Polymerase chain reaction & culture media

POLYMERASE CHAIN REACTION (PCR)

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.

• 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(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).

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.

PCR REACTION COMPONENTS

• DNA template

• Two primers

• Four normal deoxynucleosides triphosphates

• Buffer system

• DNA polymerase I

• 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.

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 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.

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.

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).

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

• 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

ADVANTAGES & DISADVANTAGES

• Advantages of PCR

• Useful non- invasive procedure.

• Sensitivity of the PCR

• Disadvantages of PCR

• False positive results (cross contamination).

• False negative results

CULTURE MEDIA

INTRODUCTION

• Any liquid, solid or gel preperation designed spcifically to support the growth,

storage or transport of microorganisms or cells

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

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.

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

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

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.

• Most culture media sterilized by autoclaving at 121 degree C for 15 min.

• Nutrients that are damaged by autoclaving sterilized separately by filtration.

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.

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.

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)

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 .

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.

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

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

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

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

Typical colony morphology

of Streptococcus sanguinis (right) and

Actinomyces odontolyticus (left)

Lactobacillus spp. will typically grow on Rogosa

agar as a sesame seed

Streptococcusgordonii are anaerobic

gram-positive cocci. clear

halo surrounding the colony

Fusobacterium nucleatumas a round, flat rhizoid,

opaque purple colony.

Porphyromonas gingivalis(green-brown) and

Prevotella intermedia(black) on a classic

nonspecific blood-agar plate.

Prevotella nigrescens forms

like, a black pigmented

colony

Parvimonas micra (small

white colony) next to

Porphyromonas gingivalis(green-brown colony)

Aggregatibacteractinomycetemcomitansgrown on a selective agar

plate containing tryptic soy,

horse serum, bacitracin and

vancomycin

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

Eubacteriumnodatum colony

morphology

strongly depends

on its substrate.

Its growth is very

slow, and it is an

obligate anaerobic

gram-positive rod

Tannerella forsythia as smooth white colocy with faded edge

Streptococcus sobrinus(colony with white halo)

Capnocytophaga

They are facultative

anaerobic rods

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

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