Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.

1. Polymerase chain
Reaction (PCR)
Mai Masri
Faculty of Science
Department of Zoology

2. Polymerase Chain Reaction
(PCR)
 One of the most powerful
tools in molecular biology
 Invented by Kary Mullis in
1983.
 This process acts as a
“copying machine” for DNA
1993 Nobel Prize in Chemistry

3. What is PCR
An in vitro (cloning) method for the
enzymatic synthesis of specific DNA
sequences.
Selective amplification of target DNA from
a heterogeneous, complex DNC/cDNA
population

4. Why PCR (amplify ) DNA?
1. Simple
2. Powerful
A. Sensitive
B. Specific
C. Reliable , fidelity
3. Fast

5. The reaction mixture
 Free nucleotides (A, G, T, C)
 DNA polymerase
 DNA (purified or a crude extract)
 Primers specific for the target DNA
 Buffer (containing magnesium)

6. The basic protocol—what’s in the
tube
Target DNA
5’ 3’
3’ 5’
primers
A
B Free
nucleotides
Taq DNA
polymerase
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Buffer
containing
magnesium

7. The basic protocol--
denaturation
Target DNA
95oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’

8. The basic protocol--annealing
~55oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’
5’
Target DNA
A
B
primers
A
B

9. The basic protocol--extension
72oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’
5’
Target DNA
Taq polymerase

10. The basic protocol--extension
72oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’
5’
Target DNA

11. The basic protocol
1. Denaturation of DNA to
single strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times

12. One One billion in about 2
hours!
 At the end of each cycle, the amount of
DNA has doubled
 In theory, each target DNA molecule is
duplicated during every cycle. This
means that 30 cycles would produce 230
(=109) molecules from a single target
molecule. In reality, the yield is lower,
especially in later cycles.
230=1,073,741,824

13. PCR - before the thermocycler
8 BORING hours per PCR!
95º C
5 min
35 times
55º C
3 min
72º C
5 min

14. standard tube, volume, cost
evaporation & heat transfer concerns
thin walled tube,  volume,  cost
 evaporation & heat transfer concerns

15.  Most enzymes would be
killed at 95oC.
 Taq was isolated from
Thermus aquaticus, a
bacteria that grows in
hot springs (~75oC) in
Yellowstone National
Park
 This organism’s enzymes
have adapted to the
high temperature, so
they can survive cycling
through the high
temperatures.

16. Primers
Target DNA
5’ 3’
3’ 5’
forward
reverse

17. Primers
 Primers provide specificity
 Must have some information about
sequence flanking your target
 Complementary to opposite strands with
3’
ends pointing towards each other
 Should have similar melting
temperatures.
 Be in vast excess.

18. Primer Design
*Typically 20 to 30 bases in length
*Annealing temperature dependent upon primer sequence
(~ 50% GC content)
*Avoid secondary structure, particularly 3’
*Avoid primer complementarity (primer dimer)
*The last 3 nucleotides at the 3` end is the substrate for
DNA polymerase - G or C
*Good free software programs available

19. Templates for PCR
 Small amount of template, in theory a
single molecule
 Do not need to isolate sequence of
interest
 DNA template need not be highly
purified
 DNA is stable in absence of nucleases

20. Templates
 Dried blood
 Semen stains
 Vaginal swabs
 Single hair
 Fingernail scrapings
 Insects in Amber
 Egyptian mummies
 Buccal Swab
 Toothbrushes

21. A simple thermocycling protocol
annealing
94ºC 94ºC
55ºC
72ºC
4ºC
3 min 1 min
45 sec
1 min
∞ hold
Initial denaturation
of DNA
1X 35X 1X
extension
denaturation

22. Typical PCR Temps/Times
hold
4o C or 10 mM EDTA
Stop reaction
5 – 10
min
70o – 75o C
Final extension
0.5 – 2
min
70o – 75o C
Primer
extension
0.5 – 1
min
45o – 65o C
Primer
annealing
0.5 – 1
min
90o – 95o C
Denature
1 – 3 min
90o – 95o C
Initial
denaturation
25 – 40
cycles

23. PCR machine

24. The PCR machine
 Very rapidly changes
the temperature
between the various
stages of the PCR
process
 Programmable for
use with many
different cycling
parameters

25. heated lid
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
Thermocyclers

26. Consumables

28. Advances due to PCR
 Generate very large quantities of
specific DNA sequences, aiding
in cloning
 Genomic sequencing
 DNA “fingerprinting”--forensic
science
 Disease diagnosis
 Paternity determination
 Mutation detection
 Taxonomy
 Many more to come….

29. 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)
Tris-HCl (10 mM, pH 8.3)
NaCl2 (sometimes
dNTPs
Taq polymerase
Primers
DNA template
Buffer
+ +
A C T G
MgCl2

30. Common PCR additives
BSA (usually at 0.1 to 0.8 µg/µL final concentration)
Stabilize Taq polymerase & overcome PCR inhibitors
DMSO (usually at 2-5% v/v, inhibitory at ≤ 10% v/v)
Denaturant - good at keeping GC rich template/primer strands from forming
secondary structures.
Glycerol (usually at 5-10% v/v)
Increases apparent concentration of primer/template mix, and often
increases PCR efficiency at high temperatures.
Stringency enhancers (Formamide, Betaine, TMAC)
Concentrations used vary by type Enhances yield and reduces non-specific
priming
Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.1–1%)
% of normal)
10
activity to ~
Taq
% SDS cuts
01
.
0
SDS (
NOT
Stabilize Taq polymerase & suppress formation of 2º structure

31. Rules of thumb for PCR conditions
Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle
profile to ensure everything is denatured prior to starting the PCR
reaction
Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x
(G+C))]ºC
If GC content is < 50% start 5ºC beneath Tm for annealing temperature
If GC content ≥ 50% start at Tm for annealing temperature
Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute.
Use 3 minutes as an upper limit without special enzymes
“Special” PCR cycling protocols
Touchdown PCR

32. PCR Problems
Taq is active at low temperatures
At low temperatures mis-priming is likely
Extension Rate
Temp
0.25 nt/sec
22o C
1.5 nt/sec
37o C
24 nt/sec
55o C
150 nucleotides in 10 min

33. “Cheap” fixes
Physical separation –”DNA-in-the-cap”
Set up reactions on ice
Hot-start PCR –holding one or more of the PCR components until the first heat
denaturation
Manually - delay adding polymerase
Wax beads
Touch-down PCR – set stringency of initial annealing temperature high,
incrementally lower with continued cycling
PCR additives
0.5% Tween 20
5% polyethylene glycol 400
betaine
DMSO
“Cures” for mis-priming