PCR is a technique used to amplify a targeted region of DNA across multiple orders of magnitude. It involves repeated cycles of heating and cooling of the DNA sample to denature the DNA strands, allow primers to anneal to the target region, and extend the primers using a DNA polymerase. Key aspects of PCR include using primers that flank the target region, a thermostable DNA polymerase like Taq polymerase, and thermal cycling to facilitate strand separation and copying. Real-time PCR allows for quantitative analysis of the amplified DNA and has advantages over traditional PCR like speed, sensitivity, and quantification ability.
The power point presentation consists of 36 slides explaining about history, principle, different steps involved and applications of DNA fingerprinting. Recent Developments and the Future prospects of DNA profiling have also been mentioned
The power point presentation consists of 36 slides explaining about history, principle, different steps involved and applications of DNA fingerprinting. Recent Developments and the Future prospects of DNA profiling have also been mentioned
There isn't one single person credited with discovering the mitochondria, as over the years a number of scientists have made important contributions to the study of the discovery of this important cellular structure:
1800s In 1857, Albert von Kölliker described what he called “granules” in the cells of muscles.
- Other scientists of the era also noticed these “granules” in other cell types.
1886 , when Richard Altman, a cytologist, identified the organelles using a dye technique, and dubbed them “bioblasts.” He postulated that the structures were the basic units of cellular activity.
1898, Carl Benda coined the term mitochondria. He derived the term from the Greek language for the words thread, mitos, and granule, chondros.
-Though mitochondria are an integral part of the cell, evidence shows that they evolved from primitive bacteria.
Ion torrent semiconductor sequencing technologyCD Genomics
Ion Torrent is the latest generation sequencing technology. Its core technology is the use of semiconductor technology in chemical and digital information to establish a direct link.
Telomere is the end part of a chromosome.its length is maintained by na enzyme called telomerase.if telomerase is lacking,many genetic diseases may result( like progeria)
There isn't one single person credited with discovering the mitochondria, as over the years a number of scientists have made important contributions to the study of the discovery of this important cellular structure:
1800s In 1857, Albert von Kölliker described what he called “granules” in the cells of muscles.
- Other scientists of the era also noticed these “granules” in other cell types.
1886 , when Richard Altman, a cytologist, identified the organelles using a dye technique, and dubbed them “bioblasts.” He postulated that the structures were the basic units of cellular activity.
1898, Carl Benda coined the term mitochondria. He derived the term from the Greek language for the words thread, mitos, and granule, chondros.
-Though mitochondria are an integral part of the cell, evidence shows that they evolved from primitive bacteria.
Ion torrent semiconductor sequencing technologyCD Genomics
Ion Torrent is the latest generation sequencing technology. Its core technology is the use of semiconductor technology in chemical and digital information to establish a direct link.
Telomere is the end part of a chromosome.its length is maintained by na enzyme called telomerase.if telomerase is lacking,many genetic diseases may result( like progeria)
The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific detection and production of large amounts of DNA. PCR-based strategies have propelled huge scientific endeavors such as the Human Genome Project. The technique is currently widely used by clinicians and researchers to diagnose diseases, clone and sequence genes, and carry out sophisticated quantitative and genomic studies in a rapid and very sensitive manner. One of the most important medical applications of the classical PCR method is the detection of pathogens. In addition, the PCR assay is used in forensic medicine to identify criminals. Because of its widespread use, it is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome
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
PCR- Steps;Applications and types of PCR (Exam point of view)Sijo A
The term PCR stands for Polymerase Chain Reaction.
It is an invitro amplification technique that allows synthesizing millions of copies of the DNA or gene of interest from a single copy.
It is called “Polymerase” because the only enzyme used in this reaction is DNA polymerase.
The PCR is invented by Kary Mullis in 1985.He received Nobel Prize in Chemistry in 1993.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
2. Polymerase Chain Reaction Methodology
A Milestone in Biomedical History
He had the idea to use a
pair of primers to bracket
the desired DNA sequence
and to copy it using DNA
polymerase, a technique
which would allow a small
strand of DNA to be
copied almost an infinite
number of times. Cetus
took Mullis off his usual
projects to concentrate on
PCR full-time
3. How DNA Works
DNA usually exists as a
double-stranded structure,
with both strands coiled
together to form the
characteristic double-helix.
