Lecture Notes
PCR (Polymerase Chain Reaction)
Polymerase chain reaction (PCR) is a technique in molecular biology used to amplify
(multiply) a single copy or a few copies of a particular region of DNA, generating thousands
to millions of copies of that particular DNA sequence. Typically, the goal of PCR is to make
enough of the target DNA region that it can be analyzed or used in some other way. For
instance, DNA amplified by PCR may be sent for sequencing, visualized by gel
electrophoresis, or cloned into a plasmid for further experiments.
The PCR was originally developed in 1985 by the American biochemist Kary Mullis. He was
awarded the Nobel Prize in Chemistry in 1993 for his pioneering work.
PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA
complementary to the template/target strand of DNA. Since DNA polymerase can add
nucleotides only onto a pre-existing 3'-OH group (-OH group attached to 3’-end of DNA),
it needs a primer, to which it can add the first and other nucleotides. At the end of the PCR
reaction, the specific sequence will be accumulated in billions of copies (amplicons).
PCR Components: There are the following components of PCR reaction:
(1) DNA Template - The sample DNA that contains the target sequence. At the beginning of
the reaction, high temperature is applied to the original double-stranded DNA molecule to
separate the strands from each other (denaturation).
(2) DNA polymerase – This enzyme synthesizes new strands of DNA complementary to the
target sequence. First and the most commonly used of DNA polymerase enzyme used in
PCR technology is Taq DNA polymerase or just Taq polymerase obtained from the
bacterium Thermus aquaticus. Taq polymerase is used in PCR, rather than other forms of the
polymerase enzymes, because it is thermostable and is not denatured at high temperature.
(3) Primers – Short pieces of single-stranded DNA that are complementary to the target
sequence. Enzyme polymerase begins to synthesise new DNA from the end of the primer.
There should be no complementarity between the two primers or the 3 ends of a single primer.
Following are the considerations that should be kept in mind while designing the primers.
a. Primer Length: The length of a primer is directly proportional to the specificity of a
PCR reaction. The length also determines the temperature at which the primers will
anneal to the template. Usually, primers are designed between 18 to 30 bases long
though the minimum length of a primer is determined by the size of the genome.
b. GC content: GC content is important as it also determines the Tm value of a sequence.
20 base pair long oligos having 50% GC content generally have a Tm value between
56-62०C. Most desirable GC content should be between 40 to 60°C. More than three
G or C nucleotides at the 3' end of the primer should be avoided as this may lead to
nonspecific priming.
c. Melting temperature (Tm): Since in a PCR reaction a set of primers is used, so effort
should be made to select the pairs that have a Tm in the range of 5°C within each other,
therefore the GC content and length must be chosen accordingly. Usually, the Tm lies
in the range of 62-70°C. Estimation of the melting and annealing temperatures of
primer: For the primer less than 25 nucleotides, the approximately Tm is calculated
using the following formula:
Tm= 4 (G + C) + 2 (A + T)
(5) Deoxynucleotides or deoxynucleotide triphosphates (dNTPs) - Single units of the bases
A, T, G and C, equipped with phosphate group. The dNTPs are building blocks for the new
DNA strands. [dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate),
dCTP (deoxycytidine triphosphate), dTTP (deoxythymidine triphosphate)].
(6) MgCl2 – Act as a cofactor for polymerase enzyme thus enhancing DNA amplification.
Magnesium is required for the DNA polymerase to function, but the specificity of any
particular PCR reaction is dependent upon the concentration of magnesium used. At low
concentration the reaction fails because the polymerase is insufficiently active. At high
concentrations, the reaction loses specificity and multiple products are produced. The optimum
concentration needs to be determined empirically for each separate PCR primer set, but will
usually be in the range of 1–5 mM.
(7) KCl – The KCl salt in the PCR buffer acts by neutralizing the charge present on the
backbone of DNA. During the elongation step of the PCR, the primer has to anneal or stick
properly to the template and this is facilitated by the KCl. Thus, it functions by reducing the
repulsion between the negatively charged DNA strands i.e., the primer and template, thereby
stabilizing the primer-template binding.
(8) PCR Buffer - Basic Components includes: Tris-HCL (pH 8.4) (maintains pH), KCl and
sometimes MgCl2.
Principle
The principle of PCR is based on the fact that at high denaturing temperatures nearing 95°C,
the two strands of the target DNA molecule separate due to breaking of A-T and G-C bonds.
At the annealing temperatures in the range of 50-65°C, the complimentary forward and reverse
primers bind at the 3’ end of the flanking regions of the separated single stranded target DNA
molecule. The Taq polymerase then extends the new DNA strand by adding dNTPs and the
double stranded molecule restructures itself at the extension temperature of 72°C. This process
is repeated several times, generating multiple copies of the target DNA molecule.
Steps:
(1) Denaturation: During the first step of PCR, called denaturation, the DNA is heated to
more than 90-95°C for 30-55 seconds, which separates the double-stranded DNA into two
separate strands. The high temperature breaks the relatively weak hydrogen bonds between
the nucleotides that form the DNA double helix.
Initial denaturation is followed by 30-35 cycles of denaturation, annealing and extension. The
number of PCR cycles depends on the amount of template DNA in the reaction mix and on the
expected yield of the PCR product.
