DNA sequencing and cloning techniques are used to determine DNA sequences and make copies of DNA fragments. There are two main DNA sequencing methods - Sanger sequencing and next generation sequencing. Sanger sequencing involves separating DNA strands, replicating them with ddNTPs to stop replication, and using gel electrophoresis to determine the sequence. Next generation sequencing is faster. DNA cloning makes identical copies of a DNA fragment by inserting it into a vector like a plasmid and introducing it into a host cell. Key tools in cloning are restriction enzymes, which cut DNA at specific sites, and ligase, which joins DNA strands. PCR is used to amplify specific DNA regions and is important for applications like DNA sequencing, genetic testing, forensics, and research
SLIDE CONTAIN BREIF NOTE ON PCR. IT CONTAINS 21 SLIDES INCLUDING, WHAT IS PCR? COMPONENTS, WORKING MECHANISM, APPLICATIONS, CONCLUSION, AND SOME REFRENCES, HISTORY ALSO
SLIDE CONTAIN BREIF NOTE ON PCR. IT CONTAINS 21 SLIDES INCLUDING, WHAT IS PCR? COMPONENTS, WORKING MECHANISM, APPLICATIONS, CONCLUSION, AND SOME REFRENCES, HISTORY ALSO
Polymerase chain reaction (PCR) is a technique in molecular biology used to
amplify (multiply) a single copy or a few copies of a piece of DNA, generating
thousands to millions of copies of that particular DNA sequence.
Polymerase Chain Reaction: Principles, Applications, and Advancements | The L...The Lifesciences Magazine
Polymerase Chain Reaction, often abbreviated as PCR, is a laboratory technique used to amplify specific segments of DNA through a series of temperature-controlled cycles.
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
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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Polymerase chain reaction (PCR) is a technique in molecular biology used to
amplify (multiply) a single copy or a few copies of a piece of DNA, generating
thousands to millions of copies of that particular DNA sequence.
Polymerase Chain Reaction: Principles, Applications, and Advancements | The L...The Lifesciences Magazine
Polymerase Chain Reaction, often abbreviated as PCR, is a laboratory technique used to amplify specific segments of DNA through a series of temperature-controlled cycles.
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
What is PCR?
History of PCR
Components of PCR
Principles of PCR
Basic Requirements
Instrumentation
PCR Programme
Advantages of PCR
Applications of PCR
Conclusion
References
PCR is a revolutionary molecular biology technique used for enzymatically replicating DNA . This technique allows a small amount of DNA molecule to be amplified many times in an exponential manner . It is commonly used in medical and biological research labs for variety of tasks such as detection of hereditary disease , identification of genetic fingerprints diagnosis of infectious disease , cloning of genes and paternity testing .
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies . The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR.
There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95°C. For example, the half-life of Taq polymerase at 95°C is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100°C. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.
At 75°C, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute!
Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Direct PCR cloning methods include TA and GC cloning, as well as TOPO® Cloning, and enable direct cloning of PCR fragments. For example, the TA cloning approach takes advantage of the 3’ A overhang naturally added to products by Taq polymerase following PCR. The resulting sticky ends then enable recombination with DNA fragments containing 3’ T overhangs, such as linearized vectors.
During indirect PCR cloning, the PCR products are modified prior to recombination with other DNA sequences. For example, in restriction cloning, restriction sites are frequently introduced via PCR to enable restriction digestion and ligation with linearized vectors. PCR mutagenesis is a technique used to generate site-directed sequence changes such as base substitutions, inserts and deletions.
To insert a single point mutation via mutagenesis, for example, PCR primers are designed that contain the desired base change, usually in the middle of the primer sequence. PCR is then performed with the mutagenic primers and a high-fidelity DNA polymerase, which results in the incorporation of the desired mutation into the original sequence.Allele-specific PCR is used to detect sequence variations and ultimately determine the genotype of an organism.
For allele-specific PCR, primers are designed to flank the region of interest. The most common application of PCR is gene expression analysis
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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2. DNA sequencing :
The process of determining the Nucleotide
sequencing of DNA which will benefit in the
following:
1) Order of nucleotides in a section of DNA
2) determine amino acid order, protein structure
and protein function
3) detect the type of mutations in genetic diseases
3. Methods of DNA sequence
1) Sangar method
2) Next generation method
4.
5.
