This document provides an overview of DNA and RNA isolation, purification, and amplification techniques. It discusses preparing genomic DNA from bacterial cells using lysis and phenol-chloroform extraction. Plasmid DNA can be purified from bacterial cells using alkaline lysis and silica binding. The document also reviews RNA isolation, which requires rapid processing to prevent RNase degradation, and describes mRNA selection using oligo-dT cellulose. Reverse transcription PCR and quantitative PCR are introduced as methods to study gene expression.
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
The document describes the process and components of emulsion PCR. Key points include:
- Emulsion PCR is used to amplify DNA in microreactors formed from water-in-oil emulsions, allowing individual DNA fragments to be amplified clonally.
- The emulsion PCR mixture contains primers, DNA polymerase, nucleotides, template DNA, and is emulsified in an oil phase containing surfactants to form water-in-oil droplets.
- The emulsion undergoes PCR cycling to amplify the DNA fragments clonally within individual droplets. The emulsion is then broken and the amplified DNA fragments can be analyzed by gel electrophoresis or used for downstream applications like sequencing.
This document provides an introduction and overview of reverse transcription PCR (RT-PCR). It discusses that RT-PCR uses the product of a reverse transcription reaction as a template for PCR amplification. The document outlines the basic principles and steps of RT-PCR, including reverse transcription of RNA to cDNA followed by PCR amplification of the cDNA. It also compares one-step vs two-step RT-PCR methods and discusses considerations like avoiding contamination of genomic DNA.
Principles of DNA isolation, PCR and LAMPPerez Eric
The document provides information about a lecture on principles of DNA isolation, purification, and polymerase chain reaction (PCR). It discusses topics like DNA location in cells, principles of DNA isolation using physical and chemical methods, components of extraction buffers, PCR principles involving DNA denaturation, primer annealing and DNA elongation, and analysis of PCR products through gel electrophoresis. The summary is:
The lecture covers principles of extracting and purifying DNA from samples, components and steps of the polymerase chain reaction (PCR) technique for amplifying DNA, and analyzing amplified DNA products through gel electrophoresis. It discusses isolating DNA from cells, components of extraction buffers, PCR steps like denaturation, annealing and elongation, and the roles of various PCR reagents
Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. Each cycle doubles the amount of target DNA. After 20-30 cycles, there can be over a billion copies of the original DNA sequence. PCR is used for a variety of applications including disease diagnosis, cloning genes, forensic analysis, and more. It is a powerful technique that has revolutionized molecular biology.
This document describes RT-PCR (reverse transcription polymerase chain reaction). It discusses that RT-PCR is used to detect RNA expression by converting RNA to cDNA using reverse transcriptase, then amplifying the cDNA using PCR. It provides details on the history and development of RT-PCR, including the discovery of reverse transcriptase. It also explains the basic procedures for one-step and two-step RT-PCR and compares the two methods.
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics.
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
The document describes the process and components of emulsion PCR. Key points include:
- Emulsion PCR is used to amplify DNA in microreactors formed from water-in-oil emulsions, allowing individual DNA fragments to be amplified clonally.
- The emulsion PCR mixture contains primers, DNA polymerase, nucleotides, template DNA, and is emulsified in an oil phase containing surfactants to form water-in-oil droplets.
- The emulsion undergoes PCR cycling to amplify the DNA fragments clonally within individual droplets. The emulsion is then broken and the amplified DNA fragments can be analyzed by gel electrophoresis or used for downstream applications like sequencing.
This document provides an introduction and overview of reverse transcription PCR (RT-PCR). It discusses that RT-PCR uses the product of a reverse transcription reaction as a template for PCR amplification. The document outlines the basic principles and steps of RT-PCR, including reverse transcription of RNA to cDNA followed by PCR amplification of the cDNA. It also compares one-step vs two-step RT-PCR methods and discusses considerations like avoiding contamination of genomic DNA.
