It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
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.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
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.
Porphyromonas gingivalis belongs to the phylum Bacteroidetes and is a nonmotile, Gram-negative, rod-shaped, anaerobic, pathogenic bacterium. It forms black colonies on blood agar.
It is found in the oral cavity, where it is implicated in certain forms of periodontal disease, as well as in the upper gastrointestinal tract, the respiratory tract, and the colon. It has also been isolated from women with bacterial vaginosis. Collagen degradation observed in chronic periodontal disease results in part from the collagenase enzymes of this species. It has been shown in an in vitro study that P. gingivalis can invade human gingival fibroblasts and can survive in them in the presence of considerable concentrations of antibiotics.P. gingivalis also invades gingival epithelial cells in high numbers, in which cases both bacteria and epithelial cells survive for extended periods of time. High levels of specific antibodies can be detected in patients harboring P. gingivalis. Dr Harshavardhan Patwal , explains the various enzymes enzyme peptidyl-arginine deiminase, which is involved in citrullination.[4] Patients with rheumatoid arthritis have an increased incidence of periodontal disease, and antibodies against the bacterium are significantly more common in these patients.
P. gingivalis is divided into K-serotypes based upon capsular antigenicity of the various types.
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
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.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
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 .
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.
2. INTRODUCTION
• PCR: Technique for in vitro (test tube) amplification of specific DNA sequences via
the temperature mediated DNA polymerase enzyme by simultaneous primer
extension of complementary strands of DNA.
• PCR: This system for DNA replication that allows a "target" DNA sequence to be
selectively amplified, several million-fold in just few hours.
3. • Coping Machine for DNA Molecule
• Invented by Kary Mullis and his colleagues in the 1983
4. DEFINITION
• It is a genetic technique that occurs in vitro which allows the enzymatic synthesis of
large quantities(amplification)of a targeted region of DNA .
• The DNA is synthesized in the same manner as that seen in vivo (in the cells ) using
a DNA polymerase (the enzymes that cells use to replicate their DNA).
5. PRINCIPLE OF PCR
• The PCR technique copies the target DNA by performing repeated cycles each
containing the following three main steps :
• 1- A denaturation or melting step to separate the two strands of DNA, this step
requires very high temp 95C for 10-20 seconds.
• 2-The Annealing step, allowing the primers to bind to the complementary sequences
on the template DNA, this step requires the temp to be dropped to 50-60 C.
• 3- The Elongation step, once the primers are bound to the template the synthesis of
DNA can start, the temperature should be increased to 70c which is the optimum
temperature for the polymerase enzyme.
6. PCR REACTION COMPONENTS
• DNA template
• Two primers
• Four normal deoxynucleosides triphosphates
• Buffer system
• DNA polymerase I
7.
8. • DNA template: is the DNA molecules that contains the DNA region (segment) to be
amplified, the segment that we are concered with is the target sequence .
• Two primers: a short segment of DNA that are complementary to the ends of each of
the sense and anti-sense strand of the DNA target, they are needed to get DNA
synthesis started .
• Deoxynucleoside triphosphates: the building blocks from which the DNA
polymerases synthesizes a new DNA strand .
• Buffer solution, providing a suitable chemical environment for optimum activity and
stability of the DNA polymerase .
• Taq polymerase: is the enzyme to manufacture the DNA copies. The PCR involves a
couple of high temperature steps so we use a heat resistant DNA polymerase, this is
extracted from heat resistant bacteria living in a hot springs at temperature up to
80°C, or another DNA polymerase with a temperature optimum at around 70 °C.
10. DENATURATION
• Denaturation: During the denaturation step, the reaction cocktail (reaction mixture)
is exposed to high temperature, usually 94-95°C for 20 secs. This high temperature
will denature the DNA—meaning the hydrogen bond between the two
complementary strands melt, unraveling the DNA molecule and exposing the
nucleotide bases. The high temperature of the denaturing step has the added
advantage of denaturing proteins (inactivating them) and disrupting cells so that
you don’t have to always start with purified DNA as your amplification template.
You can often amplify DNA directly from cell lysates—or even whole cells.
11. PRIMER ANNEALING
• During the second step of each cycle, the temperature is lowered to an annealing
temperature (50°-60° C), allowing binding (annealing) of the primers to their
complementary targets on the DNA template (one for each DNA strand). These are
designed to flank the desired target region of your DNA template and serve as the
starting points for DNA synthesis by the Taq polymerase. Each pair of primers will
have a particular annealing temperature determined by the length of the primers.
