Pcr n optimization

What is PCR ?
 The polymerase chain reaction (PCR) is a technology
in molecular biology used to amplify a single copy or a few
copies of a piece of DNA.
 Though PCR occurs in vitro, or outside of the body in a
laboratory, it is based on the natural process of DNA
replication.
 It was invented in 1983 by Dr. Kary Mullis, for which he
received the Nobel Prize in Chemistry in 1993.

Why “Polymerase” ?
 Called “polymerase” because the only enzyme used in this
reaction is DNA polymerase.
Why “Chain”?
 Called “chain” because the products of the first reaction
become substrates of the following one, and so on.

Contd….
 So the early procedures for DNA replication were :
Very inefficient
Time consuming
Required large amounts of DNA polymerase
Continual handling throughout the process

Discovery of Taq Poymerase:
 PCR method uses a suitable DNA polymerase able to
withstand the high temperatures of >90 °C required
for separation of the two DNA strands after each
replication cycle.
 DNA polymerases initially employed for in vitro
experiments presaging PCR were unable to withstand
these high temperatures.

Taq polymerase:
 In 1976 discovery of Taq polymerase_ a DNA polymerase
purified from the thermophilic bacterium, Thermus
aquaticus
 Naturally lives in hot (50 to 80 °C (122 to 176 °F))
environments such as hot springs — paved the way for
dramatic improvements of the PCR method.
 Taq polymerase :
 Stable at high temperatures
 Remaining active even after DNA denaturation

 Mullis summarized the procedure:
"Beginning with a single molecule of the genetic material
DNA, the PCR can generate 100 billion similar molecules.
It requires no more than a test tube,
a few simple reagents, and a source
of heat."

The Reaction Components:
1. Target DNA - contains the sequence to be amplified.
2. Pair of Primers -are short pieces of DNA that are
made in a laboratory.
 These are complementary to the 3' (three prime) ends
of each of the sense and anti-sense strand of the
DNA target.
 Primers are also necessary because DNA polymerase
can't attach at just any old place and start copying
away.
 It can only add onto an existing piece of DNA.

3. dNTPs - building blocks that DNA molecules are made of.
 Add a mixture of four types of nucleotides to your PCR
reaction—A's, C's, G's and T's.
 DNA polymerase grabs nucleotides that are floating in the
liquid around it and attaches them to the end of a primer.
4. Thermostable DNA Polymerase - enzyme that catalyzes the
reaction.
 It is a naturally occurring complex of proteins whose
function is to copy a cell's DNA before it divides in two.
5. Mg++ ions - cofactor of the enzyme
6. Buffer solution – maintains pH and ionic strength of the
reaction solution suitable for the activity of the enzyme

The Reaction :
PCR tube
Thermocycle

Steps of PCR:
Three major steps in a PCR, which are repeated for 20
to 40 cycles.
This is done on an automated Thermo Cycler, which
can heat and cool the reaction tubes in a very short
time.
1. Initialization step:
 Heating the reaction to a temperature of 94–96 °C for
1–9 minutes.
 Only required for DNA polymerases that require heat
activation by Hot-start-PCR.

Contd…
2. Denaturation step:
 Heating the reaction to 94–98 °C for 20–30 seconds.
 Causes DNA melting of the DNA template by
disrupting the hydrogen bonds.
3. Annealing step:
 Temperature is lowered to 50–65°C for 20–40 seconds
allowing annealing of the primers to the single-
stranded DNA template.
 Typically the annealing temperature is about 3-5
degrees Celsius below the Tm of the primers used.

Contd…
 Stable DNA-DNA hydrogen bonds are only formed when the primer
sequence very closely matches the template sequence.
 Polymerase binds to the primer-template hybrid and begins DNA
synthesis.
4. Extension/elongation step:
 Temperature at this step depends on the DNA polymerase used; Taq
polymerase has its optimum activity temperature at 75–80 °C.
 DNA polymerase synthesizes a new DNA strand complementary to
the DNA template strand by adding dNTPs.