Principles of DNA isolation, PCR and LAMPPerez Eric
The document provides information about a lecture on principles of DNA isolation, purification, and polymerase chain reaction (PCR). It discusses topics like DNA location in cells, principles of DNA isolation using physical and chemical methods, components of extraction buffers, PCR principles involving DNA denaturation, primer annealing and DNA elongation, and analysis of PCR products through gel electrophoresis. The summary is:
The lecture covers principles of extracting and purifying DNA from samples, components and steps of the polymerase chain reaction (PCR) technique for amplifying DNA, and analyzing amplified DNA products through gel electrophoresis. It discusses isolating DNA from cells, components of extraction buffers, PCR steps like denaturation, annealing and elongation, and the roles of various PCR reagents
Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. Each cycle doubles the amount of target DNA. After 20-30 cycles, there can be over a billion copies of the original DNA sequence. PCR is used for a variety of applications including disease diagnosis, cloning genes, forensic analysis, and more. It is a powerful technique that has revolutionized molecular biology.
This document describes RT-PCR (reverse transcription polymerase chain reaction). It discusses that RT-PCR is used to detect RNA expression by converting RNA to cDNA using reverse transcriptase, then amplifying the cDNA using PCR. It provides details on the history and development of RT-PCR, including the discovery of reverse transcriptase. It also explains the basic procedures for one-step and two-step RT-PCR and compares the two methods.
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics.
The document discusses various molecular cytogenetics techniques including polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and fluorescent in situ hybridization (FISH). It provides details on the principles, techniques, and applications of PCR and RT-PCR. PCR is described as a technique that amplifies specific DNA regions, allowing minute quantities to be analyzed. Key steps involve DNA denaturation, primer annealing, and fragment extension. RT-PCR involves first converting RNA to cDNA then amplifying a specific region. Both techniques have numerous diagnostic and research applications.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
PCR is a technique that amplifies specific DNA sequences. It involves denaturing DNA strands, annealing primers to the strands, and extending the primers using DNA polymerase. This cycling process exponentially amplifies the target DNA sequence. Key components are thermostable DNA polymerase from Thermus aquaticus, primers, nucleotides, buffer, and repeated heating and cooling. PCR is used in applications like disease diagnosis, forensics, and sequencing.
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 (PCR) is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of primers, DNA polymerase, and nucleotides. This enables millions of copies of the target DNA segment to be generated from a single or few DNA molecules. Key steps include DNA extraction, use of primers and DNA polymerase to enable selective replication of the target segment, and gel electrophoresis to analyze the amplified DNA fragments by size.
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
The polymerase chain reaction (PCR) is a technique used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strand is separated from its complement at a high temperature and two new strands are synthesized from the original copies at a lower temperature, thereby exponentially increasing the number of target DNA copies. Real-time PCR allows for quantification of the target DNA by detecting fluorescence at each cycle, while reverse transcription PCR is used to transcribe RNA into DNA.
PCR allows for the amplification of small amounts of DNA, generating millions of copies. It involves three steps - denaturation of the DNA template, annealing of primers, and extension of the primers by DNA polymerase. Repeating this process results in exponential growth in the number of DNA copies. DNA sequencing, such as the Sanger method, is used to determine the exact order of nucleotides in a DNA fragment and can be used to map genomes and detect genetic variations.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
Polymerase chain reaction (PCR) is a powerful method for amplifying DNA sequences. It involves denaturing DNA into single strands, annealing primers to the strands, and extending the primers to replicate the DNA. Key components of PCR include a DNA template, DNA polymerase enzyme, primers, nucleotides, and a thermocycler. The thermocycler regulates temperature changes to allow for denaturation, annealing, and elongation steps that are repeated for numerous cycles to exponentially amplify the target DNA sequence. PCR is commonly used for diagnosing diseases and detecting microorganisms, and variations like real-time PCR, microarray analysis, bridge PCR, and emulsion PCR are employed for different applications like next-generation sequencing.
The document describes the polymerase chain reaction (PCR) technique for amplifying DNA. It discusses the basic components and steps of PCR, including denaturation, annealing and extension. It also describes different PCR types such as nested PCR, RT-PCR, and applications in clinical diagnosis, forensics and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.
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
1. CENTRAL DOGMA OF MOLECULAR BIOLOGY
2. NUCLEIC ACID PREPARATION & APPLICATIONS
3. FUNDAMENTAL STEPS IN DNA PURIFICATION
4. ANALYSIS OF NUCLEIC ACIDS
5. STORAGE CONDITIONS
The document summarizes polymerase chain reaction (PCR), including its history, principles, types, and applications. It describes how PCR was invented in 1983 by Kary Mullis, allowing for the amplification of specific DNA sequences. The basic steps of PCR involve denaturation of DNA, annealing of primers, and extension of new strands by DNA polymerase. Various types of PCR are discussed, such as real-time PCR, reverse transcriptase PCR, and nested PCR. The document explains that PCR has many applications, including diagnosis of infectious diseases and detection of genetic variations.