Each single strand of DNA
is a chain of four types of
nucleotides: adenine,
cytosine, guanine, and
thymine.
4. DNA – RNA - DNA
In Molecular biology, the
polymerase chain
reaction (PCR) is a
technique to amplify a
single or few copies of a
piece of DNA across several
orders of magnitude,
generating millions or more
copies of a particular DNA
sequence.
5. Create primers
To begin synthesis of a
new strand, a short
fragment of DNA or
RNA, called a primer,
must be created and
paired with the template
strand before DNA
polymerase can
synthesize new DNA.
7. A Thermocycler is the
Backbone of PCR methodology
The method relies on
thermal cycling,
consisting of cycles of
repeated heating and
cooling of the reaction
for DNA melting and
enzymatic replication of
the DNA
8. Primers
Primers (short DNA
fragments) containing
sequences complementary
to the target region along
with a Primers after which
the method is named) are
key components to enable
selective and repeated
amplification.
9. PCR a Chain reaction
As PCR progresses, the
DNA generated is itself
used as a template for
replication, setting in motion
a chain reaction in which
the DNA template is
exponentially amplified.
PCR can be extensively
modified to perform a wide
array of genetic
manipulations.
11. Thermus aquaticus is a bacterium that lives in
hot springs and hydrothermal vents, and Taq
polymerase was identified as an enzyme able
to withstand the protein-denaturing conditions
(high temperature) required during PCR. It
replaced the DNA polymerase from E.coli
originally used in PCR . Taq's optimum
temperature for activity is 75-80°C, with a
halflife of 9 minutes at 97.5°C, and can
replicate a 1000 base pair strand of DNA in
less than 10 seconds at 72°C.
Taq polymerase fuels the
Reaction
12. Taq polymerase is a thermostable DNA
polymerase named after the
thermophilic bacterium Thermus
aquaticus from which it was originally
isolated by Thomas D. Brock in 1965. It
is often abbreviated to "Taq Pol" (or
simply "Taq"), and is frequently used in
polymerase chain reaction (PCR),
methods for greatly amplifying short
segments of DNA
Taq polymerase
13. One of Taq's drawbacks is its relatively
low replication fidelity. It lacks a 3' to 5'
exonuclease proofreading activity, and
has an error rate measured at about 1
in 9,000 nucleotides. Some thermos
table DNA polymerases have been
isolated from other thermophilic bacteria
and archaea, such as Pfu DNA
polymerase, possessing a proofreading
activity, and are being used instead of
(or in combination with) Taq for high-
fidelity amplification.
Drawbacks of Taq
polymerase
14. Taq Pol lacks 3’ to 5’ exonuclease proof reading
activity, commonly present in other polymerases.
Taq mis-incorporates 1 base in 104.
A 400 bp target will contain an error in 33% of
molecules after 20 cycles.
Error distribution will be random.
Disadvantages of
Taq Pol
16. What does PCR need?
• Template (the DNA you are exploring)
• Sequence-specific primers flanking the
target sequence, Forward & Reverse.
• Polymerases
• Nucleotides (dATP, dCTP, dGTP,
dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer
• Water, mineral oil
17. Create primers
To begin synthesis of a
new strand, a short
fragment of DNA or
RNA, called a primer,
must be created and
paired with the template
strand before DNA
polymerase can
synthesize new DNA.
20. Denaturation 93 to 95°C 1min
Annealing 50 to 55°C 45sec
Elongation 70 to 75°C 1-2min
Steps in PCR
21. How does PCR work?
• Heat (94oC) to denature DNA strands
• Cool (54oC) to anneal primers to template
• Warm (72oC) to activateTaq Polymerase,
which extends primers and replicates DNA
• Repeat multiple cycles
26. The target product is
made in the third cycle
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
5’
3’
5’
3’
5’
3’
Cycle 1
Cycle 2
Cycle 3
3’
3’
3’
3’
5’
5’
5’
5’
27. Denaturation: 94°- 95°C
Primer Annealing: 55°- 65°C
Elongation of DNA: 72°
Number of Cycles: 25-40
No target products are made until the third cycle.