Contd..
 Extension time depends on :
 DNA polymerase used
 Length of the DNA fragment to be amplified
5. Final elongation:
 Performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR
cycle to ensure that any remaining single-stranded DNA is fully extended.
6. Final hold:
 This step at 4–15 °C for an indefinite time may be employed for short-term
storage of the reaction.

PCR Stages:
 Exponential amplification:
 Initially, all the reagents employed in PCR are abundant and
the Taq polymerase is fully functional.
 In the initial phases of PCR, 100% efficient DNA
amplification, with the amount of replicated DNA doubled
per thermocycle .
 Levelling off stage:
 Reaction slows as the DNA polymerase loses activity and as
consumption of reagents such as dNTPs and primers causes
them to become limiting.
 Plateau:
 No more amplification occurs due to exhaustion of reagents
and enzyme.

Causes related to cycling times and temperatures:
Too few cycles were used:
 Using too few PCR cycles can lead to insufficient
amplification.
 Use 20-35 cycles.
 Use fewer cycles when template concentration is high,
and use more cycles when template concentration is low.
Extension time was too short:
 If the extension time is too short, there will be insufficient
time for complete replication of the target.
 Generally, use an extension time of 1 min/kb.

Annealing time was too short:
 If the annealing time is too short, primers are unable
to bind to the template.
 Use annealing time of at least 30 sec.
Annealing temperature was too high:
 If the annealing temperature is too high , primers are
unable to bind to the template.
Denaturation temperature was too low:
 If the denaturation temperature is too low, the DNA
will not completely denature and amplification
efficiency will be low.
 Use a denaturation temperature of 95ºC.

Denaturation time was too long:
 If the denaturation time is too long, DNA might be degraded.
 For the initial denaturation, use 3 min at 95.
 For the denaturation during cycling, use 30 sec at 95.
Denaturation time was too short:
 If the denaturation time is too short, the DNA will not completely
denature and amplification efficiency will be low.
 For the initial denaturation, use 3 min to activate the polymerase;
to denature the template during cycling, use 30 sec.

Causes related to PCR components :
dNTP concentration was too high:
 If the dNTP concentration is too high, Mg2+
depletion occurs.
 Each dNTP should be present at 200 µM in the final
reaction.
dNTP concentration was too low:
Each dNTP should be present at 200 µM in the final
reaction.

PCR product has high GC content (>65%):
 GC- rich PCR products are difficult to amplify. To improve amplification
increase the annealing temperature.
 For greater accuracy, optimize the annealing temperature by using a thermal
gradient.
Template was damaged or degraded or contained inhibitors:
 Template may be sheared or may contain PCR inhibitors.
 If inhibitors are suspected, dilute existing template; otherwise, use fresh
template and increase cycles.

Primers contained impurities:
 Contaminants in primers may inhibit PCR.
 Use desalted primers or more highly purified primers.
Not enough template was in the reaction:
 Insufficient amplification can result if the initial amount of template is too low.
 Increase the number of amplification cycles or, if possible increase the amount
of template.

Impure dNTPs were used:
 Contaminants in the dNTP mix can lead to incomplete
and incorrect amplification or PCR inhibition.
 Use high quality dNTPs.
Primer concentration was too high:
 Using an excessive concentration of primers can
increase the chance of primers binding nonspecifically
to undesired sites on the template or to each other.
Primer concentration was too low:
 If the primer concentration is too low, annealing may be
inefficient.

Enzyme concentration was too low:
 If the polymerase concentration is too low, not all PCR
products will be fully replicated.
 The optimal enzyme concentration depends on the
length and difficulty of the template.
Primers were designed and synthesized incorrectly
by user or manufacturer:
 Verify that primers have the correct sequence and are
complementary to the template.
 Use a primer design program to avoid repetitive
sequences, regions with high complementarity, etc.

 Target was too long:
 PCR components concentration and/or cycling condition may not be sufficient
for longer target sequences.
 Re-optimize your existing assay protocol and/or increase the duration of PCR
steps, especially the extension step.
 Water was impure:
 Water could have been contaminated during prior pipetting events.
 Use fresh nuclease-free water.
 Not enough Mg2+:
 Insufficient or omitted magnesium will result in no or reduced PCR product.
 Use 1.5 µM in the final reaction.

