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.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
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.
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
there are s many methods are used in diagnosis of human gene mutation which occur disorders ,here u get information about the diagnostic method for genetic mutation detection
whole genome analysis
history
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human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
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The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
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.
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
Explore the Illumina workflow, including sequencing by synthesis (SBS) technology, in 3-dimensional detail. Go from sample preparation, to cluster generation, to sequencing on a system flow cell with the proprietary SBS process.
there are s many methods are used in diagnosis of human gene mutation which occur disorders ,here u get information about the diagnostic method for genetic mutation detection
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
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
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
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 (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment. This slides introduces pcr importances ,uses and steps of pcr.
All about polymerase chain reaction. detailed description and explanation of instrumention, procedure, advantages, disadvantages. Also types of RtPcr..
graphical representation. explained with appropriate figrues.
Struggling with intense fears that disrupt your life? At Renew Life Hypnosis, we offer specialized hypnosis to overcome fear. Phobias are exaggerated fears, often stemming from past traumas or learned behaviors. Hypnotherapy addresses these deep-seated fears by accessing the subconscious mind, helping you change your reactions to phobic triggers. Our expert therapists guide you into a state of deep relaxation, allowing you to transform your responses and reduce anxiety. Experience increased confidence and freedom from phobias with our personalized approach. Ready to live a fear-free life? Visit us at Renew Life Hypnosis..
Navigating Challenges: Mental Health, Legislation, and the Prison System in B...Guillermo Rivera
This conference will delve into the intricate intersections between mental health, legal frameworks, and the prison system in Bolivia. It aims to provide a comprehensive overview of the current challenges faced by mental health professionals working within the legislative and correctional landscapes. Topics of discussion will include the prevalence and impact of mental health issues among the incarcerated population, the effectiveness of existing mental health policies and legislation, and potential reforms to enhance the mental health support system within prisons.
QA Paediatric dentistry department, Hospital Melaka 2020Azreen Aj
QA study - To improve the 6th monthly recall rate post-comprehensive dental treatment under general anaesthesia in paediatric dentistry department, Hospital Melaka
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
3. Polymerase Chain Reaction
(PCR)
Technique widely used in molecular biology to make
multiple copies of a specific DNA segment.
In vitro technique for amplification of a region of DNA
whose sequence is known or which lies between two
regions of known sequence.
4. Short history of PCR
1983: Dr. Kary Mullis developed PCR
1985: First publication of PCR by Cetus Corporation
appears in Science.
1986: Purified Taq polymerase first used in PCR
1988: PerkinElmer introduces the automated thermal
cycler.
1993: Dr. Kary Mullis shares Nobel Prize in Chemistry
for conceiving PCR technology.
5. Application
PCR is molecular technique to amplify segment of
DNA
Used in clinical and research laboratories for a
broad range of applications
◦ Diagnosis of genetic diseases
◦ Genetic fingerprints
◦ Detection and diagnosis of infectious diseases
◦ Detection of infection in the environment
6. Principles of PCR
Based on DNA replication in vivo
DNA is unwound to single strand, duplicated,
rewound
Amplify DNA in short period of time
Amplify DNA fragment between 0.1 to 10kbp
Some allow to amplify up to 40 kbp
7. Basic requirements for PCR
reaction
A DNA template: DNA target region to amplify.
Size of template can be <0.1 to few kilobase
Total amount of DNA for PCR is 0.05-0.1ug
A DNA polymerase: An enzyme that polymerizes new
DNA strands; heat-resistant Taq polymerase is
especially common, as it is more likely to remain intact
during the high-temperature DNA denaturation process.
Primers:
16-30 nucleotides long primers are used
Complementary to the 3' ends of each of the sense and
anti-sense strands of the DNA target;
without primers there is no double-stranded initiation
site at which the polymerase can bind
8. Cont…
Deoxynucleotide triphosphates (dNTPs): Building
blocks to synthesizes a new DNA strand.
Reaction Buffer: Providing a suitable chemical
environment for optimum activity and stability of the
DNA polymerase.
Bivalent cations: Mg++
Mg ion stimulate polymerase activity
Increase melting temperature of primer
Monovalent cations: Potassium (K) ions
9. PCR COMPONENTS
Water
10x reaction buffer
MgCl2
dNTPs
Target DNA
Forward primer
Reverse primer
Polymerase enzyme
10. Dream Taq Green PCR Master
Mix
Optimum PCR components are
directly used in form of mastermix
Contain
1. Dream Taq DNA polymerase
2. Optimized Dream Taq Green buffer
3. MgCl2
4. dNTPs
Higher yields compare to conventional
Taq DNA polymerase
11. Significance of master mix
Direct loading of PCR prodcuts on gel
High yield and high sensitivity of PCR
Amplification of long targets upto 6kb
from genomic DNA and up to 20 kb
from viral DNA
Incorporate modified nucleotides, but
doesnot incorporate dUTP
12. Procedure of PCR
Carried in reaction volume of 10-200ul
in small tubes called PCR tubes
PCR tubes 0.2-0.5mL
Placed in thermocycler
13. Main steps
Consists of series of 20-40 repeated temperature
changes called cycles
If 100% efficiency is assumed in each cycle there
will be 220 fold amplification after 20 cycle of pcr
14. Steps in PCR
Steps Temp. Duration
Denaturation 92°C to 95°C 1min
Annealing 50°C to 55°C 45sec
Elongation 70°C to 75°C 1-2min
20. Visualization of PCR products
Gel electrophoresis is employed to
check pcr products
Visualized under x-rays
Size is determined by comparing with
ladder.