Using the proper annealing temperature for your primer set is essential for efficient
and accurate amplification.
12. EXTENSION
• The reaction cocktail is now brought to the optimum reaction temperature for Taq
polymerase (65 to 85°C). During this step, the Taq will bind to each DNA strand
• and “extend” from the priming sites (add nucleotides to synthesize a complementary
strand of the targeted DNA).
13.
14. APPLICATIONS OF PCR
• GENETIC TESTING:- Where a sample of DNA is analyzed for the presence of
genetic disease mutations
• PREIMPLANTATION GENETIC DIAGNOSIS:- Where individual cells of a
developing embryo are tested for mutations
• PCR can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation
15. • Characterization and detection of infectious disease organisms by using the PCR
For eg:-
a. Human Immunodeficiency Virus (HIV).
b. Tuberculosis (T.B).
c. Brucellosis.
d. The spread of diseases' organism through populations of domestic or
wild animals can be monitored by PCR testing.
• DNA FINGERPRINT in forensic
• PARENTAL TESTING
• To study PATTERNS of GENE EXPRESSION
16. ADVANTAGES & DISADVANTAGES
• Advantages of PCR
• Useful non- invasive procedure.
• Sensitivity of the PCR
• Disadvantages of PCR
• False positive results (cross contamination).
• False negative results
18. INTRODUCTION
• Any liquid, solid or gel preperation designed spcifically to support the growth,
storage or transport of microorganisms or cells
19. PURPOSES
• To isolate bacteria in pure cultures
• To demonstrate their properties
• To obtain sufficient growth for the preparation of antigens and for other tests
• To check sensitivity to antibiotics
• To estimate visible counts
• To maintain stock
20. TYPES OF CULTURE MEDIA
Culture media have been classified in many ways:
1. Solid, semisolid and liquid.
2. Simple (basal), complex, synthetic, defined, semidefined and special media
-Special media further divided into: enriched, selective, enrichment,
indicator or differential, sugar media and transport media.
3. Aerobic media and anaerobic media.
21. LIQUID MEDIUM
• Earliest liquid medium: urine or meat broth used by Louis Pasteur
• Used for obtaining bacterial growth from blood or water when large volumes have to
be used as inoculum for preparing bulk cultures for antigens and vaccines
• Used for preparation of inoculum for biochemical reactions and antibiotic
susceptibility testing
• Difficult to isolate
• No characterisitics for identification
22. SOLID MEDIUM
• Earliest solid medium: cooked cut potato by Robert Koch (1881).
• Used gelatin (2.5-5.0%) to prepare solid media fortifying them with 1% meat extract as an essential
ingredient.
• Gelatin- Not satisfactory cause it liquefy at 24 degree C (incubation temp for most pathogenic
bacteria is 37 degree C).
• Use Agar 2% (suggested by Frau Hesse) in place of gelatin as solidifying agent for the media.
• Distinct colony morphology
• COLONY: macroscopically visible collection of millions of bacteria originating from a single
bacterial cell.
• Characteristics: easy to identify
23. AGAR-AGAR/AGAR
• Prepared from variety of seaweeds.
• No nutritive value
• Not affected by growth of bacteria
• Concentration of 1-2% usually yields a suitable gel.
• Appropriate amount of agar powder is added ti the liquid medium and dissolved by
placing mixture in a steamer at 100 degree C for 1 hour or longer.
• Mostly dissolve to give clear solution but sometimes necessary to filter off
particulate impurities.
• Melting point of bacteriological agar is 95 degree C and solidifies at 42 degree C.
• Can added to any liquid media id advantages of solid medium are required.
24. • Most culture media sterilized by autoclaving at 121 degree C for 15 min.
• Nutrients that are damaged by autoclaving sterilized separately by filtration.
25. SIMPLE MEDIA/BASAL MEDIA
• Most common in routine diagnostic labs
E.g: Nutrient Broth, Nutrient Agar
NB consist of peptone, meat extract, NaCl, water
NB + 0.5% Glucose= Glucose Broth
NB + 2% agar= Nutrient agar
Agar conc. Reduced (0.2 – 0.5%) = Semi-solid medium, if conc. raised (6%) the called hard
agar.
In semi-solid agar the motile organisms show growth in entire medium
On surface of hard agar swarming of Proteus is inhibited.
26. COMPLEX MEDIA
• They have added complex ingredients such as yeast extract or casein hydrolysate,
which consist of a mixture of many chemical species in unknown proportions.
