PCR uses primers that are complementary to the DNA region of interest in order to selectively amplify that region through repeated heating and cooling cycles. There are many types of PCR including allele-specific PCR, nested PCR, quantitative PCR, and reverse transcription PCR. Primers are important factors in PCR and should have optimal melting temperatures and minimal secondary structure or self-complementarity to avoid non-specific binding.
Restriction Fragment Length Polymorphism (RFLP)
These are bacterial enzymes used by scientists to cut DNA molecules at known locations. RFLPs (pronounced "rif lips") are used as markers on genetic maps. Typically, gel electrophoresis is used to visualize RFLPs.
Restriction Fragment Length Polymorphism (RFLP)
These are bacterial enzymes used by scientists to cut DNA molecules at known locations. RFLPs (pronounced "rif lips") are used as markers on genetic maps. Typically, gel electrophoresis is used to visualize RFLPs.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
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.
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
INTRODUCTION TO REAL TIME PCR IS GIVEN, basic principle of realtime pcr, along with the process of operating this, diagrammatic representation of the process, advantages and disadvantages o f reatimem pcr, applications of the same is also there
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.
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
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
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
Food hygiene is more than cleanliness ......
Protecting food from risk of contamination, including harmful bacteria, poison and other foreign bodies.
Preventing any bacteria present multiplying to an extent which would result in the illness of consumers or the early spoilage of the food.
Destroying any harmful bacteria in the food by thorough cooking
or processing.
Discarding unfit or contaminated food.
T-Cell Activation
• Concept of immune response
• T cell-mediated immune response
• B cell-mediated immune response
I. Concept of immune response
• A collective and coordinated response to the introduction of foreign substances in an individual mediated by the cells and molecules in the immune system.
II. T cell-mediated immune response
• Cell-mediated immunity is the arm of the adaptive immune response whose role is to combat infection of intracellular pathogens, such as intracellular bacteria (mycobacteria, listeria monocytogens), viruses, protozoa, etc.
Major Histocompatibility Complex
MHC:
• Major Histocompatibility Complex
– Cluster of genes found in all mammals
– Its products play role in discriminating self/non-self
– Participant in both humoral and cell-mediated immunity
• MHC Act As Antigen Presenting Structures
• In Human MHC Is Found On Chromosome 6
– Referred to as HLA complex
• In Mice MHC Is Found On Chromosome 17
– Referred to as H-2 complex
• Genes Of MHC Organized In 3 Classes
– Class I MHC genes
• Glycoproteins expressed on all nucleated cells
• Major function to present processed Ags to TC
– Class II MHC genes
• Glycoproteins expressed on macrophages, B-cells, DCs
• Major function to present processed Ags to TH
– Class III MHC genes
• Products that include secreted proteins that have immune functions. Ex. Complement system, inflammatory molecules
Antigen Processing and Presentation MID
Antigens and “foreignness”
• Antigens (or, more properly, immunogens) have a series of features which confer immunogenicity.
• One of these features is “foreignness.”
• So, we can infer that – most often – antigens – ultimately – originate externally.
• (There are exceptions, of course. Some cells become transformed by disease [e. g., cancer] or by aging. In such instances, the antigens have an internal origin.)
Extinction of a particular animal or plant species occurs when there are no more individuals of that species alive anywhere in the world - the species has died out. This is a natural part of evolution. But sometimes extinctions happen at a much faster rate than usual. Natural Causes of Extinction.
Difference between In-Situ and Ex-Situ conservation
Conservation of biodiversity and genetic resources helps protect, maintain and recover endangered animal and plant species. There are mainly two strategies for the conservation of wildlife: In-situ conservation and Ex-situ conservation. Although, both the strategies aim to maintain and recover endangered species, they are different from each other. Let us see how they differ from each other!
