Polymer chain reaction

1. Polymerase Chain
Reaction (PCR)
BY-Sanju sah
St. Xavier’s college, Maitighar, Kathmandu
Department of Microbiology

2. What is PCR?
• Polymerase- enzyme used in this reaction is DNA
polymerase
• Chain- products of the first reaction become substrates of
the following reaction, and so on
• Reaction- reaction of target DNA, pair of primers,
dNTPs, thermostable Taq polymerase, Mg2+, buffer,

3. The “Reaction” Components
1. Target DNA - contains the sequence to be amplified.
2. Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3. dNTPs (deoxynucleotide-triphosphates): DNA building
blocks
4. Thermostable DNA Polymerase - enzyme that catalyzes the
reaction
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

5. Kary mullis

6. • PCR stands for Polymerase Chain Reaction.
• PCR is an exponentially progressing synthesis of the defined target DNA
sequences in vitro
• It was developed in 1983 by Kary Mullis
• PCR is a biochemical process capable of amplifying a single DNA molecule
into millions of copies in a short time.
• It used to amplify the DNA segment from nanograms to micrograms.
• It uses Taq DNA polymerase isolated from thermophilic bacteria Thermus
aquaticus, which works at elevated temperature.
• Amplification is achieved by a series of three steps:
(1) Denaturation, in which double-stranded DNA templates are heated
to separate the strands;
(2) Annealing, in which short DNA molecules called primers bind to
flanking regions of the target DNA; and

7. (3) Extension, in which DNA polymerase extends the 3′ end of each primer
along the template strands.
• These steps are repeated (“cycled”) 25–30 times to exponentially produce
exact copies of the target DNA

8. PCR
process
and
steps

9. A thermal cycler for PCR

10. Basic Principles of Primer Design
Primer length:
• usually 18-28 nucleotides
Base composition:
• G/C content should be around 50%
• 3’ G/C Clamp
Melting/annealing temperature
• Preferably Tm of 55-65ºC
Avoid secondary structures (hairpin loops) and primer dimers
Design primers from unique (not repetitive) sequence
5’ and 3’ end stability

11. Primer length
• PCR Primers are short single-stranded, synthetically synthesized
oligonucleotides usually shorter than 50 nucleotides (often 18-25
nucleotides)
• Primer length should be long enough to ensure that the sequence will
be unique for the template and not so long as to affect the Tm and
annealing too radically.
• Specificity and the temperature and time of annealing are at least
partly dependent on primer length

12. Random base distribution and composition
• If the G+C% of two primers are very different, they will have different
Tms and will not anneal at the same temperature
• Ideally the primer will have a near random mix of nucleotides, a 50%
GC content and be ~20 bases long

13. G/C Clamp
• At the G/C Clamp at the 3' end of the primer should be G or C in
order to increase correct annealing at the site of addition of bases
• G-C base pairs have stronger hydrogen bonding
• Keep in mind for primer design that the 3’ ends often determine the
priming.
• If you have a primer with 5-10 nucleotides of complementarity at the
3’ end to a site, it will often prime at that site

14. Matched Primer Tm
• Approximate Tm for DNA-DNA annealing can be calculated manually
using the formula:
• Tm = 4(G + C) + 2(A + T)°C
• Tm is dependent upon ionic strength of the solution and upon the length of
the sequences to be annealed
• The shorter the oligo sequence, the lower the melting temperature
• Valid for Oligonucleotides
• between 18-24 bases
• What is an optimal annealing temperature (Tm)?
• Too high Tm: Reduced annealing, therefore less product
• Too low Tm : Unspecific annealing, unspecific product

15. No self-complementarity
• In general don’t use primers which have more than 3 bases in a stem
involving the 3’ end of the primer
• complementarity Base pairing with itself decreases the ability of a
primer to correctly anneal to the template

16. No Primer Dimers
• Primer pairs which can anneal to each other.
• If the base pairing includes one or both 3' ends, the polymerase will
simply extend the end of the primer against the other primer as
template resulting in the formation of a primer dimer
• Primer dimer formation uses up all the polymerase in the reaction and
results in a lack of other product.

17. Length and composition of amplified
sequence
• Maximum yields are generated with Taq polymerase when the length
of the product is 100-600 base pairs.
• Different polymerases have different degrees of processivity and will
have different optimal sizes.

