PCR is a method for amplifying a specific sequence of DNA from a complex mixture, without the lengthy process of cloning. It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified (target DNA).

1. Polymerase Chain Reaction and Gel Electrophoresis
• Addis Ababa University
College of Health Science
Department of Biochemistry
• By: Yohannes Gemechu
• October, 2014
1

2. Nucleic Acid Extraction
DNA Extraction
RNA Extraction
Polymerase chain Reaction
Applications, Instrumentation and reagents needed and
Procedure of PCR
Gel Electrophoresis
Applications, Instrumentation and Reagents needed and
Procedure
References 1/17/2015 2
OUTLINE

3. PCR is a method for amplifying a specific sequence of DNA from a
complex mixture, without the lengthy process of cloning.
It does require the knowledge of some DNA sequence information
which flanks the fragment of DNA to be amplified (target DNA).
From this information two oligonucleotide primers may be
chemically synthesised each complementary to a stretch of DNA to
the 3’ side of the target DNA.
Kary Mullis received a Nobel Prize in 1993 for his insight in
developing this technique.
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1.0 Polymerase Chain Reaction (PCR):

4. Used in gene recombinant DNA methodology, for example to amplify
a specific gene for cloning, sequencing, generation of DNA probes.
Diagnosis of genetic diseases, e.g hemophilia, phenylketonuria, cystic
fibrosis
Diagnosis of infectious diseases, e.g. infections with HIV, Chlamydia,
Gonorrhoea, Herpes Simplex Virus
Studies of gene expression in cells and tissues, including quantitation
of expressed mRNA
Prenatal diagnosis - such as determining the sex of fetuses for those
at risk of X-linked disorders
Applications of PCR
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5. Diagnosis of malignancies,
DNA fingerprinting, forensic and paternity investigations.
Phylogeny studies to classify species, subspecies.
Studies of ancient DNA, e.g. Neanderthals, Mammoth
Applications of PCR Cont’d
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6. DNA Extraction
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7. Methods of RNA extraction
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8.  Restriction endonucleases cleave DNA at or near specific sequences
(“restriction sites”) and naturally are involved in protecting bacteria
from invading bacterial viruses.
 The host's DNA is methylated to protect it from digestion by its
own restriction endonucleases.
 Type I restriction endonucleases recognize specific DNA sequences
but cleave the DNA randomly at sites remote from specific
sequence.
 Type III restriction endonucleases recognize specific DNA
sequences and cleave the DNA 20 to 25 bases pairs away from that
specific sequence.
 Type II restriction enzymes are the ones mostly used in
recombinant DNA methodology.1/17/2015 8
Enzymatic Treatment of DNA

9.  Enzymes that cleave the phosphodiester bonds of the DNA
backbone at or within specific nucleotide sequences.
 They make 2 cuts, one on each DNA strand.
 Cleavage sites are often at palindromic sequences.
 Mirror-like palindromes (e.g. CATTAC)or inverted repeat
palindromes (e.g. CCTAGG) exist.
 Recognition sites are usually 4 to 8 nucleotides long.
 They may produce “blunt” (smooth) or “sticky” (overhanging)
ends, depending on the enzyme.1/17/2015 9
Restriction Endonuclease Type II

10. “Sticky” and “Blunt” ends

11. Reaction mix:
1. Template DNA
2. Master Mix
• PCR Buffer
• dNTPs
• MgCl2
• Taq polymerase
• Forward Primer
• Reverse Primer
3. Thermocycler (PCR machine)
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Materials required for PCR

12. PCR amplification of DNA occurs by repeated cycles (25-30) of three
temperature dependent steps
Denaturation
The dsDNA template is denatured by briefly heating the
sample to 92-940C.
Annealing:
Oligonucleotide primers are annealed to the ssDNA template
when lowering the temperature at about 45-550C.
To ensure adequate specificity, the primers must be 20-30
nucleotides long.
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Steps of PCR

13. Primer extension (Polymerization):
DNA pol extends each primer in the 5' to 3' direction (fragment
between the primers) at a temperature of 72°C
Requires thermo-stable DNA pol that withstand repeated
exposure to high temperatures.
The rate of extension is about 50-100 nts/sec.
Thus, the time required for primer extension depends on the
length of the sequence to be amplified.1/17/2015 13
Steps of PCR Cont’d

