Polymerase Chain Reaction PCR

1. I-5- 1BY: DHARMIK MENDAPARA

2.  A method of in vitro cloning
 The most popular and widely used technique
in all fields of biological studies probably.
 Why?

3.  1. simple
 2. powerful
 A. sensitive – sensitivity
 B. specific – specificity
 C. reliable – reliability;
 3. fast

4.  Purpose: To duplicate DNA molecule
 Principle:
 Separation of DNA double-stranded template
 Primer formation
 Extension of new DNA strands by a DNA
polymerase and deoxyribonucleoside
triphosphates (dNTPs)

6.  To amplify a lot of double-stranded DNA
molecules (fragments) with same (identical)
size and sequence by enzymatic method and
cycling condition.

7.  1. Denaturation of DNA template
 2. Annealing of primers
 3. Extension of DNA molecules

8.  Specificity
 Efficiency
 Fidelity

9.  The basis of PCR is temperature changes and the effect that
these temperature changes have on the DNA.
 In a PCR reaction, the following series of steps is repeated 20-40
times
(note: 25 cycles usually takes about 2 hours and amplifies the
DNA fragment of interest 100,000 fold)
Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two strands are
separated)
Step 2: Primers Anneal
At 40C- 65C, the primers anneal (or bind to) their
complementary sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain
At 72C, DNA Polymerase extends the DNA chain by adding
nucleotides to the 3’ ends of the primers.

10.  Water
 Buffer
 DNA template
 Primers
 Nucleotides
 Mg++ ions
 DNA Polymerase

12.  Water..
 The medium for all
other components.

13.  Water
 Buffer
 Stabilizes the DNA
polymerase, DNA, and
nucleotides

14.  Water
 Buffer
 DNA template
 Contains region to be
amplified
 Any DNA desired
 Purity is required
 Should be free of
polymerase inhibitors

15.  Water
 Buffer
 DNA template
 Primers
 Specific for ends of
amplified region
 Forward and Reverse

16.  Water
 Buffer
 DNA template
 Primers
 Nucleotides
 Added to the growing
chain
 Activated NTP’s
 dATP, dGTP, dCTP, dTTP

17.  Water
 Buffer
 DNA template
 Primers
 Nucleotides
 Mg++ ions
 Essential co-factor of DNA
polymerase
 Too little: Enzyme won’t work.
 Stabilizes the DNA double-helix
 Too much: DNA extra stable, non-
specific priming, band smearing
 Used at 0.5 to 3.5 uM in the assay

18.  Water
 Buffer
 DNA template
 Primers
 Nucleotides
 Mg++ ions
 DNA Polymerase
 The enzyme that
does the extension
 TAQ or similar
 Heat-stable
 Approx 1 U / rxn

19.  Water
 Buffer
 DNA template
 Primers
 Nucleotides
 Mg++ ions
 DNA Polymerase
Summary:

20. The DNA, DNA
polymerase, buffer,
nucleoside triphosphates,
and primers are placed in
a thin-walled tube and
then these tubes are
placed in the PCR
thermal cycler
PCR Thermocycler

21.  Forensic DNA detection
 Identifying transgenic plants
 Detection and quantification of viral
infection
 Cloning
 Detection of ancient DNA
 Gene expression analysis
The best way to understand PCR is to
consider the reaction components and
how they combine to produce the best
results.

22.  How do we identify and detect a specific sequence in a genome?
 TWO BIG ISSUES:
 There are a LOT of other sequences in a genome that we’re not
interested in detecting.
 The amount of DNA in samples we’re interested in is VERY small.
PCR solves BOTH of these issues!!!