PCR (Polymerase Chain Reaction)

Contents

What is PCR?
DNA Replication vs. PCR
Basic Components of PCR
thermo-cycling protocol
3 Main Steps of PCR
Amplification
Applications
Conclusion
References
 What is PCR?

 PCR is a means to amplify a particular piece of DNA

 Amplify= making numerous copies of a segment of DNA
 PCR can make billions of copies of a target sequence of
DNA in a few hours
 PCR was invented in the 1984 as a way to make numerous
copies of DNA fragments in the laboratory
 Its applications are vast and PCR is now an integral part of
Molecular Biology
 DNA Replication vs. PCR
 PCR is a laboratory version of DNA Replication in cells
 The laboratory version is commonly called “in vitro” since it
occurs in a test tube while “in vivo” signifies occurring in a
living cell.
 Basic Components of PCR

• Template DNA (0.5 - 50 ng)

 < 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy genes

• Oligonucleotide primers (0.1 – 2.0 μM)

• dNTP’s (20 –250 μM)
• Thermostable DNA pol (0.5 – 2.5 U/rxn)
• MgCl2 (1 – 5 mM) affects primer annealing and Taq activity
• Buffer (usually supplied as 10X)
Working concentrations
KCL (10 – 50 mM) Buffer
Primers
Tris-HCl (10 mM, pH 8.3) ACTG dNTPs
Taq polymerase
NaCl2 (sometimes)
DNA template
+ + MgCl
2
 A simple thermo-cycling protocol

1X 35X 1X

94ºC 94ºC

3 min 1 min 72ºC

1 min
55ºC
Initial denaturation
of DNA 45 sec

denaturation

extension
4ºC

annealing
∞ hold
 3 Main Steps of PCR

 Step 1: Denature DNA

At 95ºC, the DNA is denatured (i.e. the two strands are
separated)

 Step 2: Primers Anneal

At 40ºC- 65ºC, the primers anneal (or bind to) their
complementary sequences on the single strands of DNA

 Step 3: DNA polymerase Extends the DNA chain

At 72ºC, DNA Polymerase extends the DNA chain by adding
nucleotides to the 3’ ends of the primers.
Polymerase Chain Reaction
Step 1:
Denaturation
dsDNA to ssDNA

Step 2:
Annealing
Primers onto template

Step 3:
Extension
dNTPs extend 2nd strand
Amplification
 Applications

Genome mapping and gene function determination

Biodiversity studies ( e.g. evolution studies)
Diagnostics ( prenatal testing of genetic diseases,
early detection of cancer, viral infections...)
Detection of drug resistance genes
Forensic (DNA fingerprinting)
 Conclusion

 PCR is not only vital in the clinical laboratory by

amplifying small amounts of DNA for STD detection,
but it is also important for genetic predisposing for
defects such as FACTOR V leiden.
 The PCR technology can also be employed in law
enforcement, genetic testing of animal stocks &
vegetable hybrids, &drug screening along with many
more areas.
 References

Biochemistry, U. Satyanarayana, 2nd edition.

Recombinant DNA Technology Page 581-583
Thank you….