18. dNTPs
• The DNA polymerases incorporate very efficiently dNTPs.
• For conventional PCR, the concentration of dNTPs are in equimolar
ratios, e.g., 200 μM each dNTP.
• Unbalanced dNTP concentrations promote a higher degree of
misincorporations by the DNA polymerase.

19. Taq polymerase
• 94 kDa protein with a 5’-3’ polymerization activity, most efficient in
the 70°–80°C range.
• Thermostable, with a half-life at 95°C of 35–40 min.
• In terms of thermal cycling, the halflife is approx 100 Cycles.
• At 75-80 °C, Taq reaches its optimal polymerization rate of about 150
nucleotides per second per enzyme molecule, and any deviations from
the optimal temperature range inhibit the extension rate of the enzyme.
• It,s drawbacks is lack of 3' to 5' exonuclease proofreading activity.
resulting in relatively low replication fidelity.

21. Magnesium chloride
• Its concentration must be optimized for every primer:template system.
• Many components of the reaction bind magnesium ion, including
primers, template, PCR products and dNTPs.
• It is necessary for free magnesium ion to serve as an enzyme cofactor
in PCR, the total magnesium ion concentration must exceed the total
dNTP concentration.
• To start the optimization process, 1.5 mM magnesium chloride is
added to PCR in the presence of 0.8 mM total dNTPs.
• This leaves about 0.7 mM free magnesium for the DNA polymerase.

22. Buffer solution
• Depending on the DNA polymerase used the optimal PCR buffer
concentration, salt concentration, and pH should be picked accordingly
to the DNA polymerase.
• PCR buffer for Taq DNA polymerase consists of 50 mM KCl and 10
mM Tris-HCl, pH 8.3, at room temperature.
• Buffer provides the ionic strength and buffering capacity needed
during the reaction.

24. Primers design
• Primers should bind to template with good specificity and strength.
• If primers do not bind to correct template, wrong sequence will get
amplified.
• Optimal primer sequences and appropriate primer concentrations are
essential for maximal specificity and efficiency in PCR.
• PCR specificity and efficiency can be greatly affected by the way
primers are designed and used.
• Even when primers are designed to have similar annealing properties,
the PCR may yield nonspecific PCR products (undesired DNA
segment amplified), low amounts of specific product, or fail
completely

25. • Complementary nucleotide sequences within a primer and between
primers should be avoided.
• If there are complimentary sequences in two primers used (one primer
for each DNA strand), the primers will hybridize with each other thus
forming primer-dimmers and will not be available for binding with
template.
• If there are complementary sequences within a primer, it will make
hairpin loop structures

26. • The primers should preferably end on a Guanine and Cytosine (GC)
sequence so that it can attach with sufficient strength with template.
This increases efficiency of priming due to stronger bonding of G and
C bases.
• Runs of three or more Cytosine (C) or Guanine (G) at the 3'-ends of
primers should be avoided. This may promote mispriming i.e non-
specific binding to G or C rich sequences in the genome other than the
target sequence.
• As Adenine and Thymine base pairs with a single H-bond so Thymine
(T) or Adenine (A) residues should be avoided at the 3’ end of primers
as this weaken the primer’s hold on the template DNA.

27. All steps for PCR primer designer
• Download DNA / RNA template - Fasta format (recommended)
• Finding conserved sequence (Alignment) - Alignment software e.g.
OMIGA / 37 Clustal W
• Design primer set - Tool from website / software
• Check primer set - Blast / oligo analyzer / 2nd structure

28. • Download DNA / RNA template
• How to look for an interested gene
• should be used by many laboratories or has already been published
• many copy gene is better than single copy gene 39 many copy gene is
better than single copy gene
• specific for only the organism(s) of interest

29. Procedure
• To prepare billions of DNA copies, many repeated cycles of DNA synthesis are
performed in one PCR tube.
• There are three major steps in a PCR, which are repeated for 30 or 40 cycles, This
is done on an automated cycler, which can heat and cool the tubes with the
reaction mixture in a very short time.
• Each cycle includes three distinct steps defined by the temperature
• All cycles are performed without intervention in a PCR machine, which can
change automatically the temperature to create the steps.
Denaturation at 94°C :
• During the heating step (denaturation), the reaction mixture is heated to 94°C for 1
min, which causes separation of DNA double stranded. Now, each strand acts as
template for synthesis of complimentary strand.