Causes related to omitted components:
dNTPs were omitted:
 Each dNTP should be present at 200 µM in the final
reaction.
Primer was omitted:
 Use well-designed primers at 0.2-1 µM in the final
reaction.
Template was omitted:
 Add template.

Enzyme was omitted or inactive:
 Use adequate units of enzyme.
 If you think the enzyme may be inactive, run PCR with fresh
polymerase from a different batch.
Mg2+ was omitted:
 Insufficient or omitted magnesium will result in no or reduced PCR
product.
 Use 1.5 µM in the final reaction.

NONSPECIFIC BAND OR PRIMER
DIMER :

Causes Related to Cycling Times and Temperatures:
 Too many cycles were used:
 Excessive cycling increases the opportunity for nonspecific amplification and
errors.
 Use fewer cycles when template concentration is low.
 when template concentration is high, and use more cycles.
 Use 20-35 cycles.
 Extension time was too long:
 Excessive extension time can allow nonspecific amplification.
 Generally, use an extension time of 1 min/kb.

Thermal cycler ramping speed is too slow:
 If the ramp speed of cycler is too slow, spurious annealing may occur due to
lower temperature and sufficient time for nonspecific binding.

Causes Related to PCR components:
 Primers contain impurities:
Contaminants in primers may inhibit PCR.
 You can try to dilute the primers to determine if inhibitory effects
exist.
 Too much primer was added:
 Using a high concentration of primers may increase the chance of
primers binding to nonspecific sites on the template or to each other.
 Use well-designed primers at 0.2-1 µM in the final reaction.

 Primers were designed or synthesized incorrectly by
user or manufacturer:
 Use a primer design program to avoid repetitive
sequences, regions with high complementarity, etc.
 Impure dNTPs were used:
 Contaminants in the dNTP mix can lead to incomplete or
incorrect amplification or PCR inhibition.
 Use high-quality dNTPs.

 Too much Mg2+ was added:
 Using high concentrations of magnesium increases the
likelihood of nonspecific primer binding and unwanted
product formation.
 Reduce the amount of magnesium in the final reaction.
 Impure water was added:
 Water could have been contaminated during prior
pipetting events.
 Use French nuclease-free water.

Causes Related to Cycling Times and
Temperatures:
 Too many cycles were used:
 Excessive cycling increases the opportunity for nonspecific amplification and
errors.
 Use 20–35 cycles.
 Extension time was too long:
 Excessive extension time can allow nonspecific amplification. Generally, use an
extension time of 1 min/kb.
 Annealing time was too long:
 Excessive annealing time may increase spurious priming. Use an annealing time
of 30 sec.

 Annealing temperature was too low:
 If the annealing temperature is too low, primers may bind
nonspecifically to the template.
 The rule of thumb is to use an annealing temperature that is
5°C lower than the Tm of the primer. For greater accuracy,
optimize the annealing temperature by using a thermal
gradient.
 Thermal cycler ramping speed is too slow:
 If the ramp speed of the cycler is too slow, spurious
annealing may occur due to lower temperature and
sufficient time for nonspecific binding.
 If ramping speed is not set at the maximum speed for the
cycler, increase to maximum ramp rate.

 Calculated primer Tmwas inaccurate:
 If the primer concentration is calculated incorrectly, the
calculated annealing temperature will also be incorrect.
 Use the lowest primer Tm when calculating the annealing
temperature.

Causes Related to PCR Components:
 Too much template was added:
 If the template concentration is too high, the polymerase
can be inhibited due to carryover of inhibitors or
inefficient denaturation.
 Reduce the number of cycles, reduce the template
concentration, and/or increase denaturation
time/temperature.
 Template contained an exonuclease or was degraded:
 Template may be sheared or contain exonuclease.
 Use a fresh template.

