POLYMERASE CHAIN Ayu Werawati (5416221078)
REACTION Ikra Nurrohman (5416221089)
Marybet T.R.H (5416221098)
(PCR) Pangartika H. (5416221105)
The most common techniques used in
molecular biology → DNA replication
Developed by Kary Mullis (1980s)
PCR principle: to amplify small quantities
of DNA to obtain millions of copies of DNA
molecules (molecular photocopying)...
... using the ability of DNA polymerase
enzyme to synthesis new strand of DNA
complementary to the template strand of
DNA
PCR depends on a series of 20 – 40
repeated cycles of DNA replication →
the number of DNA strands is doubled
after each cycle
WHAT is PCR?
COMPONENT OF
PCR
dNTPs
Primers
Single units of the base A, T,
Short pieces of single-stranded DNA (usually 20 to 30 G, and C, which are
nucleotides) that are complementary to the target essentially ‘building blocks’
sequence, and provides free 3’ hydroxyl groups to for new DNA strands.
which the polymerase can add additional dNTPs.
Primers determine the beginning and end of the
amplified region.
(deoxynucleotide triphosphates)
• dATP
• dGTP
• dCTP
• dTTP
DNA polymerase
A type of enzyme that synthesizes new strands of
DNA complementary to the target sequence by adding
nucleotides (dNTPs), started from the end of the
primer. When a dNTP binds to DNA polymerase, the
DNA template enzyme undergoes a conformational change so that
only the base on the template strand can have a
DNA sample that contains the target sequence. complementary nucleotide fit (C – G, T – A).
DNA template
Sources of DNA:
• Whole blood, blood cells, body fluid
• Skin
• Hair
• Saliva
• Microbes
• Cultured cells
• Stool
• Human / animal tissues
Steps of nucleic acids extraction:
• Mechanical disruption: using homogenizer, mortar & pestle;
depend on the starting materials
• Lysis of cells: detergent to disrupt cell membranes; protease to
degrade proteins
• Organic extraction: phenol / chloroform to denature and extract
proteins → centrifugation → aqueous phase contains water soluble
molecules (including nucleic acids)
• Nucleic acid precipitation: addition of alcohol and salt to
precipitate nucleic acids from aqueous phase
Buffer
Provide an optimal chemical environtment (pH and salt
condition) for the polymerase
MgCl2
Catalyst the reaction of the polymerase
Additives
Improve the amplification efficiency and specivity
PCR PROCESS
Double-stranded DNA melts open to single-stranded DNA
Primers anneal to their complementary
sequences
As the DNA strand separates, a primer
with a free 3’ hydroxyl group anneals to
its specific template sequences.
DNA polymerase attach at each priming site...
... add free deoxynucleotides onto the hydroxyl group via phosphoryl transfer
reaction to elongate the new strand in a 5’ to 3’ direction ...
... and synthesize the new complementary DNA
strand.
New templates Denaturation
Elongation Annealing
How to analyze?
End-Point PCR
• Conventional PCR amplicon is detected in an
end-point analysis
• Agarose gel visualization using gel electrophoresis
technique
• Mostly used to detect presence or absence of
targets, and also to estimate relative quantity
• Application: sequancing, genotyping, sequence
detection
Quantitative PCR
• Indicates how much of a specific DNA or gene is present
in the sample (detection + quantification)
• The PCR products are continuously detected
throughout the reaction cycles by either intercalating
or probe based fluorescent dyes
• The fluoresence signal increases proportionally as
product amount increases
• Much more accurate in quantifying than end-point PCR
• Application: gene expression profiling, molecular
diagnostic, genotyping
Digital PCR
• The only absolute quantitative PCR method available
• It measures the actual number of target molecules produced as
opposed to comparing the measurement to a reference
Gel Electrophoresis
Agarose gel preparation
Move the finished gel to
electrophoresis chamber
Migration of DNA
molecules from negative
to positive charged pole
Standardized molecular
Separation of double- marker to help determine the
stranded DNA by size size of DNA fragments
and charge
DNA
Staining (ethidium bromide,
Ladder
SYBR green)+ UV
transillumination
Gel electrophoresis allows
to determine the
presence and the size of
DNA (PCR product)
Real Time PCR
(Lamp, LED, laser)
Cq = quantification cycle =
cycle number at which enough
amplified product accumulates to
yield a detectable fluorescence
signal inversely correlated to the
amount of template in sample
qPCR Standard Curve
Y = a + bX;
Y = log quantity
X = Cq
Digital PCR
TYPE OF PCR
Standard PCR
Relies on standard Taq polymerase
Sufficient for application like genotyping or colony
screening which only require sequence ditection
Hot-Start PCR
Using hot-start polymerases, which reduces non-specific
amplification at the lower temperature steps of the PCR cycle,
especially during the original setup; often preffered for PCR
application that require specific detection, such as genotyping
or clinical application
High-Fidelity PCR
Using a proofreading enzyme. This method is required for
application in which target DNA needs to be accurately reproduced,
such as for cloning or sequencing
Multiplex PCR
This type of PCR can be run on many targets within one tube to
save time, effort, and reagents. Primer design and PCR
optimization is more complex
GC-Rich PCR
This type of PCR can be performed with specific
reagents developed to handle temperatures with high
GC content which are harder to amplify in general
Reverse Transcriptase PCR
• RNA detection; require reverse transcription step to
synthesize a complementary cDNA before amplification of
DNA by PCR is possible
• The cDNA is made by a RNA dependent DNA polymerase
(reverse transcriptase) using specific primer and dNTPs
then followed by amplification of the cDNA by PCR
• Two Step RT-PCR: RT-step and PCR amplification in
different tube
• One Step RT-PCR: combination of RT-step and PCR
amplification in one tube
Nested PCR
• Two pairs of primers are used for one region
• The first primer pair amplified a region as in any PCR
• The second primer pair is locates within the first PCR product
and amplified that PCR product that will be shorter than the first
one (increase sensitivity
PCR
APPLICATION
Result:
Forensic
Application
THANK YOU!
