POLYMERASE CHAIN
REACTION (PCR)
INTRODUCTION
• PCR: Technique for in vitro (test tube) amplification of specific DNA sequences via
the temperature mediate...
• Coping Machine for DNA Molecule
• Invented by Kary Mullis and his colleagues in the 1983
DEFINITION
• It is a genetic technique that occurs in vitro which allows the enzymatic synthesis of
large quantities(ampli...
PRINCIPLE OF PCR
• The PCR technique copies the target DNA by performing repeated cycles each
containing the following thr...
PCR REACTION COMPONENTS
• DNA template
• Two primers
• Four normal deoxynucleosides triphosphates
• Buffer system
• DNA po...
• DNA template: is the DNA molecules that contains the DNA region (segment) to be
amplified, the segment that we are conce...
PCR PROCEDURE
Each synthesis cycle Is composed of Three steps
• Denaturation
• Primer Annealing
• Extension
DENATURATION
• Denaturation: During the denaturation step, the reaction cocktail (reaction mixture)
is exposed to high tem...
PRIMER ANNEALING
• During the second step of each cycle, the temperature is lowered to an annealing
temperature (50°-60° C...
EXTENSION
• The reaction cocktail is now brought to the optimum reaction temperature for Taq
polymerase (65 to 85°C). Duri...
APPLICATIONS OF PCR
• GENETIC TESTING:- Where a sample of DNA is analyzed for the presence of
genetic disease mutations
• ...
• Characterization and detection of infectious disease organisms by using the PCR
For eg:-
a. Human Immunodeficiency Virus...
ADVANTAGES & DISADVANTAGES
• Advantages of PCR
• Useful non- invasive procedure.
• Sensitivity of the PCR
• Disadvantages ...
CULTURE MEDIA
INTRODUCTION
• Any liquid, solid or gel preperation designed spcifically to support the growth,
storage or transport of mi...
PURPOSES
• To isolate bacteria in pure cultures
• To demonstrate their properties
• To obtain sufficient growth for the pr...
TYPES OF CULTURE MEDIA
Culture media have been classified in many ways:
1. Solid, semisolid and liquid.
2. Simple (basal),...
LIQUID MEDIUM
• Earliest liquid medium: urine or meat broth used by Louis Pasteur
• Used for obtaining bacterial growth fr...
SOLID MEDIUM
• Earliest solid medium: cooked cut potato by Robert Koch (1881).
• Used gelatin (2.5-5.0%) to prepare solid ...
AGAR-AGAR/AGAR
• Prepared from variety of seaweeds.
• No nutritive value
• Not affected by growth of bacteria
• Concentrat...
• Most culture media sterilized by autoclaving at 121 degree C for 15 min.
• Nutrients that are damaged by autoclaving ste...
SIMPLE MEDIA/BASAL MEDIA
• Most common in routine diagnostic labs
E.g: Nutrient Broth, Nutrient Agar
 NB consist of pepto...
COMPLEX MEDIA
• They have added complex ingredients such as yeast extract or casein hydrolysate,
which consist of a mixtur...
SYNTHETIC OR DEFINED MEDIA
• Media prepared from pure chemical substances.
• Exact composition is known
• Used for special...
ENRICHED MEDIA
• Prepared to meet the nutritional requirements of fastidious organisms by addition of
substances such as b...
SELECTIVE MEDIUM
- Inhibitory substance is added to solid medium to inhibit the growth of unwanted
bacteria but permits th...
INDICATOR MEDIUM
• Contains an indicator when a particular bacteria grows which changes its color
when a particular bacter...
SUGAR MEDIUM
• Term sugar denotes to any fermentable substance such as:
Monosaccharides like pentose
Disaccharides like sa...
TRANSPORT MEDIUM
• Media used for transporting the samples.
• Delicate organisms may not survive the time taken for transp...
VARIOUS PERIODONTAL AND CARIOGENIC
APECIES GROWN ON AGAR PLATES
Streptococcus mitis are gram-
positive, culture on a blood...
Typical colony morphology
of Streptococcus sanguinis (right) and
Actinomyces odontolyticus (left)
Lactobacillus spp. will ...
Streptococcus
gordonii are anaerobic
gram-positive cocci. clear
halo surrounding the colony
Fusobacterium nucleatum
as a r...
Prevotella nigrescens forms
like, a black pigmented
colony
Parvimonas micra (small
white colony) next to
Porphyromonas gin...
It is extremely difficult to culture
Treponema denticola (spirochete) on
an agar plate and therefore not
possible to ident...
Tannerella forsythia as
smooth white colocy with
faded edge
Streptococcus sobrinus
(colony with white halo)
Capnocytophaga...
Campylobacter rectus grows
on a Hammond plate as
small, smooth opaque,
round colonies with a
black color.
Eikenella corrod...
REFERENCES
• Proteomics and Genomics (Dr. Vikash Kumar Dubey)
• Microbiology for dental students 2nd Edition (D.R. Arora)
...
Polymerase chain reaction & culture media
Polymerase chain reaction & culture media
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Polymerase chain reaction & culture media

Polymerase Chain Reaction
Published on: Mar 4, 2016
Published in: Science      
Source: www.slideshare.net


Transcripts - Polymerase chain reaction & culture media

  • 1. POLYMERASE CHAIN REACTION (PCR)
  • 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. • 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.
  • 8. PCR PROCEDURE Each synthesis cycle Is composed of Three steps • Denaturation • Primer Annealing • Extension
  • 9. 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.
  • 10. 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.
  • 11. 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).
  • 12. 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
  • 13. • 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
  • 14. ADVANTAGES & DISADVANTAGES • Advantages of PCR • Useful non- invasive procedure. • Sensitivity of the PCR • Disadvantages of PCR • False positive results (cross contamination). • False negative results
  • 15. CULTURE MEDIA
  • 16. INTRODUCTION • Any liquid, solid or gel preperation designed spcifically to support the growth, storage or transport of microorganisms or cells
  • 17. 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
  • 18. 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.
  • 19. 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
  • 20. 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
  • 21. 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.
  • 22. • Most culture media sterilized by autoclaving at 121 degree C for 15 min. • Nutrients that are damaged by autoclaving sterilized separately by filtration.
  • 23. 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.
  • 24. 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.
  • 25. 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)
  • 26. 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 .
  • 27. 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.
  • 28. 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
  • 29. 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
  • 30. 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
  • 31. 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
  • 32. Typical colony morphology of Streptococcus sanguinis (right) and Actinomyces odontolyticus (left) Lactobacillus spp. will typically grow on Rogosa agar as a sesame seed
  • 33. 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.
  • 34. 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
  • 35. 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
  • 36. Tannerella forsythia as smooth white colocy with faded edge Streptococcus sobrinus (colony with white halo) Capnocytophaga They are facultative anaerobic rods
  • 37. 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
  • 38. 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

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