The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA through a series of temperature changes and primer hybridization. The basic PCR protocol involves repeated cycles of denaturation to separate DNA strands, annealing of primers to the target sequences, and extension of the primers by a DNA polymerase. This results in exponential amplification of the target DNA region. PCR has numerous applications including DNA analysis, gene cloning, disease diagnosis, and genetic fingerprinting.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
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
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
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
A detailed description about the basic steps involved in the - PCR - Polymerase Chain Reaction, its applications,its limitations and steps to overcome it.
Polymerase chain reaction (PCR) is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules. ... The mixture is then cooled (eg, 60-65°C) so that the primers anneal (bind) to the DNA template.
The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work.
this section helps students how to prepare master mix solution and how to pcr. specially life life science fields such as biotechnology, biology, and medical laboratory
Gene cloning and polymerase chain reaction Abhay jha
In these you are able to know about the gene cloning basic steps and Polymerase chain reaction process also there is an brief description about the ideal property shown by vectors which are lambda and M13 phases and there are lots of things in these slides
Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
A workshop hosted by the South African Journal of Science aimed at postgraduate students and early career researchers with little or no experience in writing and publishing journal articles.
The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
MATATAG CURRICULUM: ASSESSING THE READINESS OF ELEM. PUBLIC SCHOOL TEACHERS I...NelTorrente
In this research, it concludes that while the readiness of teachers in Caloocan City to implement the MATATAG Curriculum is generally positive, targeted efforts in professional development, resource distribution, support networks, and comprehensive preparation can address the existing gaps and ensure successful curriculum implementation.
Thinking of getting a dog? Be aware that breeds like Pit Bulls, Rottweilers, and German Shepherds can be loyal and dangerous. Proper training and socialization are crucial to preventing aggressive behaviors. Ensure safety by understanding their needs and always supervising interactions. Stay safe, and enjoy your furry friends!
4. History
One of the most powerful tools in
molecular biology
Invented by Kary Mullis in
1983, resulting in his Nobel
Prize in Chemistry
First published account appeared
in 1985.
Awarded Nobel Prize for
Chemistry in 1993.
4/28/2012 4
5. The reaction mixture
1. DNA (purified or a crude extract)
2. Primers specific for the target DNA
3. Free nucleotides (A, G, T, C)
4. DNA polymerase
5. Buffer (containing magnesium)
6. The reaction mixture
1- DNA template that contains the DNA region (target) to be
amplified.
2- One or more primers, which are complementary to the
DNA regions at the 5' (five prime) and 3' (three prime)
ends of the DNA region.
3- A DNA polymerase such as Taq polymerase or another
DNA polymerase with a temperature optimum at around
70 C.
4/28/2012 6
7. The reaction mixture
, (dNTPs) from which the
DNA polymerase builds the new DNA
, which provides a suitable chemical environment for
the DNA Polymerase
, or ions; generally
Mg2+ is used, but Mn2+ can be utilized for PCR-mediated
DNA mutagenesis, as higher Mn2+ concentration increases
the error rate during DNA synthesis
Monovalent cation ions.
4/28/2012 7
8. Primers
• On the other hand, the length of a primer is limited by:
the maximum temperature allowed to be applied in order to melt it,
as increases with the of the primer
that are too high, i.e., above , can cause problems:
since the is at such temperatures
• The of a primer is generally from 15 to 40 ,
with a between
calculating:
Tm =4(G+C)+2(A+T)
Software
9. Primers
• The DNA fragment to be amplified is determined by
selecting primers
• Primers are :
short, artificial DNA strands
often not more than 50 and usually only 18 to 25 base
pairs long
that are complementary to the beginning or the end of the
DNA fragment to be amplified
• They anneal by adhering to the DNA template at these
starting & ending points,
where the DNA polymerase binds and begins the
synthesis of the new DNA strand
10. Primer 3' terminus
• Primer 3' terminus design is critical to PCR success
since the primer extends from the 3' end
• The 3' end should not be complementary over greater
than 3-4 bases to any region of the other primer
(or even the same primer) used in the reaction
and must provide correct base matching to the
template
• There are computer programs to help design primers
Genrunner
13. The basic protocol
1. Denaturation of DNA to single
strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
14. Procedure
The PCR process usually consists of 20 -35
each cycle consists of :
1. The has to be heated to (or 98 C if extremely thermostable
polymerases are used)
in order to separate the strands
This step is called denaturing:
it breaks apart the that connect the two DNA strands
Prior to the first cycle:
the DNA is often denatured for an to ensure that the
and the ,
have completely separated and are now
usually , but up to minutes
Also certain polymerases at this step (
15. Procedure
2. After separating the DNA strands, the temperature is
so :
the primers can themselves to the
the temperature of this stage on the and is
usually their Tm (45-60 C)
A wrong temperature during the annealing step can result in :
primers not binding to the template DNA at all
or binding at random
Time: 1-2 minutes
16. Procedure
3. Finally, the DNA polymerase has to copy the DNA strands
It starts at the annealed primer and works its way along the DNA strand
this step is called elongation
the elongation temperature depends on the DNA polymerase:
Taq polymerase elongates optimally at a temperature of 72º C
• The time for this step depends:
1. both on the DNA polymerase itself
2. and on the length of the DNA fragment to be amplified
as a rule-of-thumb, this step takes 1 minute per 1000 bp
• A final elongation step is frequently used after the last cycle
to ensure that any remaining single stranded DNA is completely copied,
this differs from all other elongation steps, only in that it is
longer, typically 10-15 minutes
17. Primers
forward
5’ 3’
Target DNA
3’ 5’
reverse
26. One One billion in about 2 hours!
• At the end of each cycle, the amount of DNA
has doubled
• By the end of 30 cycles, you will have about 1
billion molecules from the original one you
started with!!