21. Types of PCR
Allele-specific PCR
Assembly PCR
Asymmetric PCR
Convective PCR
Dial-out PCR
Digital PCR (d PCR)
Hot start PCR
In silico PCR (digital PCR, virtual PCR, electronic
PCR, e-PCR)
23. Allele-specific PCR
A PCR application in which alleles that differ by
one or more nucleotides can be distinguished on
the basis of PCR amplification.
It permits the detection of any mutation in human
DNA by analysing the PCR products directly in an
ethidium bromide-stained agarose or
polyacrylamide gel.
Used in DNA-based diagnostic techniques
involving the diagnosis of genetic and infectious
diseases.
24. Hot start PCR
Modified form of PCR 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
denaturation temperature (e.g., 95 °C)
before adding the polymerase.
25. 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.
Annealing temperatures for each of the
primer sets must be optimized to work
correctly within a single reaction.
26. Nested PCR
Increases the specificity of DNA amplification, by
reducing background due to non-specific amplification
of DNA.
Two sets 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.
27. Cont…
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.
28. Quantitative PCR (qPCR)/Real
Time PCR
Used to measure the quantity of a target sequence
(commonly in real-time).
It quantitatively measures starting amounts of DNA,
cDNA, or RNA. quantitative 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 PCR has a very high degree of
precision.
29. Reverse transcription PCR
(RT-PCR)
It is used 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 RNA transcript.
30. 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 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
33. Advantages of PCR
Fairly simple to understand and to use.
Produce Automated, fast, reliable results.
Highly sensitive.
Potential to produce millions to billions of copies
of a specific product for sequencing, cloning, and
analysis.
Broad uses.
Defined, easy to follow protocol.
34. Limitations of PCR
Smallest amount of contaminated DNA can be
amplified, resulting in misleading or ambiguous
results.
Need for target DNA sequence information
Boundary regions of DNA to be amplified must be
known.
Infidelity of DNA replication: Taq Pol – no Proof
reading– Error 40% after 20 cycles
35. Cont…
Short size and limiting amounts of
PCR product
Up to 5kb can be easily amplified .
Up to 40kb can be amplified with some
modifications.
Cannot amplify gene >100kb
Cannot be used in genome sequencing projects.
36. Things to try if PCR does not
work
A) If no product ( of correct size )
produced:
Check DNA quality.
Reduce annealing temperature.
Increase magnesium concentration.
Add dimethyl sulphoxide ( DMSO ) to assay ( at
around 10% ).
Use different thermostable enzyme.
Throw out primers - make new stocks.
37. Cont…
B) If extra spurious product bands
present:
Increase annealing temperature
Reduce magnesium concentration
Reduce number of cycles
Try different enzyme
38. Applications of PCR
Common applications of PCR in various fields can
be explained in following categories:
Medical Applications
Infectious disease Applications
Forensic Applications
Research and Molecular Genetics
39. Forensic applications
Can be used as a tool in genetic fingerprinting.
This technology can identify any one person from
millions of others in case of:
•crime scene.
•rule out suspects during police
investigation.
•paternity testing even in case of availability
of very small amount of specimens (stains of
blood, semen, hair etc.).
40. Research and molecular genetics
In genomic studies: to compare the genomes of
two organisms and identify the difference between
them.
In phylogenetic analysis: minute quantities of
DNA from any source like a fossilized material, hair,
bones, mummified tissues.
In study of gene expression analysis, PCR
based mutagenesis.
In Human genome project for aim to complete
mapping and understanding of all genes of human
beings.
41. Conclusion
PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD
detection, but it is also important for genetic
predisposing for defects.
The PCR technology can also be employed in
law enforcement, genetic testing of animal
stocks and vegetable hybrids, and drug
screening along with many more areas.
42. References
Molecular Cell Biology ( Lodish, Darnell..)
https://sciencebasedmedicine.org/wp-
content/uploads/2011/09/nested-pcr.gif
http://11e.devbio.com/ch/03/wt/031005/figure1.
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https://www.thermofisher.com/pk/en/home/bran
ds/thermo-scientific/molecular-
biology/molecular-biology-learning-
center/molecular-biology-resource-
library/basic-principles-rt-qpcr.html
Paul, N. (2010). Hot start PCR. Methods in
Molecular Biology. 630. pp. 301–318
Fundamentals of Biochem ( Voet, Voet, Pratt)