The polymerase chain reaction (PCR) is a technique used to amplify a single copy of a DNA segment across several orders of magnitude. It was developed by Kary Mullis in 1983 and involves thermal cycling to separate DNA strands and allow a DNA polymerase to replicate the strands. This results in exponential amplification of the target DNA segment. PCR is now widely used in medical research and forensic sciences to amplify specific DNA regions.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
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
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
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
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
PCR,polymerase chain reaction.Basic concept of PCR.naveed ul mushtaq
PCR.Basic concept of PCR. Steps in PCR.
Quantitative real time polymerase chain reaction.Fluorescent dyes and probes.
Advantages real-time PCR.
Real-time PCR primer
Primer design software
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
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
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
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
PCR,polymerase chain reaction.Basic concept of PCR.naveed ul mushtaq
PCR.Basic concept of PCR. Steps in PCR.
Quantitative real time polymerase chain reaction.Fluorescent dyes and probes.
Advantages real-time PCR.
Real-time PCR primer
Primer design software
Helping Farmers to Grow Healthier Crops: Use of qPCR to Monitor Plant Resista...Jonathan Clarke
1. Importance of crop diseases
2. Disease control measures
3. Use of qPCR in selected pathosystems:
• Downy mildew of opium poppy
• Verticillium wilt of olives
Lars-Gunnar Lindfors från IVL Svenska Miljöinstitutet talade om hur vi rättvist kan bedöma byggmaterial med LCA-metodiken på Betongföreningens årsmöte den 23 april 2013 i Stockholm. Detta årsmöte hade temat hållbarhet.
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 polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of different types of PCR methods, applications and optimization.
PCR is a biochemical technology in molecular biology. This technique used in DNA cloning for sequencing, gene cloning and manipulating, diagnosis of hereditary diseases.
Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
Gene cloning and polymerase chain reaction Abhay jha
In these you are able to know about the gene cloning basic steps and Polymerase chain reaction process also there is an brief description about the ideal property shown by vectors which are lambda and M13 phases and there are lots of things in these slides
Diagnostic polymerase chain reaction (PCR) is an extremely powerful, rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples.
However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids .
In general, diagnostic PCR may be divided into four steps: (1) sampling, (2) sample preparation, (3) nucleic acid amplification, and (4) detection of PCR products
Diagnostic polymerase chain reaction (PCR) is an extremely powerful, rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples.
However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids .
In general, diagnostic PCR may be divided into four steps: (1) sampling, (2) sample preparation, (3) nucleic acid amplification, and (4) detection of PCR products
Encryption in Microsoft 365 - ExpertsLive Netherlands 2024Albert Hoitingh
In this session I delve into the encryption technology used in Microsoft 365 and Microsoft Purview. Including the concepts of Customer Key and Double Key Encryption.
Elevating Tactical DDD Patterns Through Object CalisthenicsDorra BARTAGUIZ
After immersing yourself in the blue book and its red counterpart, attending DDD-focused conferences, and applying tactical patterns, you're left with a crucial question: How do I ensure my design is effective? Tactical patterns within Domain-Driven Design (DDD) serve as guiding principles for creating clear and manageable domain models. However, achieving success with these patterns requires additional guidance. Interestingly, we've observed that a set of constraints initially designed for training purposes remarkably aligns with effective pattern implementation, offering a more ‘mechanical’ approach. Let's explore together how Object Calisthenics can elevate the design of your tactical DDD patterns, offering concrete help for those venturing into DDD for the first time!
zkStudyClub - Reef: Fast Succinct Non-Interactive Zero-Knowledge Regex ProofsAlex Pruden
This paper presents Reef, a system for generating publicly verifiable succinct non-interactive zero-knowledge proofs that a committed document matches or does not match a regular expression. We describe applications such as proving the strength of passwords, the provenance of email despite redactions, the validity of oblivious DNS queries, and the existence of mutations in DNA. Reef supports the Perl Compatible Regular Expression syntax, including wildcards, alternation, ranges, capture groups, Kleene star, negations, and lookarounds. Reef introduces a new type of automata, Skipping Alternating Finite Automata (SAFA), that skips irrelevant parts of a document when producing proofs without undermining soundness, and instantiates SAFA with a lookup argument. Our experimental evaluation confirms that Reef can generate proofs for documents with 32M characters; the proofs are small and cheap to verify (under a second).
