Lecture ON Polymerase Chain Reaction. The polymerase chain reaction (PCR) is a powerful core molecular biology technique - Sometimes called "molecular photocopying. • Developed by Kary Mullis in 1985. • It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from nucleic acids of various sources. • It generates microgram (µg) quantities of DNA copies (up to billion copies) of the desired DNA (or RNA) segment. A simple PCR reaction consists of i. A DNA preparation containing the desired segment to be amplified. ii. A set of synthetic oligonucleotide primers that flank the target DNA sequence, of about 20 bases long, specific, i.e., complementary. iii. A thermostable DNA polymerase e.g., Taq isolated from the bacterium Thermus acquaticus, Pfu – Pyrococcus furiosus and Vent from Thermococcus litoralis. Pfu and Vent are more efficient than Taq polymerase. iv. Four deoxynucleoside triphosphate (dNTPs): TTP – thymidine triphosphate, dCTP – deoxycyctidine triphosphate, dATP – deoxyadenosine triphosphate and dGTP – deoxyguanosine triphosphate

1. www.covenantuniversity.edu.ng
Raising a new Generation of Leaders
Polymerase Chain Reaction PCR:
Principles and Applications
Jacob O. Popoola (Ph.D)

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Introduction to PCR
• The polymerase chain reaction (PCR) is a powerful core molecular biology
technique - Sometimes called "molecular photocopying.
• Developed by Kary Mullis in 1985.
• It is an efficient and rapid in vitro method for enzymatic amplification of specific
DNA or RNA sequences from nucleic acids of various sources.
• It generates microgram (µg) quantities of DNA copies (up to billion copies) of the
desired DNA (or RNA) segment.
• The process has been completely automated and compact thermal cyclers are
available in the market.

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A simple PCR reaction consists of
i. A DNA preparation containing the desired segment to be amplified.
ii. A set of synthetic oligonucleotide primers that flank the target DNA
sequence, of about 20 bases long, specific, i.e., complementary.
iii. A thermostable DNA polymerase e.g., Taq isolated from the
bacterium Thermus acquaticus, Pfu – Pyrococcus furiosus and Vent
from Thermococcus litoralis. Pfu and Vent are more efficient than
Taq polymerase.
iv. Four deoxynucleoside triphosphate (dNTPs): TTP – thymidine
triphosphate, dCTP – deoxycyctidine triphosphate, dATP –
deoxyadenosine triphosphate and dGTP – deoxyguanosine
triphosphate.

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Procedure of PCR
• At the start of PCR, the DNA from which a segment is to be amplified, an
excess of the two primer molecules, the four dNTPs, and the DNA
polymerase are mixed together in the reaction mixture that has appropriate
quantities of Mg2+, the following operations are now performed.
1. Denaturation: The reaction mixture is first heated to a temperature
between 90 - 98ºC (commonly 94ºC) that ensures DNA denaturation. The
duration is of this step in the first cycle of PCR is usually 2 min at 94ºC.
2. Annealing: The mixture is now cooled to a lower temp of 40 – 60ºC. This
permits annealing of the primer to the complementary sequences in the
DNA. It takes 1 min during the first as well as the subsequent cycles of
PCR.
3. Primer Extension: The temp is adjusted so that the DNA polymerase
synthesizes the complementary strands by utilizing 3’-OH of the primers;
this reaction is the same as that occurs in vivo during replication of the
leading strand of a DNA duplex.The duration is usually 2 min at 72ºC.

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7. Requirements for PCR
• Template DNA (single-strand)
• Primers: Primers range from 15 to
30 nucleotides, are single-stranded,
and are used for the
complementary building blocks of
the target sequence.
• Note; Primers allows both strands to
be copied simultaneously in both
directions.
• dNTPs (deoxyribonucleoside
triphosphates)
• A thermostable form of DNA
polymerase e.g. (Taq polymerase)
isolated from Thermus aquaticus
• Magnesium chloride: .5-2.5mM
• Buffer: pH 8.3-8.8
• A thermocycler – a thermal reactor.

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How does it work?

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Factors that influence the efficiency of PCR
PCR performance depends on several causes. The quality of primers affect specificity,
sensitivity and reliability of the PCR reaction.
1. Increase in the length of the target sequence: this declines the efficiency of
amplification.
2. Primer length: If the primers used for PCR are too long, the efficiency reduces.
3. A temperature higher than the ideal annealing temperature reduces PCR efficiency.
4. Primer sequences: if the primers used have complementary regions, primer dimers will
be formed, and this declines the PCR efficiency.
5. Addition of certain proteins e.g., bovine serum albumin enhances PCR efficiency by
protecting the DNA polymerase and by binding to PCR inhibitors.
6. Target DNA sequence with GC-rich sequences may form secondary structures in the
single strands produced by denaturation reducing PCR efficiency.

