POLYMERASE CHAIN REACTION

1. Polymerase Chain Reaction (PCR )
Suhad A. Mahdi
:MSc. Plant biotechnology
UAS,Bangalore,India

2. 35 Years of PCR and Going Strong!
Amazingly, there’s one PCR publication every ~15 minutes, and it’s still getting faster,
better, cheaper and more enabling!

3. The problem:
How do we identify and
detect a specific sequence
in a genome?

4. Paris japonica
150 Billion pb

5. The
Problem:
PCR solves BOTH of these
issues!!!

8. What’s PCR?
• A method of amplifying DNA,
• Multiplies a single, microscopic strand
of the genetic material billions of times
within hours.

9. Kary B. Mullis …….1983
Notable awards :
William Allan Award (1990)
Robert Koch Prize (1992)
Nobel Prize in Chemistry(1993)
Japan Prize (1993)

10. PCR Applications

11. Forensic DNA detection

12. Identifying transgenic plants

13. Detection and quantification of viral
infection

14. Cloning

15. Detection of ancient DNA

16. Gene expression analysis

18. PCR TYPES
RT-PCR(or Reverse Transcription PCR). It is used
to reverse-transcribe and amplifies RNA to cDNA.
Ligation-mediated PCR. ...
Methylation-specific PCR (MSP). ...
 Multiplex-PCR. ...
Variable Number of Tandem Repeats (VNTR) PCR.
Asymmetric PCR. ...
Nested PCR. ...
Quantitative PCR.

21. A Typical PCR Reaction
• Sterile Water 38.0 ul
• 10X PCR Buffer 5.0 ul
• MgCl2 (50mM) 2.5 ul
• dNTP’s (10mM each) 1.0 ul
• PrimerFWD (25 pmol/ul) 1.0 ul
• PrimerREV 1.0 ul
• DNA Polymerase 0.5 ul
• DNA Template 1.0 ul
• Total Volume 50.0 ul

22. PCR Reaction:
Buffer
–Stabilizes the DNA
polymerase, DNA, and
nucleotides
–500 mM KCl
–100 mM Tris-HCl, pH 8.3
–Triton X-100 or Tween

23. PCR Reaction:
Template DNA
–Contains region to be amplified
–Any DNA desired
–Purity not required
–Should be free of polymerase inhibitors

24. – Specific for ends of
amplified region
– Forward and Reverse
– Annealing temps
should be known .
• Depends on primer
length, GC content, etc.
– Length 15-30 pb.
– Conc 0.1 – 1.0 uM
(pMol/ul).
PCR Reaction:
Primers

25. – Added to the growing chain
– Activated NTP’s
– dATP, dGTP, dCTP, dTTP
– Stored at 10mM, pH 7.0
– Add to 20-200 uM in assay
PCR Reaction:
Nucleotides

26. – 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.
PCR Reaction:
Magnesium

27. –The enzyme that
does the extension
–TAQ or similar
–Heat-stable
–Approx 1 U
PCR Reaction:
Polymerase

28. 5'
5'
3'
3'