Polymerase chain reaction (PCR) is a nucleic acid amplification technique used in diagnostic microbiology to rapidly detect pathogens. PCR works by thermally cycling DNA to exponentially amplify target sequences using DNA polymerase. It is highly sensitive and specific, allowing detection of pathogens that cannot be cultured. Real-time PCR further allows quantification by measuring amplification over cycles. PCR has revolutionized infectious disease diagnosis by enabling rapid, sensitive detection of various microbes from clinical samples.
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.
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.
MOLECULAR TOOLS IN DIAGNOSIS AND CHARACTERIZATION OF INFECTIOUS DISEASES tawheedshafi
The future of the molecular diagnostics of infectious diseases will undoubtedly be focused on a marked increase in the amount of information detected with remarkably simplified, rapid platforms that will need complex software analysis to resolve the data for use in clinical decision-making.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Richard's entangled aventures in wonderlandRichard 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.
MOLECULAR TOOLS IN DIAGNOSIS AND CHARACTERIZATION OF INFECTIOUS DISEASES tawheedshafi
The future of the molecular diagnostics of infectious diseases will undoubtedly be focused on a marked increase in the amount of information detected with remarkably simplified, rapid platforms that will need complex software analysis to resolve the data for use in clinical decision-making.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Richard's entangled aventures in wonderlandRichard 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.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
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.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
2. Requirements for an effective diagnosis of
Infectious diseases
Diagnostic tests that are rapid, reliable and highly
sensitive & specific
Direct cultivation of pathogen
Some pathogens are not cultivable
Cell lines suitable for virus culture
Development of immunoassay and DNA-based
diagnostic methods
3. Concept of DNA based methods
Each species of pathogen carries unique DNA or RNA
sequences that differentiates from other pathogens
These methods are highly sensitive and specific
Allows rapid detection of pathogens
Finds increasing use in diagnostic microbiology Lab
5. Polymerase Chain Reaction (PCR)
Thermally controlled, enzyme mediated, invitro
amplification of the target DNA, with the help of
Primers, deoxyribonucleotide triphosphates
(dNTPs) and buffers at optimal concentrations
6. Dr. Kary Mullis, wins Nobel Prize in 1993
Nobel Prize in chemistry
for his discovery of PCR
The process was
conceptualized in 1983
Considered as one of the
milestones among
scientific techniques of
the twentieth century
7. STEPS IN PCR
EXTRACTION OF DNA
AMPLIFICATION OF TARGET DNA
DETECTION OF THE AMPLICONS
13. Post Amplification Detection Methods
By agarose gel electrophoresis
Enzymatic detection of amplified product
(capture probe)
Reverse hybridization (oligonucleotide probe)
Sequencing of the amplicons
15. M Nce Pc 1 2 3 4 5
Detection of IS6110 gene in Mycobacterium tuberculosis
IC
IS6110
700
400
250
100
bp
16. Detection of PCR products by Capture Probes
Amount of signal is directly
proportional to the
amplicons present
E.g. viral load estimation
17. DNA sequencing
Common method for analyzing amplified product
Analysis of target generated amplicon – helps to
detect pathogen
Helps to detect mutations in viruses / bacteria
Genotyping of organism
18. TYPES OF PCR
Qualitative PCR
Quantitative PCR
Nested PCR
Multiplex PCR
Reverse transcriptase PCR
Real time PCR
19. TYPES OF PCR
QUALITATIVE PCR: To detect the presence or
absence of a specific gene
QUANTITATIVE PCR (qPCR): The amount of product
synthesized during a test PCR is compared with the
amounts synthesized during PCRs with known
quantities of starting DNA
Conventional method: Agarose gel electrophoresis
Real-Time PCR: Product is measured over time
20. NESTED PCR
•Two sets of primers
•First set of primer directed
against the target gene
•Second set of primers are
internal to the first set target
Types
Single step nested PCR
Two step nested PCR
Uses: HSV, CMV, Enterovirus
M.tuberculosis
21. Multiplex PCR
Multiple primer sets for simultaneous detection of several
targets within a single PCR reaction
Clinical application
To detect microorganism causing single type of disease
Meningitis
Diarrhoeagenic E. coli
Respiratory viruses
Virulence genes of STEC
Disadvantages
Sensitivity is decreased when compared to uniplex PCR
Primers may cross react
22. Multiplex PCR for the detection of
Shiga toxigenic Escherichia coli
kb
1000
700
400
200
hly
eae
stx1
stx2
M 1 2 3 4 Pc Nc
23. Reverse transcriptase PCR (RT-PCR)
RTase enzyme is used to synthesize DNA from RNA
Types
Single step RT PCR
Single termostable DNA polymerase that also possess
significant RTase activity is used
Two step RT PCR
Separate RTase enzyme is used prior to the addition of DNA
polymerase
Uses
detection of RNA viruses
detection of microbes – rRNA – viable
to study gene expression
quantitative RT PCR – viral load
24. REAL TIME PCR
Synthesis of the amplicons is measured over time,
as the PCR proceeds through its series of cycles
Amount of amplicons formed are directly
proportional to the target DNA in the starting
material
It enables both detection and quantification of a
specific DNA sequence in the sample
By using fluorescent dye or reporter molecule
25. REAL TIME PCR
Nonspecific detector system
SYBR Green gives fluorescent signal when it binds
to double-stranded DNA
This method measures the total amount of double-
stranded DNA in the PCR
Primer dimers
26. REAL TIME PCR - Specific detector system
Reporter probe gives fluorescent signal when it hybridizes to
the PCR product - Specific
Each reporter probe has pair of labels
A fluorescent dye at one end of the oligonucleotide and a
quenching compound to the other end
Two ends of oligonucleotides base pair to one another, placing
the quencher next to the dye – No fluorescence
Hybridization between the oligonucleotide and the PCR product
disrupts base pairing quencher moves away from the dye and
fluorescent signal generated
27. Uses of Real time PCR
Quantitation of gene expression
Pathogen detection
Viral quantitation
Array verification
Drug therapy efficacy
DNA damage measurement
Quality control and assay validation
Advantages of Real time PCR
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization
28. Application of PCR in Clinical Microbiology
Detection of slow-growing or fastidious microorganisms
Detection of infectious agents that cannot be cultured
Recognition of newly emerging pathogen
Detection of RNA viruses
Diagnosis of viral encephalitis
Identification of Mycobacterium spp.
Estimation of viral load to monitor therapy
Detection of bacterial DNA for the diagnosis of septic arthritis
and reactive arthritis
Identification of antimicrobial resistance genes
29. Advantages of PCR
Speedy diagnosis
Ease of use
Sensitivity
Specificity
30. Disadvantages of PCR
Need for target DNA sequence information
Primer Designing for unexplored ones
Taq Pol – no Proof reading mech – Error 40% after 20
cycles
Short size genes Up to 40 Kb can be amplified
Technical expertise