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Types/Variations Of PCR
• Inverse PCR is used to amplify DNA with only one known sequence.
• It allows PCR to be carried out even if only one sequence is available from which primers
may be designed.
• Inverse PCR is especially useful for identifying insert positions of
various transposons and retroviruses in the host DNA. It is one of the variations of the
PCR used when only one sequence is understood to amplify DNA.
• Conventional PCR requires primers complementary to both target DNA terminals, but
Inverse PCR allows amplification, even if only one sequence from which primers can be
designed is available.
• The inverse PCR involves a series of restriction digestion followed by ligation, resulting in a
looped fragment that can then be primed for PCR through a single known sequence
section.
• Then, as with other processes of reaction in the polymerase chain, the temperature-
sensitive DNA polymerase amplifies the DNA.

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Conventional PCR
• This type is a test tube system for DNA replication that allows selective
amplification of a “target” DNA sequence of several million folds in just a few
hours.
• PCR allows the synthesis of different DNA fragments using a DNA-polymerase
enzyme which is involved in the cellular genetic material replication.
• This enzyme synthesizes a complementary DNA sequence as a small fragment
(primer) is connected to one of the DNA strands chosen to start the synthesis at
the specific site.
• Primers restrict the sequence to be repeated, and the effect is the multiplication
of billions of copies of a single DNA sequence.
• Conventional PCR is used in selective DNA isolation, amplification and
quantification of DNA,
• Also, in medical and diagnostic studies, diagnosis of infectious diseases,
• In forensic studies and detection of substances.

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Reverse Transcriptase PCR (RT-PCR)
• RT PCR is quite famous these days due to its’ tremendous usefulness in identifying
Coronavirus patients.
• Reverse Transcription PCR (RT-PCR) is a traditional PCR modification whereby RNA
molecules are first translated into complementary DNA (cDNA) molecules which can then
be amplified by PCR.
• In RT-PCR the RNA template is initially converted using reverse transcriptase to a cDNA.
• The cDNA also uses PCR to act as a blueprint for exponential amplification. RT-PCR can be
carried out either in a single tube, or in different tubes as two steps.
• The one-step method is more efficient with fewer chances of contamination and variable
incorporation.
• RT-PCR is used in testing techniques, gene injection, the treatment of genetic
disorder and cancer detection. Note: Complementary DNA (cDNA) is a DNA copy of a messenger RNA
(mRNA) molecule produced by reverse transcriptase, a DNA polymerase that can use either DNA or RNA as a
template.

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Reverse-Transcriptase Real-Time PCR (RT-qPCR)
• RT-PCR is usually aligned with Reverse Transcriptase Real-Time PCR (RT-qPCR)
generating q-PCR;
• This allows in real-time quantification of DNA after amplification.
• It is a technique that combines reverse transcription of RNA into DNA called
cDNA and amplification of specific DNA targets using (PCR).
• It is primarily used to measure the amount of a specific RNA, achieved by
monitoring the amplification reaction using fluorescence.
• Combined RT-PCR and qPCR are routinely used for analysis of gene
expression and quantification of viral RNA in research and clinical settings.

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Real-Time PCR (Quantitative PCR (qPCR))
• Quantitative PCR (qPCR), also known as real-time PCR or quantitative real-
time PCR, is a PCR-based technique which combines amplification of a target
DNA sequence with quantification of that DNA species concentration in the
reaction.
• Real-time PCR is based on the use of fluorescent dye.
• The sample’s nucleic acid concentration is quantified using the fluorescent
dye or using the oligonucleotides labelled fluorescent.
• q-PCR is used in genotyping and pathogen quantification,
• In microRNA analysis, cancer diagnosis,
• Microbial load monitoring and Genetically Modified Organisms (GMO)
diagnosis.
•

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Amplified fragment length polymorphism (AFLP) PCR
• It is a technique based on PCR which uses selective amplification of a portion of
digested DNA fragments to produce specific fingerprints for interesting genomes.
• Without prior knowledge of the genomic sequence this technique can quickly generate
large numbers of marker fragments for any organism.
• AFLP PCR uses restriction enzymes to digest genomic DNA and allows adaptors to be
applied to the fragment’s sticky ends.
• A portion of the restriction fragments is then selected for amplification using primers
complementing the adaptor sequence.
• The amplified sequences on the electrophoresis of the agarose gel are isolated and
visualized for denaturing.
• AFLP PCR is used for a variety of applications, such as the assessment of genetic
diversity within species or among closely related species,
• the inferment of population-level phylogenies and biogeographic patterns, the
generation of genetic maps and the determination of linkage between cultivars.

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Single Specific Primer-PCR (SSP-PCR)
• This allows the amplification of double-stranded DNA even when
the information about the sequence is only available at one end.
• This method, allows for the amplification of genes for which only
partial sequence information is available, and
• Permits unidirectional genome walking from known to unknown
chromosome regions.

