PCR principles

1. PCR and DNA RFLP Techniques
AYA YASSER ELWAZIR
ASS. LECTURER OF MEDICAL GENETICS – CEMCM MOLECULAR
BIOLOGY UNIT
PCR PRINCIPLES

2. How can we analyse DNA and detect gene structure and defects
when DNA is a very tiny molecule?

3. ¡ The solution came in 1983 by
Kary Mullis
¡ Invented PCR
¡ Won Nobel prize in chemistry

4. PRINCIPLES OF PCR
AGENDA
¡What is PCR?
¡Steps of PCR
¡Components of PCR

5. WHAT IS PCR?
¡ PCR stands for polymerase
chain reaction
¡ Lab technique that makes
millions of copies from a small
sample of DNA
¡ DNA is replicated over and
over again
(DNA amplification)

6. WHAT IS PCR?
Amplify area or gene of interest

7. WHAT IS PCR?
Select specific sequence Amplify selected sequence

8. WHAT IS PCR?
Amplicon easily detected by
Dyes
(Fluorescent)
Gel
electrophoresis

9. WHAT IS PCR?
DNA amplification
Genetic defects & variants Viruses Forensics
Research Evolutionary studies Prior to other techniques
Revolution in science
RFLP
Seq

10. PRINCIPLES OF PCR
AGENDA
What is PCR?
¡Steps of PCR
¡Components of PCR

11. STEPS OF PCR
The idea was to mimic DNA replication:
Double stranded
DNA
1-Unwinding by
helicase à 2 single
strands
2- Attachment of
primer
3- Addition of complementary
nucleotides by DNA polymerase
Denaturation Annealing Extension

12. STEPS OF PCR
Denaturation
¡ At a temperature of 95°C
¡ Hydrogen bonds between
the two strands are broken
¡ Takes 1- 2 min

13. ¡ Temperature reduced to 50 - 60°C
¡ Primers attach to their
complementary sequences on the
single strands
¡ Takes around ½ a minute
STEPS OF PCR
Annealing
Forward
Reverse

14. ¡ Temperature increased to 72°C
¡ DNA polymerase attaches to the 3’
end of the primer and adds
nucleotides to complete each strand
¡ Takes a few minutes
STEPS OF PCR
Extension

15. 1st cycle 2nd cycle 3rd cycle
STEPS OF PCR
30–35 cycles

16. PRINCIPLES OF PCR
AGENDA
What is PCR?
Steps of PCR
¡Components of PCR

17. COMPONENTS
¡ Template DNA
¡ Specific primers (forward – reverse)
¡ DNA polymerase (Taq polymerase)
¡ dNTPs (nucleotides:A,T, C, G)
¡ Buffer, water
5’ 3’
3’ 5’
Thermus aquaticus

18. PRINCIPLES OF PCR
AGENDA
What is PCR?
Steps of PCR
Components of PCR

19. SUMMARY
What is PCR?
Amplification of a
specific target sequence

20. Any Questions??

21. THANKYOU