Evolution Of Bacteria
Bacteria have existed from very early in the history of life on Earth. Bacteria fossils discovered in rocks date from at least the Devonian Period (419.2 million to 358.9 million years ago), and there are convincing arguments that bacteria have been present since early Precambrian time, about 3.5 billion years ago. Bacteria were widespread on Earth at least since the latter part of the Paleoproterozoic, roughly 1.8 billion years ago, when oxygen appeared in the atmosphere as a result of the action of the cyanobacteria. Bacteria have thus had plenty of time to adapt to their environments and to have given rise to numerous descendant forms.
Impact of Environment on Loss of Genetic Diversity and Speciation
Genetic variation describes naturally occurring genetic differences among individuals of the same species. This variation permits flexibility and survival of a population in the face of changing environmental circumstances. Consequently, genetic variation is often considered an advantage, as it is a form of preparation for the unexpected. But how does genetic variation increase or decrease? And what effect do fluctuations in genetic variation have on populations over time?
GENE ENVIRONMENT INTERACTION
Subtle differences in one person’s genes can cause them to respond differently to the same environmental exposure as another person. As a result, some people may develop a disease after being exposed to something in the environment while others may not.
As scientists learn more about the connection between genes and the environment, they pursue new approaches for preventing and treating disease that consider individual genetic codes.
How to store food in hot
The Good News
To maximize benefit of preservation, keep your food as fresh as possible for as long as possible. You can do this, even in the heat, by creating a “cooler” made from two basic terra cotta pots, one larger than the other. Put the smaller pot in the larger one, fill the gap with sand, and saturate the sand with water. Then cover it with a cloth. To add additional insulation from the heat, bury the pot up to its rim. The evaporation of moisture from the wet sand will cool the air around the food and help keep it fresh.
What is IUPAC naming?
In order to give compounds a name, certain rules must be followed. When naming organic compounds, the IUPAC (International Union of Pure and Applied Chemistry) nomenclature (naming scheme) is used. This is to give consistency to the names. It also enables every compound to have a unique name, which is not possible with the common names used (for example in industry). We will first look at some of the steps that need to be followed when naming a compound, and then try to apply these rules to some specific examples.
IUPAC Nomenclature
IUPAC nomenclature uses the longest continuous chain of carbon atoms to determine the basic root name of the compound. The root name is then modified due to the presence of different functional groups which replace hydrogen or carbon atoms in the parent structure.
Hybridization describes the bonding atoms from an atom's point of view. For a tetrahedral coordinated carbon (e.g. methane CH4), the carbon should have 4 orbitals with the correct symmetry to bond to the 4 hydrogen atoms.
INTRODUCTION:
Hybrid Orbitals
Developed by Linus Pauling, the concept of hybrid orbitals was a theory created to explain the structures of molecules in space. The theory consists of combining atomic orbitals (ex: s,p,d,f) into new hybrid orbitals (ex: sp, sp2, sp3).
1. Why Firefly give light during night?
2. Why atomic mass and Atomic numbers are given to elements ?
3. Why elements have been characterized and classified into different groups?
4. What is the transition of elements and what they play their role in elements stability?
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
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.
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
Search and Society: Reimagining Information Access for Radical FuturesBhaskar Mitra
The field of Information retrieval (IR) is currently undergoing a transformative shift, at least partly due to the emerging applications of generative AI to information access. In this talk, we will deliberate on the sociotechnical implications of generative AI for information access. We will argue that there is both a critical necessity and an exciting opportunity for the IR community to re-center our research agendas on societal needs while dismantling the artificial separation between the work on fairness, accountability, transparency, and ethics in IR and the rest of IR research. Instead of adopting a reactionary strategy of trying to mitigate potential social harms from emerging technologies, the community should aim to proactively set the research agenda for the kinds of systems we should build inspired by diverse explicitly stated sociotechnical imaginaries. The sociotechnical imaginaries that underpin the design and development of information access technologies needs to be explicitly articulated, and we need to develop theories of change in context of these diverse perspectives. Our guiding future imaginaries must be informed by other academic fields, such as democratic theory and critical theory, and should be co-developed with social science scholars, legal scholars, civil rights and social justice activists, and artists, among others.