6. Sanger method is done by 4 steps:
1) obtain double stranded DNA and separate it by either heating or by putting the DNA in
basic solution
2) The one stranded DNA will undergo replication with the addition of tiny amount of
(ddNTp) which stop the replication
3) Step 2 is repeated with the other 3 ddntps
4) once the 4 ddntps are done, the gel electrophoresis will begin to happen.
Evenutally, the nucletodie sequence of growing strand is detected by gel
electrophoresis. Hence, the original nucleotide sequence will be detected by the
complementary bases
7. Next generation DNA sequence
The Sangar sequencing is replaced by the Next generation DNA sequence because the
period of sequence in next generation is much faster and is done by the following steps:
1) library preparation
2) cluster amplification
3) sequencing by DNA primer and DNA polymerase
8.
9.
10. DNA cloning
is a molecular biology technique that involves the creation of identical copies of a
specific DNA fragment. The process typically includes isolating a target DNA sequence,
inserting it into a vector (such as a plasmid), and introducing the recombinant DNA into a
host organism, often a bacterium. This allows for the replication and expression of the
inserted DNA, producing multiple copies for research, gene analysis, or biotechnology
applications. DNA cloning is fundamental to genetic engineering and the study of gene
function
11.
12. pBR322 plasmid
This was one of the first plasmids used for cloning recombinant DNA molecule
By inserting the desired fragment of DNA in coded gene, the gene
will be inactivated in process called insertional inactivation which can
be used as marker.
13.
14.
15.
16. Using Restriction enzyme to make a recombinant DNA plasmid
Gene cloning and genetic engineering generally rely on enzymes called restriction endonucleases, or
restriction enzymes.
-Restriction enzymes protect the bacterial cell by cutting up foreign DNA from other organisms or phages
-Each restriction enzyme is very specific, recognizing a particular short DNA sequence, or restriction site,
and cutting both DNA strands at precise points within this restriction site.
-The DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of
methyl groups (—CH3) to adenines or cytosines within the sequences recognized by the enzymes
17. -The most commonly used restriction enzymes recognize sequences containing four to eight
nucleotide pairs. Because any sequence that is this short usually occurs (by chance) many times
in a long DNA molecule, a restriction enzyme will make many cuts in such a DNA molecule,
yielding a set of restriction fragments.
- Since restriction enzymes always cut at the same exact DNA sequence, copies of any given
DNA molecule exposed to the same restriction enzyme always yield the same set of restriction
fragments. They work in 5’ to 3’ direction
- The most useful restriction enzymes cleave the sugar-phosphate backbones in the two DNA
strands in a staggered manner
18. - The resulting double-stranded restriction fragments have at least one single-stranded end, called
a sticky end. These short extensions can form hydrogen-bonded base pairs with complementary
sticky ends on any other DNA molecules cut with the same restriction enzyme
The associations formed in this way are only temporary but can be made permanent by DNA
ligase, an enzyme that catalyzes the formation of covalent bonds that close up the sugar-
phosphate backbones of DNA strands.
The end result, , is the formation of a stable recombinant plasmid containing foreign DNA
19.
20.
21.
22.
23. Brief definition of PCR
Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify and replicate
specific segments of DNA. It involves a series of temperature-controlled cycles that denature,
anneal, and extend DNA strands, resulting in the exponential amplification of the targeted DNA
region. PCR has become a fundamental tool in various applications, including DNA sequencing,
genetic testing, medical diagnostics, forensics, and research
PCR has had a revolutionary impact on DNA analysis and research by providing a rapid and
efficient method for amplifying specific DNA sequences. This breakthrough technology enables
scientists to produce millions of copies of a targeted DNA region, even from minute samples.
PCR's speed and precision have transformed genetic research, diagnostics, and forensic analysis,
significantly accelerating the pace of scientific discovery and opening new possibilities for
understanding and manipulating genetic information
27. Steps of PCR
Denaturation: Denaturation in the context of PCR refers to the separation of the two complementary strands of DNA.
During the denaturation step of PCR, the double-stranded DNA template is subjected to a high temperature (typically
around 94-98°C). At this elevated temperature, the hydrogen bonds between the complementary base pairs (adenine with
thymine, and guanine with cytosine) break, causing the DNA strands to separate. This creates single-stranded DNA
molecules, providing the template for the next steps in the PCR process. Denaturation is a crucial step as it allows the DNA
polymerase enzyme to access and replicate the target DNA sequence during the subsequent amplification cycles
Annealing: Annealing in PCR is a step where short DNA primers bind to their complementary sequences on the single-
stranded DNA template. This occurs at a specific, lower temperature after denaturation. The primers provide a starting
point for DNA synthesis, ensuring specificity and enabling the DNA polymerase to synthesize a new DNA strand during the
subsequent extension step. Annealing is a crucial phase in the PCR cycle, facilitating the amplification of the targeted DNA
sequence.
