Polymerase Chain Reaction

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

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.

9. PCR PROCEDURE
Each synthesis cycle Is composed of Three steps
• Denaturation
• Primer Annealing
• Extension

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).

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

17. CULTURE MEDIA

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