POLYMERASE CHAIN REACTION

(PCR):
PRINCIPLE, PROCEDURE &
APPLICATION

Elliot Kidd
 Polymerase Chain Reaction (PCR):

• Used for amplifying particular segments of DNA

• Enzymatic method carried in vitro
• Quick and inexpensive
• Requires small amount of starting material
 PCR Applications:
• Forensic science: DNA finger printing, paternity testing
and criminal identification
• Diagnosis: Molecular identification of microorganisms
• Gene cloning and expression
• Gene sequencing
• Vaccine production by recombinant DNA technology
• Drug discovery
• Mutation study
• Human genome project
What do we need?
Contents of tube:
1. Template DNA
2. Buffer containing MgCl2
3. Deoxynucleosides triphosphate
(dNTPs)- dATP, dCTP, dGTP,dTTP
4. Two primers (forward & reverse)
5. DNA polymerase (Thermus
Aquatiqus polymerase) and buffer
Thermo cycler
Template DNA

Taq polymerase

Primers
dNTPs
 Optimisation of PCR Conditions
1. Template DNA
• Purity
• Concentration
2. dNTPs
• Concentration
3. Two primers (forward & reverse)
• 15-30 bp long
• Concentration (usually around 500nM)
• G+C content =40-60%
• Similar Annealing/Melting temperature (TM)
• Tm = 4(G+C) +2(A+T)
4. DNA polymerase (Taq polymerase) and buffer
• Concentration of enzyme
• [Mg2+]
Typical PCR program

Denaturation
Primer extension

Primer Annealing
 What next???

Agarose Gel Electrophoresis:

• Used to separate nucleic acids based on

their size.

• Moves negatively charged molecule

through an agarose matrix in an electric
field toward the positive pole.

• Shorter molecules move faster and

migrate farther than the longer ones.
We need…

Add: Load samples & DNA Apply voltage

1. Dye to visualise marker into gel
DNA under UV light, Visualise under UV
for example ethidium
bromide (EtBr)
2. Colour marker dye 700 bp
600 bp
(usually blue) to track 500 bp
400 bp
progress of 300 bp
electrophoresis 200 bp
100 bp
Summary Protocol for PCR
 PCR in a diagnostic setting

How do we actually genotype or diagnose someone?

• PCR is a qualitative method

•PCR alone may not tell us the presence of a particular

mutation.

• Length polymorphism such as Short Tandem repeats are

detectable.

• Point mutations require extra steps.

• Restriction Fragment Length polymorphisms
• Direct sequencing
 Restriction Enzymes
• Enzymes that cut DNA at highly specific sites
• eg HpyCH4V =

• Lots of different RE exist with unique sequences

exist
• If a single base is altered, the enzyme will not cut at the
site.
• Very useful for detecting SNPs that are within such sites
• By incubating PCR products with certain restriction
enzymes, and then running the samples on a gel – we
can genotype individuals by observing banding patterns
on an agarose gel 
Scenario 1: Single Nucleotide Polymorphisms in the
Malic Enzyme 2 gene has been associated with
adolescent onset, familial epilepsy
(Greenberg et al., 2005, Lenzen et al., 2005). For
example rs585344. A C at this position is the healthy
allele, a T is the disease allele.
Diagnosis of susceptibility alleles is very useful for
correct drug regimens and for advice to families and
their affected relatives.

How would we design a PCR experiment to detect this

SNPs?
Malic Enzyme 2(Me2)

C = Healthy
Gene: 16 exons allele
T = disease allele
5’
5’ttctaatctcagctcttcagtgacttccccgctgtaagcTtgtcaattaaaaactgc
ttcgtaattcaaggacaaattctaatctcagctcttcagtgaacactggtgtaatagta
tgtaaacgcacactggaatcaccagggagctttaaacaaattgatgcctaccttata
ccaataaaacaagaatgttccgatatgggtctccaacacccatgtttcggaatcacc
agggagctttaaaca – 3’
Malic Enzyme 2(Me2)

C = Healthy allele
Gene: 16 exons T = disease allele

5’ ttctaatctcagctcttcagtGACTTCCCCGCTGTAAGCTGtcaattaaa
aactgcttcgtaattcaaggacaaattctaatctcagctcttcagtgaacactgg
tgtaatagtatgtaaacgcacactggaatcaccagggagctttaaacaaattg
atgcctaccttataccaataaaacaagaatgttccgatatGGGTCTCCAAC
ACCCATGTTTCggaatcaccagggagctttaaaca
HpyCH4V
 Malic Enzyme 2(Me2)

Gene: 16 exons C = Healthy allele

T = disease allele
5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’
Amplicon will be 200bp long
If the T allele is present, the RE will cut the amplicon approximately in half
Primer Sequence
Forward primer 5’ GACTTCCCCGCTGTAAGCTG 3’
Reverse primer 5’ GAAACATGGGTGTTGGAGACCC 3’

Always write primers in the 5’ to 3’

direction!!!
Cycle 1
5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
95°C
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’

5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’CCCAGAGGTTGTGGGTACAAAG 5’

58°C 5’ GACTTCCCCGCTGTAAGCTG 3’
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’

5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’CCCAGAGGTTGTGGGTACAAAG 5’
72°C

5’ GACTTCCCCGCTGTAAGCTG 3’
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’
 Cycle 2 and so on
5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’

3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’
95°C
5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’

5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’CCCAGAGGTTGTGGGTACAAAG 5’
5’ GACTTCCCCGCTGTAAGCTG 3’
58°C 3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’
and
72°C 5’ GACTTCCCCGCTGTAAGCTG........... GGGTCTCCAACACCCATGTTTC 3’
3’CCCAGAGGTTGTGGGTACAAAG 5’

5’ GACTTCCCCGCTGTAAGCTG 3’
3’ CTGAAGGGGCGACATTCGAC..........CCCAGAGGTTGTGGGTACAAAG 5’
• Once PCR is completed – incubate with
restriction enzyme and buffer (in this case
HpyCH4V) at 37° C for approximately 1
hour.
• Run the samples on an agarose gel
• Evaluate the results

• What are the

genotypes for these
individuals?
 How Are Crimes Solved by PCR?
Scenario 2: The body of a woman is found behind an
abandoned warehouse on the outskirts of town. Forensic
experts carry out a technical examination of the scene
and suggest strangulation as the cause of death. The
absence of bloody footprints or weapons means there
are no obvious leads to the killer. The experts carefully
dust down the body and its surroundings for fingerprints,
hairs and clothing fibres, possibly containing skin and
bodily fluids. This material is taken back to the lab and
thoroughly examined. Skin and hair particles found under
the victim’s fingernails probably belong to the killer,
indicating that a struggle took place.
 How would PCR can be used in this scenario?

Extract DNA

Perform PCR

Agarose gel electrophoresis

 How would PCR can be used in this scenario?

• Humans share 99.9% of their DNA with each other

• Only 0.1% of total genetic sequence differ between

individuals

• Short Tandem Repeats (STR)- short (2-5 nt) sequences

that repeated many times eg “TATATATATATA”

• 1 in 10 million will have a particular STR profile

 How would PCR can be used in this scenario?

Extract DNA: Crime scene: skin

and hair under
fingernails

Perform PCR: Design primers for

STRs

Agarose gel electrophoresis:

Summary

PCR is specific to researchers needs, straightforward

but powerful.

Many applications including genotyping, mutation

detection and expression analysis.