POLYMERASE CHAIN REACTION

( Replication in vitro )

AJI SUTRISNO
What is Polymerase Chain
Reaction (PCR)?

DNA replication can also be performed

in vitro (artificially, outside the cell)

PCR is a technique to replicate DNA

in vitro.

replicate = copy = multiply = amplify

 Practical Properties of DNA

Intrinsic properties of DNA hold true even

in a test tube.
DNA heated from 90C to 95C; the two
strands separate. The nucleotides can be
identified, replicated, or transcribed.
Slowly cooling the DNA allows
complementary nucleotides to hydrogen
bond and the DNA will regain double-
stranded form.

3
Why Polymerase ?

It is called polymerase because

DNA polymerase is used to multiply the DNA.
Why Chain Reaction ?

It is called chain reaction because this

technique involves repeating different heating
and cooling cycles over and over again, as many
as 30 or more times

the products of the first reaction become

substrates of the following reaction, and so on.

Results in rapid increase in the product

History of PCR
PCR technique was invented by Kary
Mullis and co-workers in 1985.

Goal:
Make millions of copies of DNA from trace
amounts of DNA starting material.

Only specific pieces of DNA are amplified.

DNA Replication: Prokaryotic
origin of replication

1 origin of replication;
2 replication forks
Uses for PCR
Research Clinical
Gene cloning DNA fingerprinting
Crime scene
analysis
Real-time PCR Paternity testing
Archeological finds
DNA sequencing
Genetically
Cancer studies inherited diseases

GMO / GMF identification

Microorganism identification
Uses for PCR in Food Technology
DNA-based detection (PCR):
- analysis food borne pathogens
- analysis food spoilage microorganisms
- analysis genetically modified food
- allergen
- dsb
PCR specific, sensitive and rapid
CURRENT STATUS
Today, a range of pathogens can be analyzed with
rapid PCR systems, depending on the individual
system. These include
Salmonella spp., Listeria spp., Listeria
monocytogenes, Campylobacter jejuni,
Cryptosporidium parvum, E. coli O157 and E.
coli O157:H7
Additionally, PCR tests can detect spoilage
organisms in beer and can qualitatively and
quantitatively detect genetically modified organisms
(GMOs) in soy and maize, as well as used for
general GMO screening of other food types.
Impact of PCR

PCR has transformed molecular biology

through vastly extending the capacity to
identify, manipulate and reproduce DNA.

-Paul Rabinow, UC Berkeley

Making PCR, A Story of Biotechnology, University of Chicago
Press, 1996
What components you need in
PCR?
Components include:
Heat-stable DNA polymerase (ex: Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Roles of PCR Reagents
Taq polymerase
Enzyme that extends growing DNA strand
complementary to DNA template

MgCl2
Provides ions needed for enzyme reaction

dNTPs
Nucleotides (Adenine, Cytosine, Guanine,
Thymine) building blocks for new DNA strands

Buffer
Maintains optimal pH for enzyme
Roles of PCR Reagents
Primers
Anneal to single-stranded DNA template
Provide initiation site for extension of new
DNA
Forward primer
Anneals to DNA anti-sense strand
Reverse primer
Anneals to DNA sense strand

DNA template
In this case, the product of our DNA extraction
How does PCR work?
One PCR Cycle:
How does PCR work?

One PCR cycle: What the products

really looks like

Primers Template Strand

4 DNA strands
Template Strand

Biology Animation Library: http://www.dnalc.org/ddnalc/resources/pcr.html

How does PCR work?

Two cycles: What the products really

looks like

8 DNA strands
 How does PCR work?

Three cycles

16 DNA strands

Notice the production of double stranded, shortened PCR products (target sequence) that spans
the two primers.
 How does PCR work?

Four cycles

32 DNA strands
The number of DNA strands doubles after each cycle. Target sequence predominates.
Amplification
Data for PCR
How does PCR work?

After 30
cycles

Target sequence increases exponentially.

Three steps for each PCR cycle
Each PCR cycle includes:
A denaturation step (92-96oC) separates
the two DNA strands.

A primer annealing step (40-75oC) which

is a few degrees below the Tm of the
primers.

A primer extension step (72oC) which is

the optimal temperature for Taq DNA
polymerase activity.
Reaction Conditions
94oC for 2 minutes
Followed by 30 cycles of:
94oC for 40 seconds
48oC for 2 minutes
72oC for 3 minutes
Followed by 1 cycle of:
72oC for 3 minutes.

Thermocycler
Annealing Temperature
The annealing temperature (Ta) chosen for
PCR relies directly on length and composition
of the primers. Generally, an annealing
temperature is about 5C below the Tm of
primers. The optimal annealing temperature
(Ta Opt) for any given primer pair on a
particular target can be calculated as follows:
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of
product) - 25; where Tm of primer is the
melting temperature of the less stable primer-
template pair, and Tm of product is the
melting temperature of the PCR product.
PCR Thermocycler
QUESTION ???
Components for PCR
Heat-stable DNA polymerase (Taq
or Pfu polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Heat-stable DNA polymerase

Taq DNA polymerase

was isolated from
the bacterium
Thermus aquaticus.