 Primers contained impurities:
 Contaminants in primers may inhibit PCR.
 You can try to dilute the primers to determine if inhibitory
effects exist, but do not add less than 0.02 μM of each
primer.
 Primers were designed or synthesized incorrectly by user
or manufacturer:
 Use a primer design program to avoid repetitive sequences,
regions with high complementarity, etc.
 Perform a BLAST search to avoid primers that could
amplify pseudogenes or that might prime unintended
regions.

 Impure dNTPs were used:
 Contaminants in the dNTP mix can lead to incomplete or
incorrect amplification or PCR inhibition.
 Use high-quality dNTPs.
 Water was impure:
 Water could have been contaminated during prior
pipetting events.
 Use fresh nuclease-free water.

Nested PCR
 Nested PCR is used to increase the specificity
of DNA amplification.
 It reduces the contamination in products due to
the amplification of unexpected primer binding
sites.
 Two sets of primers are used in two successive
reactions.

 In the first PCR, one pair of primers is used to generate
DNA products, which may contain products amplified
from non-target areas.
 The products from the first PCR are then used as template
in a second PCR, using one ('hemi-nesting') or two
different primers whose binding sites are located (nested)
within the first set, thus increasing specificity.
 Nested PCR is often more successful in specifically
amplifying long DNA products than conventional PCR,
but it requires more detailed knowledge of the sequence of
the target.

Inverse PCR:
 Used to amplify DNA with only one known
sequence.
 One limitation of conventional PCR is that it
requires primers complementary to both
termini of the target DNA, but this method
allows PCR to be carried out even if only one
sequence is available from which primers may
be designed.

Process :
1. A target region with an internal section of known
sequence and unknown flanking regions is identified.
2. Genomic DNA is digested into fragments by
restriction enzyme.
3. Under low DNA concentrations, self-ligation is
induced to give a circular DNA product.
4. PCR is carried out as usual, with primers
complementary to sections of the known internal
sequence.
5. Finally the sequence is compared with the sequence
available in the data base.

RT(Reverse Transcription)-PCR:
 In reverse transcription polymerase chain reaction
,amplifying DNA from RNA.
 First a RNA strand (template) is reverse transcribed into its
cDNA copy using reverse transcriptase, then cDNA is
amplified using PCR.
Applications : RT-PCR is widely used in the :
 diagnosis of genetic disorders
 expression profiling (to the expression of a gene or to identify
the sequence of an RNA transcript, including transcription start
and termination sites)
 also helps in obtaining eukaryotic exon sequences from
mature mRNAs.

qPCR (Real-time PCR) :
 Quantitative PCR (qPCR) used to measure the quantity of
a target sequence (commonly in real-time).
 It quantitatively measures starting amounts of DNA,
cDNA, or RNA.
 Quantitative PCR is commonly used to determine whether
 a DNA sequence is present in a sample
 the number of its copies in the sample
 Quantitative PCR has a very high degree of precision.

Contd…
 Quantitative PCR methods use fluorescent dyes, such
as
1. Sybr Green
2. EvaGreen
3. fluorophore-containing DNA probes, such
as TaqMan ,
 These dyes used to measure the amount of amplified
product in real time.
 qPCR is also sometimes abbreviated to RT-
PCR (Real-time PCR)

Miniprimer PCR:
 Uses a thermostable polymerase that can extend from short primers
as short as 9 or 10 nucleotides.
 It permits PCR :-
Targeting to smaller primer binding regions
Used to amplify conserved DNA sequences, such as the 16S rRNA
gene

In silico PCR:
 In silico PCR also called digital PCR, virtual PCR, electronic PCR,
e-PCR.
 It refers to computational tools used to calculate theoretical PCR
results using a given set of primers to amplify DNA sequences from
a sequenced genome.
 In silico PCR was proposed as an educational tool for molecular
biology.