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
FIDO Alliance Osaka Seminar: Passkeys at Amazon.pdf
PCR and Its Types
1. 1
Types of PCR
Primers
PCR primers are important as they are complementary to the beginning and end of the
DNA fragment of interest which one needs to amplify. Primers therefore select the
boundaries of the region to be amplified by PCR. During the PCR annealing cycle, PCR
primers anneal to the complementary region of the DNA. DNA polymerase binding and
the 3’ OH of the oligo allows the synthesis of DNA to occur.
PCR Primers are short single-stranded, synthetically synthesized oligonucleotides usually
shorter than 50 nucleotides (often 18-25 nucleotides)
Important Factors To Consider When Choosing Primers:
• Optimal Tm (annealing T) ~ 50-60°C
• Oligonucleotide GC-content should be between 40-60%.
• Calculated Tm (melting temperature) for both primers used in reaction should not
differ >5°C.
• Primer annealing temperature is usually 5°C below the calculated lower Tm.
However it should be chosen empirically for individual conditions.
• Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
• 3' terminus is extremely case sensitive - it must not be complementary to any
region of the other primer used in the reaction and must provide correct base
matching to template.
• If possible, primers should start and end with 1-2 G or C residues
• Primers should NOT have any extensive secondary structure or self-
complementarity (can result in Primer-dimers).
PCR types
1. Allele-specific PCR:
A diagnostic or cloning technique which is based on single-nucleotide polymorphisms
(SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA
sequence, including differences between alleles, and uses primers whose 3' ends
encompass the SNP. PCR amplification under stringent conditions is much less efficient
in the presence of a mismatch between template and primer, so successful amplification
with an SNP-specific primer signals presence of the specific SNP in a sequence.
2. Assembly PCR or Polymerase Cycling Assembly (PCA):
Artificial synthesis of long DNA sequences by performing PCR on a pool of long
oligonucleotides with short overlapping segments. The oligonucleotides alternate
between sense and antisense directions, and the overlapping segments determine the
order of the PCR fragments, thereby selectively producing the final long DNA product.
3. Asymmetric PCR:
Amjad Khan Afridi
2. 2
Preferentially amplifies one DNA strand in a double-stranded DNA template. It is used
in sequencing and hybridization probing where amplification of only one of the two
complementary strands is required. PCR is carried out as usual, but with a great excess of
the primer for the strand targeted for amplification. Because of the slow (arithmetic)
amplification later in the reaction after the limiting primer has been used up, extra cycles
of PCR are required. A recent modification on this process, known as Linear-After-The-
Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature
(Tm) than the excess primer to maintain reaction efficiency as the limiting primer
concentration decreases mid-reaction.
4. Colony PCR :
The screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid
products. Selected colonies of bacteria or yeast are picked with a sterile toothpick or
pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix
or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony
contains the DNA fragment or plasmid of interest.
5. The Digital polymerase chain reaction simultaneously amplifies thousands of
samples, each in a separate droplet within an emulsion.
6. Helicase-dependent amplification:
similar to traditional PCR, but uses a constant temperature rather than cycling through
denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds
DNA, is used in place of thermal denaturation.
7. Hot-start PCR:
A technique that reduces non-specific amplification during the initial set up stages of the
PCR. It may be performed manually by heating the reaction components to the melting
temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have
been developed that inhibit the polymerase's activity at ambient temperature, either by the
binding of an antibody or by the presence of covalently bound inhibitors that only
dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved
with new hybrid polymerases that are inactive at ambient temperature and are instantly
activated at elongation temperature.
8. In Situ PCR (ISH):
Is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ
PCR amplification can be performed on fixed tissue or cells.
Amjad Khan Afridi
3. 3
9. Intersequence-specific PCR (ISSR):
A PCR method for DNA fingerprinting that amplifies regions between simple sequence
repeats to produce a unique fingerprint of amplified fragment lengths.
10. Inverse PCR:
Is commonly used to identify the flanking sequences around genomic inserts. It involves
a series of DNA digestions and self ligation, resulting in known sequences at either end
of the unknown sequence. Inverse PCR has numerous applications in molecular biology
including the amplification and identification of sequences flanking transposable
elements, and the identification of genomic inserts.