• Provide special nutrients.
27. SYNTHETIC OR DEFINED MEDIA
• Media prepared from pure chemical substances.
• Exact composition is known
• Used for special studies E.g metabolic requirements
• E.g. peptone water ( 1% peptone + NaCl in water)
28. ENRICHED MEDIA
• Prepared to meet the nutritional requirements of fastidious organisms by addition of
substances such as blood, serum, egg to basal medium.
• Used to grow bacteria that are exacting in their nutritional needs.
• E.g. blood agar for isolation of streptococcus, chocolate agar for isolation of Neisseria
and Haemophilus .
29. SELECTIVE MEDIUM
- Inhibitory substance is added to solid medium to inhibit the growth of unwanted
bacteria but permits the growth of wanted bacteria.
- Growth in form of colonies
- E.g. MacConkey’s medium for E. Coli, deoxycholate citrate agar (DCA) for
Salomonella and Shigella, Lowenstein-Jensen for Mycobacterium tuberculosis.
30. INDICATOR MEDIUM
• Contains an indicator when a particular bacteria grows which changes its color
when a particular bacteria grows.
• Also known as differential medium.
• E.g. Urease producing organisms like Proteus and Klebsiella
Urease producing bacteria
Urea > CO2 + NH3
NH3 (Alkaline) > Medium turns Pink
31. SUGAR MEDIUM
• Term sugar denotes to any fermentable substance such as:
Monosaccharides like pentose
Disaccharides like saccharose and lactose
Polysaccharides like insulin
Trisaccharides like raffinose
Alcohol like glycerol and sorbitol
Media consist of 1% sugar in peptone water + indicator
Contain in a small tube ( Durham’s tube) for detection of gas by the bacteria
32. TRANSPORT MEDIUM
• Media used for transporting the samples.
• Delicate organisms may not survive the time taken for transporting the specimen
without a transport medium cause normal flora overgrow pathogenic flora.
• E.g. Stuart medium
Buffered glycerol saline
33. VARIOUS PERIODONTAL AND CARIOGENIC
APECIES GROWN ON AGAR PLATES
Streptococcus mitis are gram-
positive, culture on a blood-agar
plate. A clear halo surrounding the
colonies
Veillonella parvula are
anaerobic gram-negative small cocci.
They form small transparent
colonies
Actinomyces viscosus are
microaerophilic
to anaerobic gram positive rods .
They form slimy white spherical
colonies
34. Typical colony morphology
of Streptococcus sanguinis (right) and
Actinomyces odontolyticus (left)
Lactobacillus spp. will typically grow on Rogosa
agar as a sesame seed
35. Streptococcus
gordonii are anaerobic
gram-positive cocci. clear
halo surrounding the colony
Fusobacterium nucleatum
as a round, flat rhizoid,
opaque purple colony.
Porphyromonas gingivalis
(green-brown) and
Prevotella intermedia
(black) on a classic
nonspecific blood-agar plate.
36. Prevotella nigrescens forms
like, a black pigmented
colony
Parvimonas micra (small
white colony) next to
Porphyromonas gingivalis
(green-brown colony)
Aggregatibacter
actinomycetemcomitans
grown on a selective agar
plate containing tryptic soy,
horse serum, bacitracin and
vancomycin
37. It is extremely difficult to culture
Treponema denticola (spirochete) on
an agar plate and therefore not
possible to identify this
bacteria with classic culture. Phase-
contrast microscope, the dark-field
microscope, or the electron
microscope are often used to
visualize this bacterium.
Identification and quantification is
only possible through DNA analysis.
Streptococcus mutans will grow as a
sugar cube
Eubacterium
nodatum colony
morphology
strongly depends
on its substrate.
Its growth is very
slow, and it is an
obligate anaerobic
gram-positive rod
38. Tannerella forsythia as
smooth white colocy with
faded edge
Streptococcus sobrinus
(colony with white halo)
Capnocytophaga
They are facultative
anaerobic rods
39. Campylobacter rectus grows
on a Hammond plate as
small, smooth opaque,
round colonies with a
black color.
Eikenella corrodens has a
variable colony morphology,
are anaerobic gram-negative
rods
40. REFERENCES
• Proteomics and Genomics (Dr. Vikash Kumar Dubey)
• Microbiology for dental students 2nd Edition (D.R. Arora)
• Polymerase chain reaction: A short review MT Rehman et al AKMMC J 2013 4(1)
• Carranza’s Clinical Periodontology 11th ed