The polymerase chain reaction (PCR) is a technique used to amplify a specific fragment of DNA without using living organisms. It works by cycling between high and low temperatures to denature the DNA strands, allow primers to anneal, and extend the DNA. This process rapidly increases the number of copies of the target DNA fragment. PCR requires DNA polymerase, primers, DNA template, dNTPs, buffer solution, and cations, and is commonly used in medical research for gene sequencing, disease diagnosis, and forensic analysis.
The document discusses polymerase chain reaction (PCR), including its history, principles, requirements, and applications. PCR was developed in the 1980s by Kary Mullis and allows for targeted amplification of specific DNA sequences. It involves repetitive cycles of denaturation, annealing of primers, and extension of DNA using a thermostable polymerase. PCR is a powerful technique due to its sensitivity, specificity, speed and versatility in amplifying DNA from a variety of sources for research, forensic, and diagnostic applications.
1. The document discusses different types of cloning vectors including plasmids, bacteriophages, cosmids, and phagemids that can be used to clone foreign DNA.
2. It provides details on the characteristics and components of common vectors like the pBR322 plasmid and lambda phage. Screening methods for identifying recombinant clones like blue/white selection and replica plating are also described.
3. Applications of genetic engineering using these vectors include producing recombinant proteins like insulin and hepatitis vaccines as well as studying gene function and identifying mutations associated with diseases.
In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally .
I wish for your future goal that you will shine one day inshallah .
Thank you for watching
The document discusses various molecular cytogenetics techniques including polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and fluorescent in situ hybridization (FISH). It provides details on the principles, techniques, and applications of PCR and RT-PCR. PCR is described as a technique that amplifies specific DNA regions, allowing minute quantities to be analyzed. Key steps involve DNA denaturation, primer annealing, and fragment extension. RT-PCR involves first converting RNA to cDNA then amplifying a specific region. Both techniques have numerous diagnostic and research applications.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
PCR is a technique that amplifies specific DNA sequences. It involves denaturing DNA strands, annealing primers to the strands, and extending the primers using DNA polymerase. This cycling process exponentially amplifies the target DNA sequence. Key components are thermostable DNA polymerase from Thermus aquaticus, primers, nucleotides, buffer, and repeated heating and cooling. PCR is used in applications like disease diagnosis, forensics, and sequencing.
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 (PCR) is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of primers, DNA polymerase, and nucleotides. This enables millions of copies of the target DNA segment to be generated from a single or few DNA molecules. Key steps include DNA extraction, use of primers and DNA polymerase to enable selective replication of the target segment, and gel electrophoresis to analyze the amplified DNA fragments by size.
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
The polymerase chain reaction (PCR) is a technique used to amplify specific DNA fragments. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strand is separated from its complement at a high temperature and two new strands are synthesized from the original copies at a lower temperature, thereby exponentially increasing the number of target DNA copies. Real-time PCR allows for quantification of the target DNA by detecting fluorescence at each cycle, while reverse transcription PCR is used to transcribe RNA into DNA.
PCR allows for the amplification of small amounts of DNA, generating millions of copies. It involves three steps - denaturation of the DNA template, annealing of primers, and extension of the primers by DNA polymerase. Repeating this process results in exponential growth in the number of DNA copies. DNA sequencing, such as the Sanger method, is used to determine the exact order of nucleotides in a DNA fragment and can be used to map genomes and detect genetic variations.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
Polymerase chain reaction (PCR) is a powerful method for amplifying DNA sequences. It involves denaturing DNA into single strands, annealing primers to the strands, and extending the primers to replicate the DNA. Key components of PCR include a DNA template, DNA polymerase enzyme, primers, nucleotides, and a thermocycler. The thermocycler regulates temperature changes to allow for denaturation, annealing, and elongation steps that are repeated for numerous cycles to exponentially amplify the target DNA sequence. PCR is commonly used for diagnosing diseases and detecting microorganisms, and variations like real-time PCR, microarray analysis, bridge PCR, and emulsion PCR are employed for different applications like next-generation sequencing.
The document describes the polymerase chain reaction (PCR) technique for amplifying DNA. It discusses the basic components and steps of PCR, including denaturation, annealing and extension. It also describes different PCR types such as nested PCR, RT-PCR, and applications in clinical diagnosis, forensics and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.