The inverse PCR method includes a series of digestions and self-ligations with the DNA
being cut by a restriction endonuclease. This cut results in a known sequence at either end
of unknown sequences.
Inverse PCR Steps
1) Target DNA is lightly cut into smaller fragments of several kilobases by
restriction endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate
backbone to reform. This gives a circular DNA ligation product.
3) Target DNA is then restriction digested with a known endonuclease.
This generates a cut within the known internal sequence generating a
linear product with known terminal sequences. This can now be used for
PCR (polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now
known internal sequences.
11. Ligation-mediated PCR :
Uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments
of the target DNA. PCR primers that anneal to the linker sequences are then used to
amplify the target fragments. This method is deployed for DNA sequencing, genome
walking, and DNA footprinting A related technique is Amplified fragment length
polymorphism, which generates diagnostic fragments of a genome.
12. Methylation-specific PCR (MSP):
Is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in
genomic DNA.Target DNA is first treated with sodium bisulfite, which converts
unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR
primers. Two amplifications are then carried out on the bisulfite-treated DNA: One
primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and
the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP
Amjad Khan Afridi
4. 4
used in Q-PCR provides quantitative information about the methylation state of a given
CpG island.
13. Long PCR:
Is a PCR is which extended or longer than standard PCR, meaning over 5 kilobases
(frequently over 10 kb). Long PCR is usually only useful if it is accurate. Thus, special
mixtures of proficient polymerases along with accurate polymerases such as Pfu are often
mixed together. Applications of Long PCR Long PCR is often used to clone larger
genes or large segments of DNA which standard PCR cannot.
14. Miniprimer PCR:
Uses a thermostable polymerase (S-Tbr) that can extend from short primers
("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to
smaller primer binding regions, and is used to amplify conserved DNA sequences, such
as the 16S (or eukaryotic 18S) rRNA gene.
15. Multiplex Ligation-dependent Probe Amplification (MLPA):
permits multiple targets to be amplified with only a single primer pair, thus avoiding the
resolution limitations of multiplex PCR (see below).
16. Multiplex-PCR:
Consists of multiple primer sets within a single PCR mixture to produce amplicons of
varying sizes that are specific to different DNA sequences. By targeting multiple genes at
once, additional information may be gained from a single test run that otherwise would
require several times the reagents and more time to perform. Annealing temperatures for
each of the primer sets must be optimized to work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length, should be different enough to form distinct
bands when visualized by gel electrophoresis.
17. Nested PCR:
increases the specificity of DNA amplification, by reducing background due to non-
specific amplification of DNA. Two sets (instead of one pair) of primers are used in two
successive PCRs. In the first reaction, one pair of primers is used to generate DNA
products, which besides the intended target, may still consist of non-specifically
amplified DNA fragments. The product(s) are then used in a second PCR with a set of
primers whose binding sites are completely or partially different from and located 3' of
each of the primers used in the first reaction. Nested PCR is often more successful in
specifically amplifying long DNA fragments than conventional PCR, but it requires more
detailed knowledge of the target sequences.
18. Overlap-extension PCR:
Amjad Khan Afridi
5. 5
A genetic engineering technique allowing the construction of a DNA sequence with an
alteration inserted beyond the limit of the longest practical primer length.
19. Quantitative PCR (Q-PCR):
Used to measure the quantity of a PCR product (commonly in real-time). It
quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly
used to determine whether a DNA sequence is present in a sample and the number of its
copies in the sample. Quantitative real-time PCR has a very high degree of precision.
QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-
containing DNA probes, such as TaqMan, to measure the amount of amplified product in
real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR.
QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly
refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.
20. Reverse Transcription PCR (RT-PCR):
For amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into
cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling,
to determine the expression of a gene or to identify the sequence of an RNA transcript,
including transcription start and termination sites. If the genomic DNA sequence of a
gene is known, RT-PCR can be used to map the location of exons and introns in the gene.