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Miniprimer PCR
• A new PCR method is called Miniprimer PCR using “miniprimers” of engineered
polymerase and 10-nucleotides.
• This approach is used to show sequences of novel 16S rRNA genes which would not
have been identified with normal primers.
• Miniprimer PCR uses a polymerase enzyme that is thermostable and can extend
from short primers (9 or 10 nucleotides).
• This method allows PCR targeting to smaller binding regions and is used to
amplify highly conserved sequences of DNA, such as the rRNA gene 16S (or
eukaryotic 18S).

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Allele-specific PCR
• This is based on allele-specific primers that can be used to analyze polymorphism of
single nucleotides.
• Also called the (amplification refractory mutation system) ARMS-PCR is the allele-specific
PCR, corresponding to the use of two different primers for two different alleles.
• One is the mutant set of refractory (resistant) primers to the normal PCR, and the other is
the normal set of primers that are refractory to the mutant PCR reaction.
• The 3 ‘ends of these primers are modified so that the normal allele can be amplified by
one set of the primers while others amplify the mutant allele.
• This mismatch allows for amplification of a single allele by the primer.
• It is commonly used in the diagnosis of single point gene defects, such as sickle cell
anemia and thalassemia.
• It is also used for the precise identification of genotypes from the ABO blood stream.

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Assembly PCR
• PCR assembly is a method of assembling large oligonucleotides from multiple shorter
fragments of DNA.
• The size of the oligonucleotides used in PCR is 18 base pairs, while PCR lengths of up
to 50bp are used in the assembly to ensure correct hybridization.
• The oligonucleotides bind to complementary fragments during the PCR processes and
are then loaded with polymerase enzyme.
• Thus, each cycle of this PCR decreases arbitrarily the length of various fragments,
depending on which oligonucleotides locate each other.
• Assembly PCR is used to improve the yield of the desired protein and can also be used
to make large quantities of RNA for structural or biochemical studies.

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Alu PCR
• Alu PCR is a simple and easy technique of DNA fingerprinting, based on simultaneous examination of
multiple genomic loci accompanied by repetitive elements of Alu.
• Alu components are small stretches of DNA initially distinguished by the activity of the endonuclease
limiting Arthrobacter luteus (Alu).
• Alu elements are one of the most abundant transposable elements found throughout the
human genome and have been used as genetic markers and play a role in evolution.
• In Alu PCR, two complementary fluorochrome-labeled primers are used to perform the PCR and the PCR
products are then analyzed.
• Several genetically inherited human diseases and various forms of cancer have been used to insert alu.
• This PCR thus plays a crucial role in the detection of these diseases and mutations.

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Repetitive sequence-based PCR
• It is a modified PCR technique that uses primers that target non-coding,
interspersed repetitive sequences throughout the bacterial genome.
• These non-coding, repeated sequence blocks can serve as multiple genetic
targets for oligonucleotide organisms, allowing individual bacterial strains to
produce specific DNA profiles or fingerprints.
• The main application of rep-PCR is in the typing of various bacteria by
molecular strain.
• It is also used to discriminate epidemiologically against diverse pathogens.

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Variable Number of Tandem Repeats (VNTR) PCR
• In forensic science, they are important markers for the
individualization.
• Fragments are amplified in VNTR PCR which showed little
variation within a species but showed differences between
species.
• It can successfully amplify from a very small amount of
genomic deoxyribonucleic acid (DNA).

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PCR Applications
1. PCR is used to amplify Human-specific DNA sequences / gene
of interest.
2. PCR amplification is used to monitor cancer therapy
3. PCR is also used to detect bacterial and viral infections
4. In detection of mutations in cancers and inherited disorders. –
screening particular genes for mutations
5. Also used for sex determination of prenatal cells.
6. DNA fingerprinting and Forensics
7. PCR helps in linkage analysis using single sperm cells.
8. Also used in genetic diversity and molecular evolution studies.

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Video of PCR.

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References
1. Garafutdinov RR, Galimova AA, Sakhabutdinova AR. The influence of
quality of primers on the formation of primer dimers in PCR. Nucleosides
Nucleotides Nucleic Acids. 2020;39(9):1251-1269. doi:
10.1080/15257770.2020.1803354. Epub 2020 Aug 17. PMID: 32799617.
2. Aun, O., & Schönswetter, P. (2012). Amplified fragment length
polymorphism: an invaluable fingerprinting technique for genomic,
transcriptomic, and epigenetic studies. Methods in molecular biology
(Clifton, N.J.), 862, 75–87. https://doi.org/10.1007/978-1-61779-609-8_7
3. Cardelli, Maurizio. (2011). Alu PCR. Methods in molecular biology (Clifton,
N.J.). 687. 221-9. 10.1007/978-1-60761-944-4_15.
4. T.A.BROWN. (2010). GENE CLONING & DNA ANALYSIS (6th ed.). A John
Wiley & Sons, Ltd,Publication.