14. Steps of PCR
Steps of PCR
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15. Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
5’3’
3’5’
3’5’
5’
5’3’
5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’
PCR
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16. Melting
94 oC
Temperature
100
0
50
T i m e
5’3’
3’5’
PCR Cont’d
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17. PCRMelting
94 oC
Temperature
100
0
50
T i m e
3’5’
5’3’
Heat
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18. PCRMelting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94 oC
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19. PCRMelting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
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20. PCRMelting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
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21. PCRMelting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat
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22. PCRMelting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’
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23. Fragments of
defined length
PCRMelting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’
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24. DNA Between The Primers Doubles With
Each Thermal Cycle
0
Cycles
Number
1
3
8
2
4
1
2
4
16
5
32
6
64
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25. 1/17/201525

26. • It is 2n x y
• Where:
y = the starting number of copies &
n = the number of thermal cycles
E.g. If we start with 1 copy, how many copies are made in 30 cycles?
o 2n x y
o 230 x 1
o 1,073,741,824 x 1
o 1,073,741,824
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Theoretical yield

27. 1/17/201527

28. 2.0. Gel Electrophoresis
 It refers to migration of all charged solutes in a medium under the
influence of electrical field
 Application of electrophoresis
 To determine the presence or absence of PCR products
 To quantify the size of PCR products
 Separating DNA of different Lengths
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29. 1. Gel casting apparatus
a. Casting tray,
b. Comb
c. Support
2. Support medium
It may be paper or gel made up of agarose, cellulose acetate, starch, or
polyacelamide
3. Buffers
 Types of buffers
Running/Electrophoresis buffer:
 Usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
Instrumentation & reagents of electrophoresis
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30. Loading buffer/sample buffer
It contains something dense (glycerol or sucrose) to allow the
sample to "fall" into the sample wells
It also contains one or two tracking dyes, which migrate in the gel
to allow visualize how far the electrophoresis run.
For example, bromophenol blue & xylene cyanol are used.
The DNA samples are loaded into the wells of the agarose as 10-
20% glycerol solutions1/17/2015 30
Instrumentation & reagents of electrophoresis Cont’d

31. 4. Stains
a. Ethidium bromide,
 a fluorescent dye used for staining nucleic acids.
b. Coomassie Brilliant Blue,
Staining for proteins
Transilluminator (Uv-light box)
 Used to visualize etbr-stained DNA in gels.
6. Power supplies
 It is a deriving force
 To supply electric current1/17/2015 31
Instrumentation & reagents of electrophoresis Cont’d

32. 1/17/2015 32
Electrophoresis procedure

33. 1/17/2015
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34. Gel Electrophoresis
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35. RunGelUvlightbox
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36. 1/17/2015 36
 The intensity of the band is proportional to
the amount of DNA found in the band
The upper band has far less
DNA when compared to the lower
band. The intensity of the bands
are proportional to the amount of
DNA at that position in the gel

37. f. INTERPRETATION:
The sizes of the various fragments can be identified by including a
“ladder” in the gel
A ladder is a mixture of DNA fragments of known size
It is usually run beside the unknown sample so that the sizes of
various DNA fragments in the sample can be identified
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38. Sizing a Gel Product
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Base
Pairs
(bp)
4000
3000
2000
1600
1000
500
1Kbp Sample
ladder
2000 bp
1000 bp

39. The electric field: an increase in voltage will therefore increase the
rate of migration proportionally
Size of DNA: the rate of migration decreases for larger molecules,
due to the increased frictional & electrostatic forces exerted by the
surrounding medium.
Concentration of the gel
• By increasing the agarose concentration the smaller DNA fragments
will give a clearer separation
• By lowering the agarose concentration the larger fragments of DNA
will give a clearer separation
• By optimizing the % agarose one can clearly separate a mixture of
similar DNA fragments
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Factors affecting electrophoresis

40. References
Wilson K. and Walker J . Principles and techniques of biochemistry and
molecular biology. Cambridge University press, UK.7th ed.
http://www.slideshare.net/harshit172/agarose-gel-electrophoresis-
25523393.
http://www.powershow.com/view/14e926-
YmYwM/Agarose_Gel_Electrophoresis_powerpoint_ppt_presentation.
http://www.slideshare.net/AYSHA007/copy-of-pcr
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41. THANK YOU
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