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
1. CENTRAL DOGMA OF MOLECULAR BIOLOGY
2. NUCLEIC ACID PREPARATION & APPLICATIONS
3. FUNDAMENTAL STEPS IN DNA PURIFICATION
4. ANALYSIS OF NUCLEIC ACIDS
5. STORAGE CONDITIONS
The document summarizes polymerase chain reaction (PCR), including its history, principles, types, and applications. It describes how PCR was invented in 1983 by Kary Mullis, allowing for the amplification of specific DNA sequences. The basic steps of PCR involve denaturation of DNA, annealing of primers, and extension of new strands by DNA polymerase. Various types of PCR are discussed, such as real-time PCR, reverse transcriptase PCR, and nested PCR. The document explains that PCR has many applications, including diagnosis of infectious diseases and detection of genetic variations.
The polymerase chain reaction (PCR) is a technique used to amplify a specific fragment of DNA without using living organisms. It works by cycling between high and low temperatures to denature the DNA strands, allow primers to anneal, and extend the DNA. This process rapidly increases the number of copies of the target DNA fragment. PCR requires DNA polymerase, primers, DNA template, dNTPs, buffer solution, and cations, and is commonly used in medical research for gene sequencing, disease diagnosis, and forensic analysis.
The document discusses polymerase chain reaction (PCR), including its history, principles, requirements, and applications. PCR was developed in the 1980s by Kary Mullis and allows for targeted amplification of specific DNA sequences. It involves repetitive cycles of denaturation, annealing of primers, and extension of DNA using a thermostable polymerase. PCR is a powerful technique due to its sensitivity, specificity, speed and versatility in amplifying DNA from a variety of sources for research, forensic, and diagnostic applications.
1. The document discusses different types of cloning vectors including plasmids, bacteriophages, cosmids, and phagemids that can be used to clone foreign DNA.
2. It provides details on the characteristics and components of common vectors like the pBR322 plasmid and lambda phage. Screening methods for identifying recombinant clones like blue/white selection and replica plating are also described.
3. Applications of genetic engineering using these vectors include producing recombinant proteins like insulin and hepatitis vaccines as well as studying gene function and identifying mutations associated with diseases.
In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally .
I wish for your future goal that you will shine one day inshallah .
Thank you for watching
How to Fix the Import Error in the Odoo 17Celine George
An import error occurs when a program fails to import a module or library, disrupting its execution. In languages like Python, this issue arises when the specified module cannot be found or accessed, hindering the program's functionality. Resolving import errors is crucial for maintaining smooth software operation and uninterrupted development processes.
Main Java[All of the Base Concepts}.docxadhitya5119
This is part 1 of my Java Learning Journey. This Contains Custom methods, classes, constructors, packages, multithreading , try- catch block, finally block and more.
বাংলাদেশের অর্থনৈতিক সমীক্ষা ২০২৪ [Bangladesh Economic Review 2024 Bangla.pdf] কম্পিউটার , ট্যাব ও স্মার্ট ফোন ভার্সন সহ সম্পূর্ণ বাংলা ই-বুক বা pdf বই " সুচিপত্র ...বুকমার্ক মেনু 🔖 ও হাইপার লিংক মেনু 📝👆 যুক্ত ..
আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
তাই একজন নাগরিক হিসাবে এই তথ্য গুলো আপনার জানা প্রয়োজন ...।
বিসিএস ও ব্যাংক এর লিখিত পরীক্ষা ...+এছাড়া মাধ্যমিক ও উচ্চমাধ্যমিকের স্টুডেন্টদের জন্য অনেক কাজে আসবে ...
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
ISO/IEC 27001, ISO/IEC 42001, and GDPR: Best Practices for Implementation and...PECB
Denis is a dynamic and results-driven Chief Information Officer (CIO) with a distinguished career spanning information systems analysis and technical project management. With a proven track record of spearheading the design and delivery of cutting-edge Information Management solutions, he has consistently elevated business operations, streamlined reporting functions, and maximized process efficiency.
Certified as an ISO/IEC 27001: Information Security Management Systems (ISMS) Lead Implementer, Data Protection Officer, and Cyber Risks Analyst, Denis brings a heightened focus on data security, privacy, and cyber resilience to every endeavor.