Paper: https://eprint.iacr.org/2023/1886
Why You Should Replace Windows 11 with Nitrux Linux 3.5.0 for enhanced perfor...SOFTTECHHUB
The choice of an operating system plays a pivotal role in shaping our computing experience. For decades, Microsoft's Windows has dominated the market, offering a familiar and widely adopted platform for personal and professional use. However, as technological advancements continue to push the boundaries of innovation, alternative operating systems have emerged, challenging the status quo and offering users a fresh perspective on computing.
One such alternative that has garnered significant attention and acclaim is Nitrux Linux 3.5.0, a sleek, powerful, and user-friendly Linux distribution that promises to redefine the way we interact with our devices. With its focus on performance, security, and customization, Nitrux Linux presents a compelling case for those seeking to break free from the constraints of proprietary software and embrace the freedom and flexibility of open-source computing.
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
Pushing the limits of ePRTC: 100ns holdover for 100 daysAdtran
At WSTS 2024, Alon Stern explored the topic of parametric holdover and explained how recent research findings can be implemented in real-world PNT networks to achieve 100 nanoseconds of accuracy for up to 100 days.
Removing Uninteresting Bytes in Software FuzzingAftab Hussain
Imagine a world where software fuzzing, the process of mutating bytes in test seeds to uncover hidden and erroneous program behaviors, becomes faster and more effective. A lot depends on the initial seeds, which can significantly dictate the trajectory of a fuzzing campaign, particularly in terms of how long it takes to uncover interesting behaviour in your code. We introduce DIAR, a technique designed to speedup fuzzing campaigns by pinpointing and eliminating those uninteresting bytes in the seeds. Picture this: instead of wasting valuable resources on meaningless mutations in large, bloated seeds, DIAR removes the unnecessary bytes, streamlining the entire process.
In this work, we equipped AFL, a popular fuzzer, with DIAR and examined two critical Linux libraries -- Libxml's xmllint, a tool for parsing xml documents, and Binutil's readelf, an essential debugging and security analysis command-line tool used to display detailed information about ELF (Executable and Linkable Format). Our preliminary results show that AFL+DIAR does not only discover new paths more quickly but also achieves higher coverage overall. This work thus showcases how starting with lean and optimized seeds can lead to faster, more comprehensive fuzzing campaigns -- and DIAR helps you find such seeds.
- These are slides of the talk given at IEEE International Conference on Software Testing Verification and Validation Workshop, ICSTW 2022.
Climate Impact of Software Testing at Nordic Testing DaysKari Kakkonen
My slides at Nordic Testing Days 6.6.2024
Climate impact / sustainability of software testing discussed on the talk. ICT and testing must carry their part of global responsibility to help with the climat warming. We can minimize the carbon footprint but we can also have a carbon handprint, a positive impact on the climate. Quality characteristics can be added with sustainability, and then measured continuously. Test environments can be used less, and in smaller scale and on demand. Test techniques can be used in optimizing or minimizing number of tests. Test automation can be used to speed up testing.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Generative AI Deep Dive: Advancing from Proof of Concept to ProductionAggregage
Join Maher Hanafi, VP of Engineering at Betterworks, in this new session where he'll share a practical framework to transform Gen AI prototypes into impactful products! He'll delve into the complexities of data collection and management, model selection and optimization, and ensuring security, scalability, and responsible use.
PHP Frameworks: I want to break free (IPC Berlin 2024)Ralf Eggert
In this presentation, we examine the challenges and limitations of relying too heavily on PHP frameworks in web development. We discuss the history of PHP and its frameworks to understand how this dependence has evolved. The focus will be on providing concrete tips and strategies to reduce reliance on these frameworks, based on real-world examples and practical considerations. The goal is to equip developers with the skills and knowledge to create more flexible and future-proof web applications. We'll explore the importance of maintaining autonomy in a rapidly changing tech landscape and how to make informed decisions in PHP development.