At 30 cycles there are 1,073,741,764 target
copies (~1×109).
PCR Cycles Review
28. Primers can have self-annealing regions within each primer (i.e. hairpin and
fold back loops)
A primer may be self-complementary and be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
The 3´ end of the primer is base-paired,
preventing it annealing to the target DNA.
Primers That Form
Hairpins
30. Need for target DNA sequence information
Primer Designing for unexplored ones.
Boundary regions of DNA to be amplified must be known.
Infidelity of DNA replication.
Taq Pol – no Proof reading mech – Error 40% after 20
cycles
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.
Limitations of PCR
31. • Pfu DNA Polymerase from Pyrococcus
furiosus possesses 3' to 5' exonuclease
proofreading activity.
• The error rate is only 3.5% after 20 cycles
• More amount of primer is added to avoid
primer dimering.
• For unexplored genes, primers used in closely
related species are used.
How to overcome
Difficulties?
33. The Real Time assays are proving to better
technologies
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization
Now a new gold standard for rapid diagnosis of
virus infection in the acute phase samples.
New Technologies – Real
Time Assays
34. Real-time polymerase chain reaction, also called
quantitative real time polymerase chain reaction (Q-
PCR/qPCR) or kinetic polymerase chain reaction, is a
laboratory technique based on the polymerase chain
reaction, which is used to amplify and simultaneously
quantify a targeted DNA molecule. It enables both detection
and quantification (as absolute number of copies or relative
amount when normalized to DNA input or additional
normalizing genes) of a specific sequence in a DNA sample.
Real-time polymerase
(qPCR) chain reaction
35. All real time PCR systems rely upon the
detection and quantization of fluorescent
reporter, the signal of which increases in direct
proportion of the amount of PCR product in a
reaction.
Real Time Reporters
36. qPCR Syber Green
USING SYBER®
GREEN
The simplest and economical
format the reporter is the
double strand DNA specific
dye SYBR ® Green
Called as Molecular Probe.
37. How SYBR Green
works
SYBR green binds to
double stranded DNA
and upon excitation
emits light
Thus as PCR product
accumulates the
fluoresce increases
39. Other Alternatives
Two most popular
alternatives to SYBR green
are TaqMan® and
Molecular Beacons.
Both technologies depend
on hybridization probes
relying on fluorescence
resonance energy transfer.(
FRET) and quantization
43. Documentation of
Amplification
The light emitted from
the dye in the excited
state is received by a
computer and shown on
a graph display, such as
this, showing PCR
cycles on the X-axis
and a logarithmic
indication of intensity on
the Y-axis.
45. Molecular Beacons –
qPCR
Molecular beacons are designed
to adopt a hairpin structure while
free in solution, brining the
fluorescent dye and quencher in
close proximity. When a molecular
beacon hybridizes to a target the
fluorescent dye emits light upon
irradiation, and rebind to target in
every cycle for signal
measurement.
46. qPCR Method
Molecular Beacons
Molecular beacons are
short segments of
single-stranded DNA .
The sequence of each
molecular beacon must
be customized to detect
the PCR product of
interest.
47. Quantitation of gene expression
Pathogen detection
Viral quantitation
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Genotyping
qPCR applications
48. Reverse transcription polymerase chain reaction
(RT-PCR) is a variant of polymerase chain reaction
(PCR), commonly used in molecular biology to
generate many copies of a DNA sequence, a
process termed "amplification". In RT-PCR,
however, RNA strand is first reverse transcribed into
its DNA complement using the enzyme reverse
transcriptase, and the resulting cDNA is amplified
using traditional or real-time PCR..
Reverse transcription
polymerase chain reaction
(RT-PCR)
49. Not to be confused with
qPCR
Reverse transcription
PCR is not to be
confused with real-time
polymerase chain
reaction (qPCR), which
is also sometimes
(incorrectly) abbreviated
as RT-PCR.
50. Multiplex PCR
TaqMan probes and
Molecular beacons
allow multiple DNA
species to be measured
in the same sample (
Multiplex PCR) since
fluorescent dyes with
different emission
spectra may be
attached to different
probes
51. Several viral infections can be detected in acute phase
serum samples.