His expertise extends across a diverse spectrum of reporting, database, and web development applications, underpinned by an exceptional grasp of data storage and virtualization technologies. His proficiency in application testing, database administration, and data cleansing ensures seamless execution of complex projects.
What sets Denis apart is his comprehensive understanding of Business and Systems Analysis technologies, honed through involvement in all phases of the Software Development Lifecycle (SDLC). From meticulous requirements gathering to precise analysis, innovative design, rigorous development, thorough testing, and successful implementation, he has consistently delivered exceptional results.
Throughout his career, he has taken on multifaceted roles, from leading technical project management teams to owning solutions that drive operational excellence. His conscientious and proactive approach is unwavering, whether he is working independently or collaboratively within a team. His ability to connect with colleagues on a personal level underscores his commitment to fostering a harmonious and productive workplace environment.
Date: May 29, 2024
Tags: Information Security, ISO/IEC 27001, ISO/IEC 42001, Artificial Intelligence, GDPR
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This document provides an overview of wound healing, its functions, stages, mechanisms, factors affecting it, and complications.
A wound is a break in the integrity of the skin or tissues, which may be associated with disruption of the structure and function.
Healing is the body’s response to injury in an attempt to restore normal structure and functions.
Healing can occur in two ways: Regeneration and Repair
There are 4 phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. This document also describes the mechanism of wound healing. Factors that affect healing include infection, uncontrolled diabetes, poor nutrition, age, anemia, the presence of foreign bodies, etc.
Complications of wound healing like infection, hyperpigmentation of scar, contractures, and keloid formation.
1. DNA and RNA isolation and purification (course
readings 10 and 11)
I. Genomic DNA preparation overview
II. Plasmid DNA preparation
III. DNA purification
• Phenol extraction
• Ethanol precipitation
IV. RNA work
2. What do we need DNA for?
•Detect, enumerate, clone genes
•Detect, enumerate species
•Detect/sequence specific DNA regions
•Create new DNA “constructs” (recombinant DNA)
What about RNA?
•Which genes are being transcribed?
•When/where are genes being transcribed?
•What is the level of transcription?
5. Genomic DNA prep: removing proteins and RNA
Add the enzyme RNase to degrade RNA in the aqueous
layer
Need to mix gently! (to avoid shearing breakage of the
genomic DNA)
chloroform
6. 2 ways to concentrate the genomic DNA
70% final conc.
“spooling” Ethanol precipitation
7. Genomic DNA prep in plants --
how get rid of carbohydrates?
CTAB:
Cationic
detergent
(MC 6.61-6.62)
(low ionic
conditions)
N+
CH3
Br-
CH3
CH3
C16H33
8. Plasmids: vehicles of recombinant DNA
Bacterial cell
genomic DNA plasmids
Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate
9. Plasmid purification: alkaline lysis
Alkaline
conditions
denature
DNA
Neutralize:
genomic DNA
can’t renature
(plasmids
CAN because
they never
fully
separate)
10. DNA purification: silica binding
Binding occurs in presence of high
salt concentration, and is disrupted
by elution with water
11. DNA purification: phenol/chloroform extraction
1:1 phenol : chloroform
or
25:24:1 phenol : chloroform : isoamyl alcohol
Phenol: denatures proteins, precipitates form at
interface between aqueous and organic layer
Chloroform: increases density of organic layer
Isoamyl alcohol: prevents foaming
12. 1. Aqueous volume (at least 200 microliters)
2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube
4. Repeat steps 2 and 3 until there is no precipitate at
phase interface
5. (extract aqueous layer with 2 volumes of chloroform)
Phenol extraction
13. Ethanol depletes the hydration shell surrounding DNA…
• Allowing cations to interact with the DNA phosphates
• Reducing repulsive forces between DNA strands
• Causing aggregation and precipitation of DNA
• Aqueous volume (example: 200 microliters)
-- add 22 microliters sodium acetate 3M pH 5.2
-- add 1 microliter of glycogen (gives a visible pellet)
-- add 2 volumes (446 microliters) 100% ethanol
-- mix well, centrifuge at high speed, decant liquid
-- wash pellet (70% ethanol), dry pellet, dissolve in
appropriate volume (then determine DNA concentration)
Ethanol precipitation (DNA concentration)
15. Isolation of RNA -- Course reading 11
DNA --------------> mRNA --------------> protein
Lots of information in mRNA:
When is gene expressed?