Ligation-mediated PCR:
 This technique uses small DNA linkers ligated to the DNA of interest
and multiple primers annealing to the DNA linkers .
 It has been used for
DNA sequencing
Genome walking
DNA foot printing

Hot-start PCR:
 A technique that reduces non-specific amplification during the initial set up stages
of the PCR.
 It may be performed manually by heating the reaction components to the
denaturation temperature (e.g., 95 °C) before adding the polymerase.
 Specialized enzyme systems have been developed that inhibit the polymerase's
activity at ambient temperature, either by the binding of an antibody or by the
presence of covalently bound inhibitors that dissociate only after a high-temperature
activation step.

60
Helicase-dependent amplification:
 Similar to traditional PCR, but uses a constant
temperature rather than cycling through denaturation
and annealing/extension cycles.
 DNA helicase, an enzyme that unwinds DNA, is used
in place of thermal denaturation.

Allele-specific PCR:
 Diagnostic or cloning technique based on single-nucleotide polymorphisms (SNPs) .
 Requires prior knowledge of a DNA sequence, including differences between alleles, and
uses primers whose 3' ends encompass the SNP.
 PCR amplification under stringent conditions is much less efficient in the presence of a
mismatch between template and primer so successful amplification with SNP-specific primer
signals presence of the specific SNP in a specific sequence.

Multiplex-PCR:
 Multiplex-PCR uses several pairs of primers annealing to different target
sequences.
 This permits the simultaneous analysis of multiple targets in a single sample.
 For example,
 in testing for genetic mutations,
 six or more amplifications might be combined. In the standard protocol for DNA
Fingerprinting, the targets assayed are often amplified in groups of 3 or 4.

Applications:
1. Medical Applications:
 The first application of PCR was for genetic testing, where a sample of DNA is
analyzed for the presence of genetic disease mutations.
 PCR can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation.
 Many forms of cancer involve alterations to oncogenes . By using PCR-based tests
to study these mutations.

2. Infectious Disease Applications:
 The human immunodeficiency virus (or HIV), is a difficult target to find and
eradicate.
 PCR tests have been developed that can detect as little as one viral genome among
the DNA of over 50,000 host cells.
 Infections can be detected earlier, donated blood can be screened directly for the
virus, newborns can be immediately tested for infection, and the effects of antiviral
treatments can be quantified.

 Some disease organisms, such as that for tuberculosis, are difficult to sample from patients
and slow to be grown in the laboratory.
 PCR-based tests have allowed detection of small numbers of disease organisms (both live or
dead), in convenient samples.
 The spread of a disease organism through populations of domestic or wild animals can be
monitored by PCR testing.
 The sub-types of an organism that were responsible for earlier epidemics can also be
determined by PCR analysis.

3. Forensic applications:
 In its most discriminating form, genetic fingerprinting can uniquely discriminate
any one person from the entire population of the world.
 Minute samples of DNA can be isolated from a crime scene, and compared to that
from suspects.
 Less discriminating forms of DNA fingerprinting can help in parental testing,
where an individual is matched with their close relatives.
 DNA from unidentified human remains can be tested, and compared with that
from possible parents, siblings, or children

Research Applications:
 The task of DNA sequencing can also be assisted by PCR. Known segments of
DNA can easily be produced from a patient with a genetic disease mutation.
 PCR has numerous applications to the more traditional process of DNA cloning. It
can extract segments for insertion into a vector from a larger genome, which may
be only available in small quantities.

 Sequence-tagged sites is a process where PCR is used as an indicator that a
particular segment of a genome is present in a particular clone.
 An exciting application of PCR is the phylogenic analysis of DNA from ancient
sources, such as that found in the recovered bones of Neanderthals, or from frozen
tissues of mammoths.
 A common application of PCR is the study of patterns of gene expression. Tissues
(or even individual cells) can be analyzed at different stages to see which genes
have become active, or which have been switched off.

71
Summary
blood, chorionic
villus, amniotic
fluid, semen, hair
root, saliva
68,719,476,736 copies Gel Analysis,
Restriction
Digestion,
Sequencing

72
Conclusion:
•The speed and ease of use, sensitivity, specificity and robustness of PCR
has revolutionised molecular biology .
•PCR the most widely used and powerful technique with great spectrum of
research and diagnostic applications.

Pcr n optimization

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