequence on the target
DNA.
Primers are chosen such that one is complimentary to the one
strand at one end of the target sequence and that the other is
complimentary to the other strand at the other end of the target
sequence.
19
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
20. Step 3: Extension or Primer Extension
20
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
extension
extension
DNA polymerase catalyzes the extension of the strand in the 5-
3 direction , starting at the primers, attaching the appropriate
nucleotide (A-T, C-G)
21. GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
21
• The next cycle will begin by denaturing the new DNA
strands formed in the previous cycle.
22. GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
22
The size of the DNA Fragment Produced in PCR is Dependent
on the Primers.
• The PCR reaction will amplify the DNA section between the
two primers.
• If the DNA sequence is known , primers can be developed to
amplify any piece of an organism’s DNA.
23. PCR has become a very powerful tool in molecular
biology
23
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
• One can amplify fragments of interest in an organism’s DNA
by choosing the right primers.
• One can use the selectivity of the primers to identify the
likelihood of an individual carrying a particular allele of a
gene.
• One can start with a single sperm cell or strand of hair and
amplify the DNA sufficiently to allow for DNA analysis and a
distinctive band on an agarose gel.
24. More about Primers
• PCR primers are short , single stranded DNA molecules (15-40 bp)
• They are manufactured commercially and can be ordered to match
any DNA sequence.
• Primers are sequence specific, they will bind to a particular
sequence in a genome.
• As you design primers with a loner length (16-40 bp), the primers
become more selective.
• DNA polymerase requires primers to initiate replication.
• Primers bind to their complementary sequence on the target DNA.
• A primer composed of only 3 letter, ACC, for example, would be
very likely to encounter its compliment in a genome.
• As the size of the primer is increased , the likelihood of, for
example, a primer sequence of 35 base letters repeatedly
encountering a perfect complementary section on the target DNA
become remote.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY) 24
25. APPLICATION OF PCR: GENETIC DISEASE
• Primers can be created that will only bind and amplify certain
alleles of genes or mutations of genes.
• This is the basis of genetic counselling and PCR is used as part
of the diagnostic tests for genetic diseases.
• Some diseases that can be diagnosed with the help of PCR :
Huntington’s disease
Cystic fibrosis
Human immunodeficiency virus.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
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26. 26
Huntington’s Disease (HD)
HD is a genetic disorder characterized by abnormal body
movements and reduced mental abilities.
HD is caused by a mutation in the Huntingtin (HD) gene
In non-HD individuals, the HD gene is “expanded”
In non-HD individuals, the HD gene has a pattern called
trinucleotide repeats with “CAG” occurring in repetition less
than 30 times.
In HD individuals, the “CAG” trinucleotide repeat occurs
more than 36 times in the HD gene.
The DNA is amplified via PCR and sequenced ( a technique
by which the exact nucleotide sequence is determined) and
the number of trinucleotide repeats is then counted.
GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
27. Cystic Fibrosis (CF)
CF is a genetic disease characterized by severe breathing
difficulties and a predisposition to infections.
CF is caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CTFR) gene.
In non-CF individuals, the CTFR gene codes for a protein that
is a chloride ion channel and is involved in the production of
sweat, digestive juices and mucus.
In CF individuals, mutations in the CTFR gene leads to thick
mucous secretions in the lungs and subsequent persistent
bacterial infections.
The presence of CTFR mutations in a individual can be
detected by performing PCR and sequencing on that
individual’s DNA.
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GCOP, AMRAVATI (PHARMACOGNOSY AND PHYTOCHEMISTRY)
29. Application of PCR technology in crop improvement
1. To increase the number of very few number of DNA template.
2. Isolation of a orthologous gene sequence by using degenerate
primers designed from closely related plant species gene sequence
alignments.
3. Amplification of gene from cDNA
4. Synthesis of complementary DNA (cDNA) from RNA isolated
from the crop using modified process of PCR called Reverse-
transcriptase PCR.
5. Identification of genetically modified crops for the presence of
transgene using PCR.
6. Screening and differentiation of diseased with viral pathogen and
healthy plants using viral gene specific primers.
7. PCR is used for DNA sequencing to determine unknown PCR-
amplified sequences, which helps in gene discovery.
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30. APPLICATION OF PCR IN SHORT
1. Direct sequencing of amplified DNA
2. Engineering of new DNA sequences
3. Site – directed mutagenesis
4. Detection of gene expression
5. Amplification of specific sequences from cDNA and genome
libraries.
6. Identification of transgenics
7. Plant genetic transformation and detection of transgenics at the
tissue and whole plant levels.
8. Determination of changes in a particular gene sequence resulting
from tissue culture (e.g. somaclonal variation)
9. A modification of PCR technology known as “inverse PCR” has
been used to determine the T DNA copy number in transgenic
plants generated by Agrobacterium – mediated transformation.
10. While the expression of foreign DNA in transformation
experiments can be determined by RTPCR.
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![A SEMINAR
ON
APPLICATION OF PCR IN PLANT GENOME ANALYSIS
Submitted by- Miss. Trupti A. Raut
Roll No.: 15 (M. pharmacy First Year)
[Pharmacognosy & Phytochemistry]
Guided by : Archana Mohod Mam
1
GOVERNMENT COLLEGE OF PHARMACY, AMRAVATI](https://image.slidesharecdn.com/applicationofpcrseminar-221013114356-02a4daf6/85/APPLICATION-OF-PCR-SEMINAR-ppt-1-320.jpg)