Increasing used in for early and accurate detection of
all most human viruses including
Measles, Mumps, Herpes simplex
viruses, Rota viruses Noro
virus,Influenzae virus type A and B,
Respiratory Syncitical virus, SARS,
Dengue Japanese Encephalitis,
Hepatitis B and C, West Nile,
Chikungunya,HIV, Avian flu virus,
Uses of Automated
qPCR
52. Multiplex PCR in Real Time
Multiplex real time
quantitative RT-PCR
assays have been
developed for simultaneous
detection identification and
quantification of HBV, HCV
and HIV-1 In plasma and
Serum samples.
53. Directly
Sybr green
Quality of primers critical
Indirectly
In addition to primers, add a
fluorescently labeled
hybridization probe
How are you going to
measure the PCR product
54. Negative control (no DNA)
checks reagents for contamination
No reverse transcriptase control
detects if signal from contaminating DNA
Positive control
checks that reagents and primers work
especially importance if trying to show absence of
expression of a gene
Importance of
controls
55. Same copy number in all cells
Expressed in all cells
Medium copy number advantageous
correction more accurate
Reasonably large introns
No pseudo gene
No alternate splicing in region you want to PCR
Standards
56. Loop mediated isothermal amplification is a simple,
rapid, specific and cost effective nucleic acid
amplification method characterized by use of 8
distinct regions on the target gene.
The amplification proceeds at a constant
temperature using strand displacement reaction.
Loop Mediated Isothermal
Amplification (LAMP)
57. LAMP
Amplification and detection
of gene can be completed in
a single step, by incubating
the mixture of samples,
DNA polymerase, primers
with strand displacement
activity and substrates at a
constant temperature of
630c.
58. LAMP (Loop mediated isothermal
amplification)
Originally reported by Notomi et al in 2000 of EIKEN Chemical Co. Ltd.,
Japan
(http://www.eiken.co.jp/en/)
As of 17th April 2013, PubMed database has listed more than 820 articles
on this topic
59. Bst DNA polymerase with strand displacement
activity at 65℃
The whole amplification to 109 – 1010 copies is
finished within 15 to 60 min at 65℃, isothermally
Amplification and detection of gene can be
completed in a single step
No need for a step to denature double stranded
into a single stranded form
60. The amplification efficiency is extremely high
Reduced total cost- not require special reagents
or sophisticated equipments
Amplified products have a structure consisting
of alternately inverted repeats of the target
sequence on the same strand
Amplification can be done with RNA templates
following the same procedure as with DNA
templates, simply through the addition of
reverse transcriptase (RT-LAMP)
61. LAMP
Compared with PCR, and
real time PCR, the LAMP
has advantages of reaction
simplicity and detection
sensitivity.
The higher sensitivity and
specificity of LAMP reaction
is attributed to continuous
amplification under
isothermal condition
employing six primers
recognizing eight distinct
regions of the target.
62. LAMP functions on isothermal amplification.
LAMP does not require any thermal cycler and thus
can be performed even with water bath/heating block
LAMP method does not require sophisticated
temperature control devices
Cost effective
Advantages of LAMP
63. Continued…
2- Amplification
Is of two types;
A. Non-cycling Amplification
B. Cycling Amplification
A. Non-cycling Amplification:
Generation of stem loop DNA with dumbbell-shaped structure at
both ends.
B. Cycling Amplification:
Dumbbell-shaped DNA is quickly amplified by the use of loop
primers.
66. Continued…
3- Detection
i. Visual Detection
Turbidity - Magnesium pyrophosphate
Fluorescence – Calcein
ii. Gel Electrophoresis
Lane 1 and 3 has target DNA.
Lane 2 and 4 has non-target DNA.
Lane M has DNA Ladder.
67. { {
LAMP vs. PCR
Isothermal Reaction.
Isothermal Temperature (60-
65⁰C).
Doesn’t require expensive
thermocycler.
Detection limit is greater.
Amplification specificity is
higher as uses 4/6
oligonucleotides.
Visualization of DNA could be
done through eyes, gel
electrophoresis and
turbidimeter.
Cyclic Reaction.
Variable Temperature.