What is timing of gene expression?
What is the level of gene expression?
(but what does an mRNA measurement really say about
expression of the protein?)
16. RNA in a typical eukaryotic cell:
10-5 micrograms RNA
80-85% is ribosomal RNA
15-20% is small RNA (tRNA, small nuclear RNAs)
About 1-5% is mRNA
-- variable in size
-- but usually containing 3’ polyadenylation
17. The problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular
transesterification
RNA is susceptible to nearly ubiquitous RNA-degrading
enzymes (RNases)
RNases are released upon cell lysis
RNases are present on the skin
RNases are very difficult to inactivate
-- disulfide bridges conferring stability
-- no requirement for divalent cations for activity
18. Common sources of RNase and how to avoid them
Contaminated solutions/buffers
USE GOOD STERILE TECHNIQUE
TREAT SOLUTIONS WITH DEPC (when possible)
MAKE SMALL BATCHES OF SOLUTIONS
Contaminated equipment
USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS
BAKE GLASSWARE, 300°C, 4 hours
USE “RNase-free” PIPET TIPS
TREAT EQUIPMENT WITH DEPC
19. Top 10 sources of RNAse contamination
(Ambion Scientific website)
1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)
10) Enzyme preparations
20. Inhibitors of Rnase
DEPC: diethylpyrocarbonate
alkylating agent, modifying proteins and nucleic acids
fill glassware with 0.1% DEPC, let stand overnight at
room temp
solutions may be treated with DEPC -- add DEPC to
0.1%, then autoclave (DEPC breaks down to CO2 and ethanol)
21. Inhibitors of Rnase
Vanadyl ribonucleoside complexes
competitive inhibitors of RNAses, but need to be removed
from the final preparation of RNA
Protein inhibitors of RNAse
horseshoe-shaped, leucine rich protein, found in
cytoplasm of most mammalian tissues
must be replenished following phenol extraction steps
24. Purifying RNA: the key is speed
Break the cells/solubilize components/inactivate RNAses by the
addition of guanidinium thiocyanate (very powerful denaturant)
Extract RNA using phenol/chloroform (at low pH)
Precipitate the RNA using ethanol/LiCl
Store RNA:
in DEPC-treated H20 (-80°C)
in formamide (deionized) at -20°C
25. Selective capture of mRNA: oligo dT-cellulose
Oligo dT is linked to cellulose matrix
RNA is washed through matrix at high salt concentration
Non-polyadenylated RNAs are washed through
polyA RNA is removed under low-salt conditions
(not all of the non-polyadenylated RNA gets removed
26. Other methods to capture mRNA
Poly(U) sepharose chromatography
Poly(U)-coated paper filters
Streptavidin beads:
•A biotinylated oligo dT is added to guanidinium-
treated cells, and it anneals to the polyA tail of mRNAs
•Biotin/streptavidin interactions permit isolation of the
mRNA/oligo dT complexes
27. How good is the RNA prep?
The rRNA should form 2 sharp bands in ethidium
bromide-stained gels (but mRNA will not be visible
Use radiolabelled poly dT in a pilot Northern
hybridization--should get a smear from 0.6 to 5 kb on
the blot
Use a known, “standard” gene probe (e.g. GAPDH in
mammalian cells) in Northern hybridization--there
should be a sharp band with no degradation products
28. In vitro amplification of DNA by PCR
I. Theory of PCR
II. Components of the PCR reaction
III. A few advanced applications of PCR
a) Reverse transcription PCR (for RNA
measurements)
b) Quantitative real-time PCR
c) PCR of long DNA fragments
d) Inverse PCR
e) MOPAC (mixed oligonucleotide priming)