This talk is aimed at encouraging a more independent approach to using PHP frameworks, moving towards a more flexible and future-proof approach to PHP development.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
2. INTRODUCTION
The polymerase chain reaction (PCR) is a biochemical technology
in molecular biology to amplify a single or a few copies of a piece
of DNA across several orders of magnitude, generating thousands to millions
of copies of a particular DNA sequence.
It was developed by Kary Mullis in 1983. PCR is now a common and often
indispensable technique used in medical and biological research labs for a
variety of applications. These include DNA cloning for sequencing, DNA-
based phylogeny, or functional analysis of genes; the diagnosis of hereditary
diseases; the identification of genetic fingerprints (used in forensic
sciences and paternity testing); and the detection and diagnosis of infectious
diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along
with Michael Smith for his work on PCR.
3. INVENTOR OF PCR Kary Mullis (1983)
Nobel Prize Winner in Chemistry(1993)
4. An Introduction..
The method relies on thermal cycling, consisting of cycles of repeated heating
and cooling of the reaction for DNA melting and enzymatic replication of the
DNA. Primers (short DNA fragments) containing sequences complementary to
the target region along with a DNA polymerase (after which the method is
named) are key components to enable selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is
exponentially amplified. PCR can be extensively modified to perform a wide
array of genetic manipulations.
5. Introduction contd..
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq
polymerase, an enzyme originally isolated from the bacterium Thermus
aquaticus. This DNA polymerase enzymatically assembles a new DNA strand
from DNA building-blocks, the nucleotides, by using single-stranded DNA as a
template and DNA oligonucleotides (also called DNA primers), which are
required for initiation of DNA synthesis. The vast majority of PCR methods
use thermal cycling, i.e., alternately heating and cooling the PCR sample to a
defined series of temperature steps. These thermal cycling steps are
necessary first to physically separate the two strands in a DNA double helix at
a high temperature in a process called DNA melting. At a lower temperature,
each strand is then used as the template in DNA synthesis by the DNA
polymerase to selectively amplify the target DNA. The selectivity of PCR
results from the use of primers that are complementary to the DNA region
targeted for amplification under specific thermal cycling conditions
6. PCR Principles and Procedure
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most
PCR methods typically amplify DNA fragments of between 0.1 and 10kilo base
pairs (kb), although some techniques allow for amplification of fragments up
to 40 kb in size. The amount of amplified product is determined by the
available substrates in the reaction, which become limiting as the reaction
progresses.
A basic PCR set up requires several components and reagents. These
components include:
DNA template that contains the DNA region (target) to be amplified.
Two primers that are complementary to the 3' (three prime) ends of each of
the sense and anti-sense strand of the DNA target.
7. PCR Principles and Procedure
Taq polymerase or another DNA polymerase with a temperature optimum at
around 70 °C.
Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate
groups), the building-blocks from which the DNA polymerase synthesizes a
new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum
activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but
Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher
Mn2+ concentration increases the error rate during DNA synthesis[8]
Monovalent cation potassium ions.
8. DNA POLYMERASE
Necessary for reproduction of new cells
Duplicates DNA
More than one DNA polymerases exist in different organisms
9. Properties of DNA polymearse
DNA polymerase needs Mg++ as cofactor
Each DNA polymerase works best under optimal temperature, pH and salt concentration
PCR buffer provides optimal pH and salt condition
DNA strands are anti-parallel
One strand goes in 5’ 3’
The complementary strand is opposite
DNA polymerase always moves in one direction (from 5’ 3’)
DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain
dNTP follow standard base pairing rule
The newly generated DNA strands serve as template DNA for the next cycle
PCR is very sensitive
Widely used
10. Taq DNA polymerase
Derived from Thermus aquaticus
Heat stable DNA polymerase
Ideal temperature 72C
11. PCR principles and procedure contd..
The PCR is commonly carried out in a reaction volume of 10–200 μl in small
reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler
heats and cools the reaction tubes to achieve the temperatures required at
each step of the reaction (see below). Many modern thermal cyclers make use
of the Peltier effect, which permits both heating and cooling of the block
holding the PCR tubes simply by reversing the electric current. Thin-walled
reaction tubes permit favorable thermal conductivity to allow for rapid
thermal equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube. Older thermocyclers lacking a
heated lid require a layer of oil on top of the reaction mixture or a ball of
wax inside the tube.