Taq polymerase is Hot springs at Yellowstone

stable at the high National Park, Wyoming.
http://waynesword.palomar.edu/lmexer3b.htm

temperatures (~95oC)
used for denaturing
DNA.
Properties of DNA polymerases
Value for indicated DNA polymerase
Property KOD DNA Pfu DNA Taq DNA
polymerase polymerase polymerase
Origin Archaea Archaea Bacteria

Optimum temperature
75 75 75
(C)
Optimum pH at 75C 6.5 6.5 8.0-8.5
Thermostability 95C,12 h; 95C, 6h;
95C, 1.6 h
(half-life) 100C,3.0 h 100C, 2.9h
5'3' exonuclease
- - +
activity
3'5' exonuclease
+ + -
activity
Terminal transferase
- - +
activity
Processivity (bases) >300 <20 ND
Elongation rate (bases/s) 106-138 25 61
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA
oligonucleotides)
Deoxynucleotides dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Primers
Two oligonucleotides of different
sequences.

Typically 18-25 nucleotides long.

Primers complementary base pair

(hybridize or anneal) to template DNA.

General Example of Primers

 Example of Primers

3 TACTTCAAACCTTTATAAAC 5
5 CACCATGAAGTTTGGAAATATTTG 3
(Forward Primer)
(Reverse Primer)
3 TTTTAGCTTTACTTAAATGG 5
5 AAAATCGAAATGAATTTACC 3
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides dATP, dTTP,
dCTP, dGTP
DNA template
Mg++, buffer components, and water
Deoxynucleic Acids
dATP, dTTP, dGTP and dCTP should be
present in equal amounts.

10X dNTP mix is the least stable

component.
Store frozen in small aliquots
Keep dNTPs on ice!
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Template DNA

1ng-1ug template DNA

Higher concentrations for total genomic
Lower concentrations for plasmid DNA
Template DNA
Always add template DNA last to your
reaction vial to avoid contamination.

Always run controls

(+) cloned template (if available)
(-) water only control
(-) vector only control (pGEM)
(-) forward primer control
(-) reverse primer control
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and
water
Mg++, Buffer, and Water
Mg+2 is an essential cofactor for Taq &
Pfu DNA polymerase activity. Final
[Mg+2] = 1.5mM

10X PCR buffer=100mM Tris, pH 8.3 +

500mM KCl.
Mg++, Buffer, and Water
Water should be ultrapure (MilliQ water)
with no salts or DNA contamination.

Template DNA and primers should be

resuspended in MilliQ water to avoid
high concentrations of EDTA.
PCR mix (an example)
PCR components Amount
Template DNA (5-200 ng) variable
1 mM dNTPs (200 uM final) 10 uL
10 X PCR buffer 5 uL
25 mM MgCl2 (1.5 mM final) 3 uL
20 uM forward primer (20 pmoles final) 1 uL
20 uM reverse primer (20 pmoles final) 1 uL
5 units/uL Taq DNA polymerase (1.5 units) 0.3 uL
Water Variable
Final Volume 50 uL
Considerations
Contamination can easily lead to
erroneous results
Avoid contaminating with DNA or PCR
product
DNA stocks, PCR reagents
Gloves, tips, pipetters, benches

Carefully measure reagent quantities

Use appropriate cycling conditions
 16S rDNA Isolation and Amplification
Forward Primer
BSF8/27 : AGAGTTTGATCCTGGCTCAG PCR
95 2 min
Reverse Primer

}
95 30 sec
1512aR : ACGGTTACCTTGTTACGACTT 55 1 min 30 cycles
72 2 min
72 5 min

4500 bp

1500 bp

16S rDNA
pGEM-16S rDNA
PCR applications

Primer name Sequence (5'-3') Reference

8F AGA GTT TGA TCC TGG CTC AG [8][9]

U1492R GGT TAC CTT GTT ACG ACT T same as above

928F TAA AAC TYA AAK GAA TTG ACG GG [10]

336R ACT GCT GCS YCC CGT AGG AGT CT as above

1100F YAA CGA GCG CAA CCC
1100R GGG TTG CGC TCG TTG
337F GAC TCC TAC GGG AGG CWG CAG
907R CCG TCA ATT CCT TTR AGT TT
785F GGA TTA GAT ACC CTG GTA
805R GAC TAC CAG GGT ATC TAA TC
533F GTG CCA GCM GCC GCG GTA A
518R GTA TTA CCG CGG CTG CTG G
Phylogenetic tree
Any questions?????

THANK YOU