30. Annealing step (50-70ºC):
• At 54°C, short DNA pieces called primers bind at complementary
sites of the template DNA.
• The primers define the target sequence, which is the specific region of
DNA that will be copied.
• Annealing temperature is calculated from the primer composition (the
number of nucleotides as well as number of guanine and cytosine).
Normally, it is needed to calculate the optimal annealing temperature
for each primer.
• The annealing temperature can be measured by calculating the melting
temperature of the strand-primer.

31. • Tm = [4*(C+G) + 2*(A+T)] °C
• Time: 1 min.
• The length and GC-content (guanine-cytosine content) of the primer
should be sufficient for stable binding with template.
• Guanine pairs with cytosine with three hydrogen bonding adenine
binds with thymine with two hydrogen bonds. Thus, higher GC
content results in stronger binding.
• In case GC content is less, length may be increased to have stronger
binding (more number of H bonding between primer and template).

32. Extension at 72°C :
• In this phase of PCR, the DNA synthesis would occur. The primer is
extended by the Taq polymerase in presence of nucleotide. The Taq
polymerase is thermostable and works efficiently at elevated temperature
i.e. 72 °C.
• The polymerase adds nucleotide (dNTP's) complimentary to template on 3’
–OH of primers thereby extending the new strand.
• Time: 2 mins
Final hold:
• First three steps are repeated 35-40 times to produce millions of exact
copies of the target DNA.
• Once several cycles are completed, during the hold step, 4–15 °C
temperature is maintained for short-term storage of the amplified DNA
sample.

35. PCR-an exponential cycle
• As both strands are copied during PCR, there is an exponential
increase of the number of copies of the gene.
• Suppose there is only one copy of the desired gene before the PCR
starts, after one cycle of PCR, there will be 2 copies, after two cycles
of PCR, there will be 4 copies, after three cycles there will be 8 copies
and so on.

36. PCR
cycle

37. PCR
steps
Vs.
Temperature

38. Factors affecting the accuracy and time for PCR:
Annealing temperature:
• As discussed above, the annealing temperature needs to be maintained.
• If the temperature is too low then there can be mismatch pairing.
• The mismatched pairing can lead to decrease in the quality of the PCR
product.
Length of the primer used:
• The primer used needs to be unique.
• If the primer used has a very short sequence then there are chances that it
would bind randomly to any non-target sequence.
• If the sequence of the primer is long then its specificity to bind target
sequence increase.
• However, if the primer is too long, then it would take a long time to find
and anneal to the target sequence.

39. Types of PCR
Conventional PCR
• This defined as a normal PCR process.
• Here the primers bind specifically to each other with 2 DNA strands.
• Primers also limit the sequence to be replicated and a particular DNA
sequence is amplified with billions of copies.
• All that needed for the PCR process are PCR tubes, DNA polymerase,
buffer, and target DNA, primers.
• The whole process takes place within 35-40 minutes repeatedly and
viewed by gel electrophoresis technique.

40. Multiplex PCR
• Multiplex PCR is an adaptation of PCR which allows simultaneous
amplification of many sequences.
• consists of multiple primer sets within a single PCR mixture to
produce amplicons of varying sizes that are specific to different DNA
sequences.
• By targeting multiple genes at once, additional information may be gained
from a single test-run that otherwise would require several times the
reagents and more time to perform.
• Here in multiplex PCR the base pair lengths should be different to form
distinct bands because varying sizes of different DNA genes are targeted in
a single reaction to avoid higher expenses, time consumption and
recognizes many pathogens at once.

41. • Annealing temperatures for each of the primer sets must be optimized
to work correctly within a single reaction, and amplicon sizes. i.e.,
their base pair length should be different enough to form distinct bands
when visualized by gel electrophoresis.
• This technique is used for diagnosis of different diseases in the same
sample, it can detect different pathogens in a single sample.
• Several multiplex PCR’s have been described for the simultaneous
detection of M.tuberculosis and Brucella species.

42. Nested PCR
• Nested PCR increases the specificity of DNA amplification, by
reducing background due to non-specific amplification of DNA.
• Two sets of primers are used in two successive PCRs.
• In the first reaction, one pair of primers is used to generate DNA
products, which besides the intended target, may still consist of non-
specifically amplified DNA fragments.
• The product(s) are then used in a second PCR with a set of primers
whose binding sites are completely or partially different from and
located 3' of each of the primers used in the first reaction.
• Nested PCR is often more successful in specifically amplifying long
DNA fragments than conventional PCR, but it requires more detailed
knowledge of the target sequences.