Extension:Extension in PCR is the stage where DNA polymerase synthesizes a new DNA strand complementary to the
single-stranded DNA template. This occurs at a higher temperature than annealing, typically around 72°C. The enzyme adds
nucleotides to the primer, elongating the DNA strand. This process replicates the target DNA sequence and completes one
cycle of the polymerase chain reaction. Extension ensures the exponential amplification of the desired DNA region,
contributing to the overall success of the PCR process.
28.
29. Types of PCR
- Standard PCR:
- Basic amplification of DNA.
- **Real-Time PCR (qPCR):**
- Continuous monitoring for real-time analysis.
- **Reverse Transcription PCR (RT-PCR):**
- Amplifying RNA to study gene expression.
- **Nested PCR:**
- Increased specificity with two rounds of amplification.
- **Multiplex PCR:**
- Simultaneous amplification of multiple targets.
- **Hot Start PCR:**
- Minimizing non-specific amplification.
30. Applications of PCR
DNA Sequencing:
DNA sequencing is a technique used to determine the exact order of nucleotides in a DNA molecule. It
involves preparing a DNA sample, initiating a sequencing reaction to synthesize complementary strands,
separating the fragments by size, and analyzing the order of labeled nucleotides. This process provides
valuable insights into genetic information, aiding research in genetics, medicine, and evolutionary biology.
Advances in sequencing technologies have significantly increased the speed and efficiency of this
fundamental molecular biology tool
Determining the order of nucleotides:
Molecular diagnostics involves the detection and analysis of specific genetic material, proteins, or other
molecular markers to diagnose diseases and conditions. In brief, this approach employs techniques such as
PCR, DNA sequencing, and other molecular assays to identify genetic variations, pathogens, or abnormal
molecular patterns associated with various disorders. Molecular diagnostics provides precise and rapid
diagnostic information, enabling personalized treatment strategies and improved disease management.
Applications include identifying genetic disorders, detecting infectious diseases, and assessing cancer
biomarkers, contributing to more accurate and targeted medical interventions
31. Detecting genetic disorders and infectious diseases:
PCR, a vital tool in forensic science, amplifies trace amounts of DNA from crime scene samples. After
extracting DNA, PCR selectively replicates specific regions, enabling the creation of a DNA profile. This
unique profile aids in the identification of individuals and facilitates the comparison of crime scene evidence
with known samples, contributing significantly to criminal investigations. PCR's sensitivity and accuracy
make it instrumental in analyzing even minute biological material, helping forensic experts link suspects to
crime scenes and establish crucial connections in criminal cases
Medical Diagnosis - Detecting Mutations and Disease Markers Using PCR:
PCR is pivotal in medical diagnosis, precisely detecting mutations and disease markers. Beginning with the
extraction of genetic material from patient samples, PCR selectively amplifies specific DNA regions
associated with the condition of interest. The amplified DNA is then analyzed, providing crucial insights for
healthcare professionals to confirm diagnoses, assess disease progression, and tailor personalized
treatment strategies based on an individual's genetic profile. PCR's accuracy and efficiency contribute
significantly to advancing diagnostic capabilities in the field of medicine.
32. Pharmacogenomics:
PCR, a key component in personalized medicine, amplifies specific DNA regions from patient samples. This
genetic profiling enables the identification of variations and markers associated with diseases. Utilizing this
information, healthcare professionals tailor treatment plans, selecting interventions based on an individual's
genetic makeup. PCR's role extends to disease prevention and monitoring treatment responses, offering a
personalized approach to healthcare that maximizes effectiveness and minimizes adverse effects
Studying Gene Mutations and Treatment Responses in Cancer Through PCR:
PCR is pivotal in cancer research, focusing on gene mutation analysis and treatment responses. By
amplifying specific DNA regions, PCR helps identify mutations in cancer-related genes, guiding personalized
treatment strategies. Additionally, PCR monitors changes in genetic markers during treatment, offering real-
time insights into how cancer cells respond to therapies. This precision contributes to tailored interventions,
improving our understanding of cancer biology and enhancing the effectiveness of cancer treatments
33. Made by:
Ahmed Afifi – Badr Mohamed – Mazen
Khaled – Ahmed Mohsen – Mahmoud
Adham
Under supervision
Ms.Noah