12. Procedure For PCR
Typically, PCR consists of a series of 20-40 repeated temperature changes,
called cycles, with each cycle commonly consisting of 2-3 discrete
temperature steps, usually three . The cycling is often preceded by a single
temperature step (called hold) at a high temperature (>90°C), and followed
by one hold at the end for final product extension or brief storage. The
temperatures used and the length of time they are applied in each cycle
depend on a variety of parameters. These include the enzyme used for DNA
synthesis, the concentration of divalent ions and dNTPs in the reaction, and
the melting temperature (Tm) of the primers.
Initialization step: This step consists of heating the reaction to a temperature
of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which
is held for 1–9 minutes. It is only required for DNA polymerases that require
heat activation by hot-start PCR.
13. Procedure For PCR cont.…
Denaturation step: This step is the first regular cycling event and consists of
heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of
the DNA template by disrupting the hydrogen bonds between complementary
bases, yielding single-stranded DNA molecules.
Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40
seconds allowing annealing of the primers to the single-stranded DNA
template. Typically the annealing temperature is about 3-5 degrees Celsius
below the Tm 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.
14. Procedure cont..
Extension/elongation step: The temperature at this step depends on the DNA
polymerase used; Taq polymerase has its optimum activity temperature at 75–
80 °C, and commonly a temperature of 72 °C is used with this enzyme. 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, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand.
The extension time depends both on the DNA polymerase used and on the
length of the DNA fragment to be amplified. As a rule-of-thumb, at its
optimum temperature, the DNA polymerase will polymerize a thousand bases
per minute. Under optimum conditions, i.e., if there are no limitations due to
limiting substrates or reagents, at each extension step, the amount of DNA
target is doubled, leading to exponential (geometric) amplification of the
specific DNA fragment.
15. Procedure For PCR cont.…
Final elongation: This single step is occasionally performed at a temperature
of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any
remaining single-stranded DNA is fully extended.
Final hold: This step at 4–15 °C for an indefinite time may be employed for
short-term storage of the reaction.
16. PCR Stages..
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled
(assuming 100% reaction efficiency). The reaction is very sensitive: only minute
quantities of DNA need to be present.[13]
Levelling off stage: The reaction slows as the DNA polymerase loses activity and
as consumption of reagents such as dNTPs and primers causes them to become
limiting.
Plateau: No more product accumulates due to exhaustion of reagents and
enzyme.
17. PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to
contamination causing amplification of spurious DNA products. Because of
this, a number of techniques and procedures have been developed for
optimizing PCR conditions. Contamination with extraneous DNA is addressed
with lab protocols and procedures that separate pre-PCR mixtures from
potential DNA contaminants. This usually involves spatial separation of PCR-
setup areas from areas for analysis or purification of PCR products, use of
disposable plasticware, and thoroughly cleaning the work surface between
reaction setups. Primer-design techniques are important in improving PCR
product yield and in avoiding the formation of spurious products, and the
usage of alternate buffer components or polymerase enzymes can help with
amplification of long or otherwise problematic regions of DNA. Addition of
reagents, such as formamide, in buffer systems may increase the specificity
and yield of PCR. Computer simulations of theoretical PCR results (Electronic
PCR) may be performed to assist in primer design.
18. Application of PCR
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective
amplification of a specific region of DNA. This use of PCR augments many
methods, such as generating hybridization
probes for Southern or northern hybridization and DNA cloning, which require
larger amounts of DNA, representing a specific DNA region. PCR supplies these
techniques with high amounts of pure DNA, enabling analysis of DNA samples
even from very small amounts of starting material.