43. • Nested PCR is used because it intends to reduce the contaminations in
products due to the amplification of unexpected primer binding sites.
• It has drawbacks like risk of contamination and needs great care while
being performed.
• These contaminations can be controlled by adding ultra-pure oil of
two mixtures, and by using primers designed to anneal at different
temperatures.
• Nested/seminested PCR were used in identifying Brucella in human
blood samples.
• This method is more specific but has disdvantages like ‘primer and
dimerisation’ cross reaction.

44. Reverse Transcription PCR (RT-PCR)
• It is used for amplifying DNA from RNA.
• Reverse transcriptase reverse transcribes RNA into cDNA, which is
then amplified by PCR.
• RT-PCR is widely used in expression profiling, to determine the
expression of a gene or to identify the sequence of an RNA transcript,
including transcription start and termination sites.
• If the genomic DNA sequence of a gene is known, RT-PCR can be
used to map the location of exons and introns in the gene.

45. • This type of PCR has been useful for diagnosis of RNA viruses, as
well as for evaluation of antimicrobial therapy.
• It has also been used to study gene expression in vitro, due to the
obtained cDNA retains the original RNA sequence.
• The main challenge of using this technique is the sample of mRNA,
because this is considered difficult to handle by low level and
concentration of mRNA of interest and low stability at room
temperature together with sensitivity to action of ribonucleases and pH
change

47. Real Time PCR
• RT-PCR also known as quantitative PCR is used to amplify and
simultaneously quantify a target DNA.
• It differs from standard PCR in a way that it can detect the amplified
product as the reaction progresses with time but in standard PCR the
amplified product is detected at the end of the reaction by agarose gel
electrophoresis.
• It quantitatively measures starting amounts of DNA, cDNA, or RNA.

48. • Quantitative PCR has a very high degree of precision. Quantitative
PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or
fluorophore-containing DNA probes, such as TaqMan, to measure the
amount of amplified product in real time.
• The increase in the DNA in each cycle reflects a propotional increase
in emitted fluorescence and also propotional to the hybridisation of
probes.
• It is also sometimes abbreviated to RT-PCR (real-time PCR) but this
abbreviation should be used only for reverse transcription PCR. qPCR
is the appropriate contractions for quantitative PCR (real-time PCR).

49. Advantages
• The technique is highly sensitive with the potential to produce
millions to billions of copies of a specific product for sequencing,
cloning, and analysis.
• qRT-PCR shares the same advantages as the PCR, with an added
advantage of quantification of the synthesized product, therefore, it
has its uses to analyze alterations of gene expression levels in tumors,
microbes, or other disease states.
• The sequencing of unknown etiologies of many diseases are being
figured out by the PCR.
• The technique can help identify the sequence of previously unknown
viruses related to those already known and thus give us a better
understanding of the disease itself.

50. Limitations
• One major limitation of PCR is that prior information about the target
sequence is necessary in order to generate the primers that will allow
its selective amplification.
• Like all enzymes, DNA polymerases are also prone to error, which in
turn causes mutations in the PCR fragments that are generated.
• Another limitation of PCR is that even the smallest amount of
contaminating DNA can be amplified, resulting in misleading or
ambiguous results.
• Reagents should be dispensed into single-use aliquots. Pipetters with
disposable plungers and extra-long pipette tips should be routinely
used.

51. Applications
PCR is now a common and often indispensable technique used
in clinical and research laboratories for a broad variety of applications.
These include:
• DNA cloning for sequencing, gene cloning and manipulation, gene
mutagenesis; construction of DNA-based phylogenies, or functional
analysis of genes
• diagnosis and monitoring of hereditary diseases
• amplification of ancient DNA for study
• analysis of genetic fingerprints for DNA profiling (for example,
in forensic science and parent testing);
• and detection of pathogens in nucleic acid tests for the diagnosis
of infectious diseases (Viral, Bacterial, Protozoal, Fungal etc)

52. APPLICATIONS OF PCR
PCR can be applied in many fields
• In diagnosis therapy
• In nucleic acid detection assays
• In medical field
• In agricultural sciences
• In mycology-parasitology
• In dentistry
• In virological diagnostics

53. • In molecular systematic evolution
• In cancer therapy
• In therapy resistant assessment
• PCR-as biomarker
• In forensic medicine
• In virology
• In bacteriology
• In phytopathology
• In PCR-fingerprinting
• In the detection of microbilogical gene