pcr requirement, working mechanism, and its application

2. INTRODUCTION
• Pcr technique was developed by Kary mullis,
califormia (1985)
• This technique has made it possible to
synthesize large quantities of DNA fragments
without its cloning
• It is ideally suited where the quantity of
biological specimen available is very low such
as a single fragment of hair or a tiny blood stain
left at the site of a crime

3. Requirements
 DNA Templates: any source that contain one or more target
DNA molecules to be amplified can be taken as template
 Primer: A pair of oligonucleotides of about 180-300
nucleotides with similar G+C content act as a primer
The primers are designed to anneal on opposite strands of target
sequence so that they will extended towards each other by
addition of nucleotide to their 3’ end
 Enzymes: Enzyme is thermostable called Taq polymerase
isolated from Thermus aquaticus and vent polymerase from
thermococcus litoralis
It survives at 95⁰c for 1-2 mins & has a half life of more than 2 hrs

4. WORKING MECHANISM OF
PCR
Denaturation:
 The target DNA (sequence of 100-5000
bases) to be amplified is heated
denaturated (95⁰c for 15 sec) to separate its
complementary strands.
 This process is called melting of target
DNA
 A separation each strand act as template
for DNA synthesis

5. Primer annealing:
 Annealing of two oligonucleotide primers to
the denatured DNA strands
 Since nucleotide sequence of each of
oligonucleotide primer is complementary to
3’end of single stranded template
 Primers are added in excess and the
temperature lowered to about 68⁰c for 60 sec

7. Primer extension:
 Nucleotide tri-phosphate (dATP, dGTP, dCTP,
dTTP) and a thermostable DNA polymerase
are added to the reaction mixture
 The DNA polymerase accelerate the
polymerization process of primer at 68⁰c
reasulting in synthesis of copies of target DNA
sequence
 The concentration of Mgᶧᶧ is maintained between
1 & 4 Mm

8.  Thus in the first step the target DNA is copied from
the primer sites for various distance untill the start
of second cycle
 However, after completion of one cycle the targeted
sequence on both strands are copied and four
strands are produced
 Now, the first cycle is repeated which yields eight
copies from four strands
 The second cycle is started with heating of double
stranded DNA to result in single stranded DNA

9.  Similarly, the third cycle produces 16 strands.
This cycle is repeated about 50 times
 Theoretically, 20 cycles (each of 3 steps) will
produce about one million copies of target DNA
sequence & 30 cycles will produce one billon
copies
 For the working of PCR about 10-100
picomoles of primers are required
 The PCR machine carryout 25 cycles & amplify
DNA 10⁵ times in 75 minutes

10. APPLICATION OF PCR
 Diagnosis of pathogen
 Diagnosis of specific mutation
 Prenatal diagnoasis
 DNA fingerprinting
 In research
 In molecular archeaology
(palaentology)
 Diagnosis of plant pathogen