Denaturation (95⁰C)
Annealing (50-60⁰C)
Polymerization (72⁰C)
• Require thermocycler.
• Detection limit is lower.
• Amplification specificity is
lower than that of LAMP.
• Visualization of DNA is done
through gel electrophoresis.
Loop-mediated Isothermal
Amplification - LAMP
Polymerase Chain Reaction - PCR
68. { {
Could be done using crude
DNA samples.
Loop primers accelerate
reaction rate.
Need pure DNA samples
for amplification.
No loop primer.
LAMP vs. PCR
Loop-mediated Isothermal
Amplification - LAMP
Polymerase Chain Reaction - PCR
69. Lesser False Positives in
LAMP
In LAMP both amplification
and detection occur
simultaneously during the
exponential phase without
going through the plateau
phase where the non
spurious amplification leads
to lower sensitivity and false
positivity.
70. Loop Mediated Isothermal
Amplification in Clinical Diagnosis
LAMP technology proving to
be ideal in detection of DNA
or RNA of the pathogenic
organisms
Proving to be highly efficient
in diagnosis of Viral and
Bacterial infections,
LAMP is capable of
detecting the presence of
pathogenic agents earlier
than PCR
71. LAMP proving an efficient
Technology
A one step single tube real time
accelerated reverse transcription
loop mediated isothermal
amplification (RT-LAMP) assays
for rapid detection of some
recently emerged viral pathogen
eg West Nile, SARS, Dengue,
Japanese encephalitis
Chikungunya Norwalk, H5N1
highly pathogenic avian
influenza., and CMV,HPV,VZV
73. One Step RT-PCR Kit
The QIAGEN One Step
RT-PCR Kit is designed
for easy and sensitive
one-step RT-PCR using
any RNA template. A
unique enzyme
combination and specially
developed reaction buffer
ensure efficient reverse
transcription and PCR in
one tube.
74. RT-PCR in one step
The Robus™ T I Kit is base
RobusT RT-PCR Kits
perform cDNA
synthesis and PCR
amplification of cDNA
successively in a
single tube during a
continuous thermal
cycling
75. Clinical diagnostics: detection and
quantification of infectious microorganisms,
cancer cells and genetic disorders
Capable of amplifying long targets, up to 6.0 kb
One-tube system allows rapid, sensitive and
reproducible analysis of RNA with minimal risk
of sample contamination
Amplifies products from a wide variety of total
RNA or mRNA sources
Uses and Advantages in
Testing by PCR Methods
76. Advantages
Molecular methods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&D is absolutely necessary
77. Advantages
Molecular methods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
R&D is absolutely necessary
79. ESTABLISHMENT OF A PCR
LABORATORY
To perform PCR for the
repetitive detection of a
specific sequence, three
distinct laboratory areas
are required. The
specific technical
operations, reagents
,and personnel
considerations
80. PCR contamination be considered as a form of
infection. If standard sterile techniques that
would be applied to tissue culture or
microbiological manipulations are applied to PCR,
then the risk of contamination will be greatly
reduced. Above all else, common sense should
prevail.
Prevention of
Contamination in PCR
Laboratory
81. The single most important source of PCR
product contamination is the generation of
aerosols of PCR amplicons that is associated
with the post-PCR analysis. Methods for
eliminating this aerosol range from physical
design of laboratories and use of specific
pipettes to chemical and enzymatic
approaches. The choice of method is often
dependent on the frequency of amplification of
a target amplicon and the relative amounts and
concentrations of the amplicons created by the
PCR.
Avoiding
contamination
82. PCR laboratory
Sample handling
DNA preparation
Clean room
Stock solutions
Laboratory
Mixing site
Thermocycler
Amplification
Detection
Documentation
QC & QA
Quality control & assurance
R & D
(Research and development)
Alternatives: - commercial kits
- robots + kits
No alternative
85. Contamination
PCR allows the
production of more than
10 million copies of a
target DNA sequence from
only a few molecules of
DNA. The sensitivity of
PCR means that the
sample used for PCR
should not be
contaminated with any
other DNA’s that may
reside in the laboratory
environment.
86. PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA in
infectious diseases, but it is also important
for genetic predisposing for defects in
Genetic disorders.
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.
Conclusion