230=1,073,741,824
27. The basic protocol—what’s in the tube
5’ 3’
Target DNA
3’ 5’
A
B Free
primers nucleotides
Mg2+ Mg
2+
Buffer
Taq DNA Mg2+ containing
Mg2+
polymerase Mg2+ magnesium
Mg2+
28. 4/28/2012 Free Template from www.brainybetty.com 28
29. Optimising the PCR Reaction
C G Denaturation -
- Annealing -
Primer extension -
--
Ramp -
dNTP -
DNA Template DNA -
PCR -
-
Tm -
30. PCR optimization
1. For the preparation of reaction mixture, a laminar flow
cabinet with UV lamp is recommended
2. Fresh gloves should be used for each PCR step
3. As well as displacement pipettes with aerosol filters
4. The reagents for PCR should be prepared separately and used
solely for this purpose
5. Aliquots should be stored separately from other DNA
samples
6. A control reaction (inner control), omitting template
DNA, should always be performed, to confirm :
a. the absence of contamination
b. or primer multimer formation
31. Applications of PCR
1. the detection of hereditary diseases
2. the identification of genetic fingerprints
3. the diagnosis of infectious diseases
4. the cloning of genes
5. paternity testing
6. and DNA computing
32.
33. How It Works
• Heating/cooling
• Capillary surface area
Intake
• Single chamber
– holds 32 capillaries
• Photohybrids measure
fluorescence at
530, 640 and 705nm
41. The use of multiple, unique primer sets within a single PCR reaction ,
to produce amplicons of varying sizes specific to different DNA sequences
• By targeting multiple genes at once,
additional information may be elicited from a single test run that otherwise
:
would require several times the reagents and technician time to perform
• Annealing temperatures for each of the primer sets ,
must be optimized to work correctly within a single reaction
and amplicon sizes should be separated by enough difference,
in final base pair length to form distinct bands via gel electrophoresis
42. Multiplex PCR
• PCR reactions can be devised in which several
targets are amplified simultaneously often used
in diagnostic applications.
44. Nested PCR
is intended to reduce the contaminations in
products due to the amplification of
unexpected primer binding sites
• Two sets of primers are used in two successive
PCR runs
the second set intended to amplify a secondary
target within the first run product
• This is very successful, but requires more
detailed knowledge of the sequences involved
45. RT-PCR
RT-PCR (Reverse Transcription PCR) is the method
used to amplify, isolate or identify a known sequence
from a cell or tissues RNA library
• Essentially normal PCR preceded by transcription by
Reverse transcriptase (to convert the RNA to cDNA)
this is widely used in :
1. expression mapping, determining when and where
certain genes are expressed
2. detection of RNA viruses
46. Colony PCR
Bacterial clones (E.coli) can be screened for
the correct ligation products
• Selected colonies are picked with a sterile
toothpick from an agarose plate,
and dabbed into the master mix or sterile
water,
primers (and the master mix) are added
the PCR protocol has to be started with an
extended time at 95ºC
48. Uses of PCR
PCR can be used for a broad variety of experiments and analyses:
1. Genetic fingerprinting
• is a forensic technique used to identify a person by comparing his or
her DNA with a given sample
• An example is blood from a crime scene being genetically compared
to blood from a suspect
• The sample may contain only a tiny amount of DNA ,
(obtained from a source such as blood, semen, saliva, hair, or other
organic material)
49. Uses of PCR
2. Detection of hereditary diseases
• The detection of hereditary diseases in a given genome is a
long and difficult process, :
which can be shortened significantly by using PCR
• Each gene in question can easily be amplified through PCR by
using the appropriate primers :
and then sequenced to detect mutations
50. Uses of PCR
3. Viral diseases
• can be detected using PCR through amplification of
the viral DNA
• This analysis is possible right after infection,
which can be from several days to several months
before actual symptoms occur
• Such early diagnoses give physicians a significant
lead in treatment
• Treatment evaluation, viral load
• Genotyping, viruses, bacteria
51. Uses of PCR
4. Mutagenesis
• Mutations can be introduced into copied DNA sequences,
in two fundamentally different ways in the PCR process
• Site-directed mutagenesis allows the experimenter to introduce a mutation
at a specific location on the DNA strand
• Usually, the desired mutation is incorporated in the primers used for the
PCR program
• Random mutagenesis, is based on the use of error-prone polymerases in the
PCR process
the location and nature of the mutations cannot be controlled
• One application of random mutagenesis is :
to analyze structure-function relationships of a protein
• By randomly altering a DNA sequence:
one can compare the resulting protein ,
with the original and determine the function of each part of the protein
52. Uses of PCR
• 5. Genotyping of specific mutations
• Through the use of allele-specific PCR,
one can easily determine which allele of a mutation or
polymorphism an individual has
• Here, one of the two primers is common,
and would anneal a short distance away from the mutation,
while the other anneals right on the variation
• The 3' end of the allele-specific primer is modified,
to only anneal if it matches one of the alleles