Polymerase chain reaction
Technique involves a single copy or a few copies of a piece of DNA across several orders OF
magnitude, generating
millions of copies of a particular or targeted DNA sequence.
PCR uses thermal cycler (regulates temperature and time) for continuous denaturation and
amplification of DNA
molecule using dNTPs and thermostable DNA Polymerase
DENATURATION
Splitting of duplex DNA to its single strands by complete hydrogen bonds breakage is termed as denaturation.
Denaturation temperature in PCR can be defined as- “ The temperature of irreversible strand separation of a
homogenous population of DNA molecule
Denaturation temperature of a dsDNA is determined by its G+C content and length of the template. Generally
denaturation is performed at 93- 95 °C (usually > 90 °C). 95 °C is highest temperature that Taq polymerase can
endure for 30 cycles.
Denaturation sometime performed for 5-10 minutes before starting the denaturation of first cycle (Initial denaturation),
in order to increase the probability that long molecule of template DNA are fully denatured
ANNEALING
It is a critical step in PCR and depend on primers deployed in reaction.
Annealing temperature ranges from 50-70 °C. In this step two synthetic oligonucleotide primers bind with flanking
complementary sequence of target region of template DNA.
Annealing temperature (Ta ) depend on melting temperature (Tm) of oligonucleotide primers. Annealing temperature is typicall
about 3-5 °C lower than calculated melting temperature of oligonucleotide primers
EXTENSION
Extension at 3’ end of primer is performed at or near the optimum temperature of thermostable DNA polymerase (72-78°C) .
Extension is performed for 1 min for every 1000 bp of product.
In the first two cycles, extension from one primer proceed beyond the sequence complementary to the binding site of other
primer. In the next (third) cycle, first mol. will start to produce whose length is equal to segment of DNA delimited by binding
site of primers
TOUCHDOWN PCR
Determination of Tm (melting temperature) from Wallace and other equations are approximations and may not work
under actual conditions
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a type of PCR which offer
simple and rapid mean of optimization of PCR conditions, specificity and sensitivity.
Otherwise lengthy optimization procedure or redesigning of primers.
It employ higher initial annealing temperature than calculated melting temperature of primer being used.
Touchdown increases specificity of the reaction at higher temperatures and increases the efficiency towards the end b
lowering the annealing temperature
It is particularly useful in increasing the specificity of PCR, it uses a cycling program where the annealing temperature
gradually reduced (e.g. 1-2°C /every second cycle).
Korbie, Darren J., and John S. Mattick. "Touchdown PCR for increased specificity and sensitivity in PCR amplification."
Nature protocols 3.9 (2008): 1452-1456. The initial annealing temperature should be several degrees above the
estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated
annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing
temperature.
The primer will anneal at the highest temperature which is leastpermissive of nonspecific binding that it is able to
tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most like
that this is the sequence of interest. In touchdown PCR, the annealing step initially set 5-10 °C higher than
calculated Tm of primer and in subsequent cycles, the annealing temp. is gradually decreased by a small amount so th
by end of PCR, the annealing temperature will 2-5 °C below the calculated Tm of primers.
Technique involves a single copy or a few copies of a piece of DNA across several orders OF
magnitude, generating
millions of copies of a particular or targeted DNA sequence.
PCR uses thermal cycler (regulates temperature and time) for continuous denaturation and
amplification of DNA
molecule using dNTPs and thermostable DNA Polymerase
DENATURATION
Splitting of duplex DNA to its single strands by complete hydrogen bonds breakage is termed as denaturation.
Denaturation temperature in PCR can be defined as- “ The temperature of irreversible strand separation of a
homogenous population of DNA molecule
Denaturation temperature of a dsDNA is determined by its G+C content and length of the template. Generally
denaturation is performed at 93- 95 °C (usually > 90 °C). 95 °C is highest temperature that Taq polymerase can
endure for 30 cycles.
Denaturation sometime performed for 5-10 minutes before starting the denaturation of first cycle (Initial denaturation),
in order to increase the probability that long molecule of template DNA are fully denatured
ANNEALING
It is a critical step in PCR and depend on primers deployed in reaction.
Annealing temperature ranges from 50-70 °C. In this step two synthetic oligonucleotide primers bind with flanking
complementary sequence of target region of template DNA.
Annealing temperature (Ta ) depend on melting temperature (Tm) of oligonucleotide primers. Annealing temperature is typicall
about 3-5 °C lower than calculated melting temperature of oligonucleotide primers
EXTENSION
Extension at 3’ end of primer is performed at or near the optimum temperature of thermostable DNA polymerase (72-78°C) .
Extension is performed for 1 min for every 1000 bp of product.
In the first two cycles, extension from one primer proceed beyond the sequence complementary to the binding site of other
primer. In the next (third) cycle, first mol. will start to produce whose length is equal to segment of DNA delimited by binding
site of primers
TOUCHDOWN PCR
Determination of Tm (melting temperature) from Wallace and other equations are approximations and may not work
under actual conditions
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a type of PCR which offer
simple and rapid mean of optimization of PCR conditions, specificity and sensitivity.
Otherwise lengthy optimization procedure or redesigning of primers.
It employ higher initial annealing temperature than calculated melting temperature of primer being used.
Touchdown increases specificity of the reaction at higher temperatures and increases the efficiency towards the end b
lowering the annealing temperature
It is particularly useful in increasing the specificity of PCR, it uses a cycling program where the annealing temperature
gradually reduced (e.g. 1-2°C /every second cycle).
Korbie, Darren J., and John S. Mattick. "Touchdown PCR for increased specificity and sensitivity in PCR amplification."
Nature protocols 3.9 (2008): 1452-1456. The initial annealing temperature should be several degrees above the
estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated
annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing
temperature.
The primer will anneal at the highest temperature which is leastpermissive of nonspecific binding that it is able to
tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most like
that this is the sequence of interest. In touchdown PCR, the annealing step initially set 5-10 °C higher than
calculated Tm of primer and in subsequent cycles, the annealing temp. is gradually decreased by a small amount so th
by end of PCR, the annealing temperature will 2-5 °C below the calculated Tm of primers.