19. Application of PCR cont….
Other applications of PCR include DNA sequencing to determine unknown PCR-
amplified sequences in which one of the amplification primers may be used in
Sanger sequencing, isolation of a DNA sequence to expedite recombinant
DNA technologies involving the insertion of a DNA sequence into a plasmid or
the genetic material of another organism. Bacterial colonies (E. coli) can be
rapidly screened by PCR for correct DNA vector constructs.[18] PCR may also be
used for genetic fingerprinting; a forensic technique used to identify a person
or organism by comparing experimental DNAs through different PCR-based
methods.
Some PCR 'fingerprints' methods have high discriminative power and can be
used to identify genetic relationships between individuals, such as parent-
child or between siblings, and are used in paternity testing (Fig. 4). This
technique may also be used to determine evolutionary relationships among
organisms.
20. Application of PCR cont..
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze
extremely small amounts of sample. This is often critical for forensic analysis, when
only a trace amount of DNA is available as evidence. PCR may also be used in the
analysis of ancient DNA that is tens of thousands of years old. These PCR-based
techniques have been successfully used on animals, such as a forty-thousand-year-
old mammoth, and also on human DNA, in applications ranging from the analysis of
Egyptian mummies to the identification of a Russian tsar and the body of British
king Richard III.
Quantitative PCR methods allow the estimation of the amount of a given sequence
present in a sample—a technique often applied to quantitatively determine levels
of gene expression. Real-time PCR is an established tool for DNA quantification that
measures the accumulation of DNA product after each round of PCR amplification.
21. Application of PCR cont..
PCR in diagnosis of diseases
PCR permits early diagnosis of malignant diseases such
as leukaemia and lymphomas, which is currently the highest-developed in
cancer research and is already being used routinely. PCR assays can be
performed directly on genomic DNA samples to detect translocation-specific
malignant cells at a sensitivity that is at least 10,000 fold higher than that of
other methods.
22. Application of PCR cont..
PCR also permits identification of non-cultivatable or slow-growing
microorganisms such as mycobacteria, anaerobic bacteria,
or viruses from tissue culture assays and animal models. The basis for PCR
diagnostic applications in microbiology is the detection of infectious agents
and the discrimination of non-pathogenic from pathogenic strains by virtue of
specific genes.
Viral DNA can likewise be detected by PCR. The primers used need to be
specific to the targeted sequences in the DNA of a virus, and the PCR can be
used for diagnostic analyses or DNA sequencing of the viral genome. The high
sensitivity of PCR permits virus detection soon after infection and even before
the onset of disease. Such early detection may give physicians a significant
lead time in treatment. The amount of virus ("viral load") in a patient can also
be quantified by PCR-based DNA quantitation techniques
23. Disadvantages Of PCR
Extremely liable to contamination, High degree of operator skill
required. Not easy to set up a quantitative assay.
A positive result may be difficult to interpret, especially with latent
viruses such as CMV, where any seropositive person will have virus
present in their blood irrespective whether they have disease or not.
These problems are being addressed by the arrival of commercial closed
systems such as the Roche Cobas Amplicor which requires minimum
handling. The use of synthetic internal competitive targets in these
commercial assays has facilitated the accurate quantification of results.
However, these assays are very expensive.
24. In Summary PCR Applications
Classification of
organisms
Genotyping
Molecular archaeology
Mutagenesis
Mutation detection
Sequencing
Cancer research
Detection of pathogens
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
25. Disadvantages Of PCR
Need equipment
Taq polymerase is expensive
Contamination
Internal control
False reactions
Cross-reactions
Capacity building needed
Unspecific amplification
26. Types Of PCR
Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.
Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses
primers external to the sequence of interest. The second PCR uses the product of the
first PCR in conjunction with one or more nested primers to amplify the sequence
within the region flanked by the initial set of primers.
Inverse PCR : Used to amplify DNA of unknown sequence that is adjacent to known
DNA sequence.
Quantitative PCR: Product amplification with respect to time, which is compared
with standard DNA.
Hot Start PCR : : Used to optimize the yield of the desired amplified product in PCR
and simultaneously to suppress nonspecific amplification.
27. QUANTITATION OF mRNA
northern blotting
ribonuclease protection assay
in situ hybridization
RT-PCR
most sensitive
can discriminate closely related mRNAs
technically simple
but difficult to get truly quantitative results using conventional PCR
Why real time PCR ?