Polymerase Chain Reaction &

types

Nandith P B
Research Scholar
 PCR- Introduction
• Polymerase chain reaction (PCR) is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of DNA
across several orders of magnitude, generating thousands to millions of
copies of a particular DNA sequence

• Developed in 1983 by Kary Mullis (Nobel Prize in Chemistry in

1993)

• PCR methods rely on thermal cycling, which involves exposing the

reactants to cycles of repeated heating and cooling, permitting
different temperature-dependent reactions

• Most PCR methods amplify DNA fragments of between 0.1 and 10

kilo base pairs (kbp)

•Number of DNA copies formed after a given number of cycles is 2^n

 PCR- Steps
• Denaturation: 94–98 °C for 20–30 seconds
• This causes DNA melting or denaturation of the double-stranded DNA template
by breaking the hydrogen bonds between complementary bases, yielding two
single-stranded DNA molecules

• Annealing: 50–65 °C for 20–40 seconds

• Hybridization of the primer to the strand.
• Two different primers are typically included in the reaction mixture
• It is critical to determine a proper temperature for the annealing step because
efficiency and specificity are strongly affected by the annealing temperature.
• A typical annealing temperature is about 3–5 °C below the Tm (Melting
temperature ) of the primers used. Tm = 2 X (A+T) + 4 X (G+C)

• Elongation: 75–80 °C
• In this step, the DNA polymerase synthesizes a new DNA strand complementary
to the DNA template strand

• Final elongation: 70–74 °C for 5–15 minutes

• To ensure that any remaining single-stranded DNA is fully elongated
 PCR- Steps (Contd….)
The temperatures used and the length of time they are applied in each
cycle depend on

•Enzyme used for DNA synthesis

•Concentration of bivalent ions

• dNTPs in the reaction

•Melting temperature (Tm) of the primers

 PCR Set-up
• DNA template that contains the DNA target region to amplify

• DNA polymerase an enzyme (Thermus aquaticus) that polymerizes new

DNA strands

• Two DNA primers that are complementary to the 3' (three prime) ends of
each of the sense and anti-sense strands of the DNA target

• Deoxynucleotide Triphosphates (dNTPs) which act as building blocks

from which the DNA polymerase synthesizes a new DNA strand

• Buffer solution providing a suitable chemical environment for optimum

activity and stability of the DNA polymerase

• Bivalent cations typically magnesium (Mg) or manganese (Mn) ions;

Mg2+ is the most common
 DNA Extraction

Basic Principle

Lyse = Break cell open to free DNA or RNA

Bind = Bind the Nucleic Acid to the Membrane

Wash = Wash off all other impurities such as proteins, sugars, salts
etc. from the membrane-DNA complex

Elute = Finally the pure DNA on the membrane is harvested or

eluted off the membrane
Purified
 PCR Set-up

PCR Reaction Set-up

 PCR Machine
• Laboratory apparatus most commonly used to amplify segments of
DNA via the polymerase chain reaction (PCR)
• The device has a thermal block with holes where tubes holding the
reaction mixtures can be inserted
• The cycler then raises and lowers the temperature of the block in
discrete, pre-programmed steps
 PCR

Semi Q
Q PCR
PCR
 What is Electrophoresis?
•Electrophoresis is a method to separate nucleic acids or proteins
in a matrix of agarose / acrylamide-bis-acrylamide.
•The proteins and nucleic acids are separated by charge and/or
size using an electric field.

Agarose Gel Electrophoresis

Semi Q PCR- Result
Lanes: 1 2 3

Lane 1: 1Kb DNA Ladder

Lane 2: Control PCR Product
Lane 3: PCR Product
 Q PCR
• A real-time polymerase chain reaction (Real-Time PCR), also
known as quantitative polymerase chain reaction (qPCR)

• Technique based on the polymerase chain reaction (PCR)

• It monitors the amplification of a targeted DNA molecule during

the PCR, i.e. in real-time, and not at its end, as in conventional
PCR.
 Dye Based Q PCR
• Non-specific fluorescent dyes that intercalate with any double-
stranded DNA

Probe Based Q PCR

• Sequence-specific DNA probes consisting of oligonucleotides
that are labelled with a fluorescent reporter which permits
detection only after hybridization of the probe with its
complementary sequence
Probe Based Q PCR
 Q PCR- Result

SYBR Green fluorescence chart produced Melting curve produced at the end of

in real-time PCR. real-time PCR.
 Variants of PCR
• Multiplex-PCR
• simultaneous analysis of multiple targets in a single sample
• Nested PCR
• used to increase the specificity of DNA amplification
• Quantitative PCR
• used to measure the specific amount of target DNA (or RNA) in a sample
•Hot-start PCR
• technique performed manually by heating the reaction components to the DNA melting
temperature (e.g:95 °C) before adding the polymerase. In this way, non-specific
amplification at lower temperatures is prevented
• Gradient PCR
• Used to optimize the annealing temperature
• RT PCR
• RNA template is first converted into a complementary DNA (cDNA). The cDNA is
then used as a template for exponential amplification using PCR
Multiplex-PCR
Nested-PCR
RT-PCR
 Medical Applications

Infectious disease
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by
bacteria or viruses. Example: CHIKUNGUNIYA, DENGUE, ZIKA, TB

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as

mycobacteria, anaerobic bacteria, or viruses

The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the
discrimination of non-pathogenic from pathogenic strains by virtue of specific genes

Tissue Typing
DNA-based test that can detect the presence or absence of antigens by determining whether cells have
the genes for the antigens
 General Applications

• Amplification of small amount of DNA for further analysis.

• Isolation of a particular gene of interest from a tissue sample.

• Production of DNA for sequencing.

• Analysis of mutations

• Diagnosis of monogenic diseases (single gene disorders)

• Detection of micro organisms that are difficult to culture.

• Crucial forensic evidence where sufficient DNA sample is not available.

THANK YOU