Molecular Cloning, p. 8.1-8.24
29. What is PCR?
• Polymerase Chain Reaction--first described in
1971 by Kleppe and Khorana, re-described and
first successful use in 1985
• Allows massive amplification of specific sequences
that have defined endpoints
• Fast, powerful, adaptable, and simple*
• Many many many applications
* usually
30. Why amplify specific sequences?
• To obtain material for cloning and sequencing, or
for in vitro studies
• To verify the identity of engineered DNA
constructs
• To monitor gene expression
• To diagnose a genetic disease
• To reveal the presence of a micro-organism
• To identify an individual
• Etcetera, etcetera
31. What you need for PCR:
1. Template DNA that contains the “target
sequence”
2. Primers: short oligonucleotides that define the
ends of the target sequence
3. Thermostable DNA polymerase
4. Buffer, dNTPs
5. A thermal cycler
32. Denaturation: denature template strands (94°C for
2-5 minutes), can also add your DNA polymerase at
this temp. for a “hot start” (adding DNA pol to a hot
tube can prevent false priming in the initial round of
DNA replication)
Annealing: The default is usually 55°C. This
temperature variable is the most critical one for
getting a successful PCR reaction. This is the best
variable to start with when trying to optimize a PCR
reaction for a specific set of primers. Annealing
temperatures can be dropped as low as 40-45°C,
but non-specific annealing can be a problem
A typical PCR program:
33. Extension: generally 72°C, this is the operating
temperature for many thermostable DNA
polymerases.
Number of cycles: Depends on the number of
copies of template DNA and the desired amount
of PCR product. Generally 20-30 cycles is
sufficient.
A typical PCR program:
34. How it works:
a simple PCR reaction, first cycle
(Can also be
Single-stranded)
94°C
50°C
72°C
Cycles of
denaturation,
primer annealing,
and primer
extension by DNA
polymerase
35. a simple PCR reaction, second cycle
like
first
cycle
new
reactions
36. a simple PCR reaction, third cycle
PCR animation:
http://www.dnai.org/b/index.html
http://www.dnalc.org/ddnalc/resources/shockwave/pcran
whole.html
37. Choosing primers:
• Should be 18-25 (17-30?) nucleotides in length (giving
specificity)
• Calculated melting temperature varies depending on the
method used (55-65°C using the Wallace Rule, eg. see MC),
but should be nearly identical for both primers
• Avoid inverted repeat sequences and self-complementary
sequences in the primers, avoid complementarity between
primers (‘primer dimers’)
• Have a G or C at the 3’ end (a G/C “clamp”)
• Many computer programs exist for helping meet these
criteria (ex: Biology Workbench, workbench.sdsc.edu)
38. Thermostable DNA polymerases
• Isolated from thermophilic bacteria and archaea (T.
aquaticus is a bacterium, not an archaeon)
• Bacterial enzymes (e.g. Taq) good for routine reactions
and small PCR products, fidelity of replication is
somewhat low
• Archaeal enzymes (e.g. Pfu) also good for routine
reactions and best for cloning: 3’--5’ exonuclease activity
provides very high fidelity, and enzymes are very stable to
heat
(See Molecular Cloning table 8-1)
39. Thermal cyclers
Standard: heat block, “ramp” times fairly long (10 -20
seconds to change temperature), 30 cycle PCR lasts 2-3
hours.
Advantage: easily automated, heat blocks can PCR
up to 384 samples at a time
Disadvantage: relatively slow
New: reactions are being sped up significantly
--capillary tubes heated and cooled by blasts of air--
30 cycle-PCR done in >30 minutes (harder to scale up)
--fluid flow cells: channels force liquid through
temperature gradients, very fast (but still not widely
available)
40. Sources of problems in PCR
• Inhibitors of the reaction from the the template
DNA preparation (protease, phenol, EDTA, etc)
• Cross-contamination by DNA from sources
other than the template added
– if this becomes a problem:
• Work in a laminar flow hood (decontaminate using
UV light 254 nm)
• Use PCR dedicated pipettors (with barrier tips),
PCR dedicated reagents
• Centrifuge tubes before opening them to prevent
spattering, pipet contamination
41. Controls to include in difficult PCRs:
Bystander
DNA
template
DNA
Target
DNA
Specific
primers
Positive
controls
1 + - + +
2 - - + +
Negative
controls
3 - - - +
4 + - - +
Bystander DNA:
not recognized
by primers
Target DNA:
known to
contain primer
recognition
sequences
42. Hot Start of PCR reactions
• Witholding some component of the reaction until the
denaturing temperature is reached (94°C)
• This helps prevent non-specific priming, which may
occur at the low temperatures (room temp.) -- the
non-specific priming could give artifactual
amplification as PCR block temperature rises
A) Wait until 94°C to add enzyme --or--
B) Use enzyme bound to an inactivating enzyme
antibody that releases at high temperature --or--
C) Use wax beads containing Mg++ that can only be
released at high temp.