The 5' end of a gene (corresponding to the transcription start site) is typically identified
by RACE-PCR (Rapid Amplification of cDNA Ends).
21. Solid Phase PCR:
Encompasses multiple meanings, including Colony Amplification (where PCR colonies
are derived in a gel matrix, for example), 'Bridge PCR' (primers are covalently linked to a
solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied
in the presence of solid support bearing primer with sequence matching one of the
aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR
can be improved by employing high Tm and nested solid support primer with optional
application of a thermal 'step' to favour solid support priming).
22. Thermal Asymmetric Interlaced PCR (TAIL-PCR):
For isolation of an unknown sequence flanking a known sequence. Within the known
sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures;
a degenerate primer is used to amplify in the other direction from the unknown sequence.
23. Touchdown PCR (Step-down PCR):
A variant of PCR that aims to reduce nonspecific background by gradually lowering the
annealing temperature as PCR cycling progresses. The annealing temperature at the
initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at
Amjad Khan Afridi
6. 6
the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures
give greater specificity for primer binding, and the lower temperatures permit more
efficient amplification from the specific products formed during the initial cycles.
24. Universal Fast Walking:
For genome walking and genetic fingerprinting using a more specific 'two-sided' PCR
than conventional 'one-sided' approaches (using only one gene-specific primer and one
general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving
lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-
dependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and
3'RACE LaNe.
25. Variable Number of Tandem Repeats (VNTR) PCR :
Targets areas of the genome that exhibit length variation. The analysis of the genotypes
of the sample usually involves sizing of the amplification products by gel electrophoresis.
Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the
basis for DNA Fingerprinting databases such as CODIS.
Pretreatments and extensions
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of
modification:
• Normally PCR primers are chosen from an invariant part of the genome, and
might be used to amplify a polymorphic area between them. In Allele-specific
PCR the opposite is done. At least one of the primers is chosen from a
polymorphic area, with the mutations located at (or near) its 3'-end. Under
stringent conditions, a mismatched primer will not initiate replication, whereas a
matched primer will. The appearance of an amplification product therefore
indicates the genotype.
• InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting
that uses primers selected from segments repeated throughout a genome to
produce a unique fingerprint of amplified product lengths. The use of primers
from a commonly repeated segment is called Alu-PCR, and can help amplify
sequences adjacent (or between) these repeats.
• Primers can also be designed to be 'degenerate' - able to initiate replication from a
large number of target locations. Whole genome amplification (or WGA) is a
group of procedures that allow amplification to occur at many locations in an
unknown genome, and which may only be available in small quantities. Other
techniques use degenerate primers that are synthesized using multiple
nucleotides at particular positions (the polymerase 'chooses' the correctly matched
primers). Also, the primers can be synthesized with the nucleoside analog inosine,
Amjad Khan Afridi
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which hybridizes to three of the four normal bases. A similar technique can force
PCR to perform Site-directed mutagenesis.
• Normally the primers used in PCR are designed to be fully complementary to the
target. However, the polymerase is tolerant to mis-matches away from the 3' end.
Tailed-primers include non-complementary sequences at their 5' ends. A
common procedure is the use of linker-primers, which ultimately place
restriction sites at the ends of the PCR products, facilitating their later insertion
into cloning vectors.
• An extension of the 'colony-PCR' method (above), is the use of vector primers.
Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a
single set of primers are designed for the areas of the vector flanking the insertion
site. Amplification occurs for whatever DNA has been inserted.
• PCR can easily be modified to produce a labeled product for subsequent use as a
hybridization probe. One or both primers might be used in PCR with a radioactive
or fluorescent label already attached, or labels might be added after amplification.
These labeling methods can be combined with 'asymmetric-PCR' (above) to
produce effective hybridization probes.
Do not read success stories, you will get only message..
Read failure stories, you will get some ideas to get success.
In the blink of an eye, every thing can change . …….
So forgive often and love with all your heart…….
You never know when you may not have that chance again.
Amjad Khan Afridi