43. Touchdown PCR
• Useful if your primers are not 100% complementary to your
template DNA (e.g. degenerate oligos), or when there are
multiple members of the gene family you are amplifying
• Allows you to selectively amplify only the best sequences
(with the least mismatches) while minimizing non-specific
PCR products
• Start with 2 cycles at an annealing temperature about 3°C
higher than the calculated primer melting temperatures.
• Progressively reduce the annealing temperature by 1°C at 2
cycle intervals
45. III. Special applications for PCR
A. Reverse transcription PCR (for RNA
measurements)
B. Quantitative (real-time) PCR
C. PCR of long DNA fragments
D. Whole genome amplification
E. Inverse PCR
E. MOPAC (mixed oligonucleotide priming)
46. Amplification of RNA (monitor gene expression):
reverse transcription PCR (RT-PCR)
Step 1: generate a 1st strand cDNA using reverse transcriptase
(catalyzes synthesis of DNA from an RNA template)
Step 2: normal PCR (from cDNA) using gene-specific primers
A)
B)
C)
47. Quantitative
(real time)
PCR
The more target DNA there
is, the more probe anneals,
the more it is cleaved (by
Taq’s 5’-3’ exonuclease
activity)
Fluorescence measurements
are done simultaneously with
PCR cycles, yields an
instantaneous measurement of
product levels
48. Quantitative
Real Time
(QRT) PCR
Position of the
steep part of the
curve varies
depending on
the amount of
template DNA or
RNA, can
measure
variation over 5
or 6 orders of
magnitude
more
template less
template
49. Another quantitative measure of double stranded DNA in a
PCR reaction: binding of SYBR Green Dye
From the Molecular Probes website
(www.probes.com)
Non-fluorescing SYBR
green dye
Fluorescing SYBR
green dye
50. Use of a quenching dye to reduce measurement of
“primer dimer” artifacts in QRT-PCR
QSY quencher dye: it
absorbs fluorescence from
sybr green dyes in the
vicinity--prevents
accumulation of signal
from primer dimers
51. Always do your controls!
QRT-PCR using Sybr green dye fluorescence
(From the Invitrogen website)
Standard curve: what is “threshold” for specific number of DNA
molecules?
52. PCR of long sequences (>2 kb)
Long DNAs are difficult to amplify
– Breakage of the DNA
– Non-processive behavior of DNA
polymerase
– Misincorporation by error prone DNA
polymerases
53. Changes to protocol that assist in long PCR
– Make sure DNA is exceedingly clean
– Use DNA polymerase “cocktail”: Taq for it’s
high activity, and Pfu for its proofreading
activity (it can actually correct Taq’s
mistakes)
– Increase time of extension reaction (5-20
minutes, compared to the standard 1
minute for short PCRs)
PCR of long sequences (>2 kb)
54. •Amplified product longer than 3 kilobases with high fidelity
•10 times fewer mutations than with conventional PCR
•Taq DNA pol (no proofreading) plus an archaeal DNA pol
(does proofreading)
•Betaine (amino acid analogue with several small
tetraalkyammonium ions)--reduces non-specific amplification
products--reduces non-complementary primer-template
interactions? (unknown how it works)
55. Whole genome amplification: multiple
displacement amplification (MDA)
Applications: forensics, embryonic disease diagnosis,
microbial diversity surveys, etc.
How it works:
Strand-displacement amplification used by rolling-
circle replication systems.
Phi29 DNA polymerase (very low error rate) and
random hexamer primers, low temperature! (30°C)
56. Whole genome amplification : multiple displacement
amplification (MDA)
20-30 micrograms human DNA can be recovered from 1-10
copies of the human genome
Distribution of products appears to be random sampling of
the available template (and this is good!)
58. “Vectorette” PCR
First primer: known sequence
Vectorette primer: only in vectorette-ligated sequence--it
cannot anneal until there is a single round of primer
extension from the specific primer
http://www.bio.psu.edu/People/Faculty/Akashi/vectPCR.html
59. MOPAC: Mixed oligonucleotide primed
amplification of cDNA
If you only have a protein sequence, and you want to clone
the gene for the protein:
1. Design oligonucleotides based on deduced mRNA (and
DNA) sequence (but since multiple codons can encode
the same amino acid, this gets complicated quickly)
2. program your oligo synthesizer to make primer sets that
are randomized for the degenerate positions of each
codon
3. use universal nucleotides like inosine, which base pairs
with C, T, and A (limits degeneracy)
4. Do your PCR and hope for the best