(2) Annealing: (Binding Primers to the DNA Sequence)
PCR does not copy the all of the DNA in the sample. It copies only a very specific sequence
DNA, targeted by the PCR primers. These primers are oligonucleotide (short pieces of
synthetic DNA) that bind, or anneal, only to sequences on either side of the target DNA region.
Two primers (forward and reverse primers) are used in step 2 - one for each of the newly
separated single DNA strands. The primers bind to the beginning of the sequence that will be
copied, marking off the sequence for step three. Time & temperature depends on base
composition, length & concentration of primers.
• Annealing temperature is 5°C below melting temperature of primers.
• Increasing annealing temperature enhances discrimination against incorrectly annealed
primers & reduces misextension of incorrect nucleotides.
(3) Extension: Extension step occurs at 72°C for 30-55 seconds by adding complimentary
dNTPs to the new strands. Nucleotides are added to the annealed-primers by the DNA
polymerase (Taq polymerase) to create a new strand of DNA complementary to each of the
single template strands. After the last cycle, the samples are usually incubated at 72°C for 5-
15 minutes to fill-in the protruding ends of newly synthesized PCR products.
Post amplification detection
The amplified PCR product is observed as a fluorescent pink band by agarose gel
electrophoresis following ultraviolet transillumination of the agarose gel stained in Ethidium
bromide (EtBr) solution. EtBr has been used since many years for the visualization of nucleic
acids in agarose gel. EtBr is also a potent mutagen, causing mutations in the living cell.
Applications:
• Molecular cloning
• DNA sequencing
• Forensics
• Amplification of unknown sequences
• Clinical pathology
• Genetic diagnosis
• Characterizing unknown mutations
• Quantitative PCR of RNA/ DNA.
Types of PCR:
• Multiplex PCR
• Nested PCR
• Reverse Transcriptase PCR (RT- PCR)
• Real time-PCR or quantitative PCR (qPCR)
Multiplex PCR: Multiplex- PCR mix makes use of multiple primer sets within a single PCR
mixture to produce amplicons of varying sizes which are specific for different sequences of
DNA. In a single tube set there will be specific primers of different targets. The base pair length
of the amplicons, should be different enough to segregate well and form distinct bands when
visualized by gel electrophoresis.
Multiplex- PCR can detect different pathogens in a single sample.
Nested PCR:
• Nested polymerase chain reaction (Nested PCR) is a modification of PCR intended to
reduce non-specific binding in products due to the amplification of unexpected primer
binding sites.
• Nested polymerase chain reaction involves two sets of primers, used in two
successive runs of polymerase chain reaction, the second set intended to amplify a
secondary target within the first run product.
• The target DNA undergoes the first run of polymerase chain reaction with the first set
of primers (shown in green). The product from the first reaction undergoes a second
run with the second set of primers (shown in red). It is very unlikely that any of the
unwanted PCR products contain binding sites for both the new primers, ensuring the
product from the second PCR has little contamination from unwanted products of
primer dimers, and alternative primer target sequences.
Reverse Transcription-PCR (RT- PCR):
• Reverse transcription polymerase chain reaction (RT-PCR), a variant
of polymerase chain reaction (PCR), commonly used in molecular biology to detect
RNA expression.
• This technique enables quantitative detection of levels of RNA expression by
creating complimentary DNA (cDNA) from RNA with the help of reverse
transcriptase enzyme, followed by further amplification of cDNA using standard PCR.
• RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest
into its DNA complement through the use of reverse transcriptase. Subsequently, the
newly synthesized cDNA is amplified using traditional PCR.
• In addition to the qualitative study of gene expression, quantitative PCR can be utilized
for quantification of RNA, in both relative and absolute terms, by incorporating qPCR
into the technique.
• It is frequently used in the expression analysis of single or multiple genes, and
expression patterns for identifying infections and diseases.
✓ **Real time-PCR or quantitative PCR (qPCR):
Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of
nucleic acids. It monitors the amplification of a targeted DNA molecule during the PCR (i.e.,
in real time), not at its end, as in conventional PCR.
• Real-time PCR process is generally carried out for the detection of fluorescence emitted
by the excited molecules which increase as the reaction proceeds and detection of signal
in real time allows quantification of starting material. The signal of which increases in
direct proportion to the amount of PCR product in a reaction.
• It is also called Quantitative real time polymerase chain reaction. First introduced in
1992 by Higuchi and co-workers.
Applications of real time PCR:
• Gene expression analysis: - Cancer research and drug research
• Disease diagnosis and management: - Viral and bacterial quantification.
• Food testing: - Percent GMO food.
• Animal and plant breeding: - Gene copy number
• Validation of DNA microarray results.
• Variation analysis including SNP discovery and validation.
Real-time PCR advantages
• Quantitation of amplified product in real time process rather than end point
detection.
• There is no requirement of gel electrophoresis for detection.
Real-time PCR disadvantages: -
* Setting up requires high technical skill and support.
* High equipment cost
* Runs are more expensive than conventional PCR
Real time PCR chemistries
They allow detection via the generation of a fluorescent signal. Chemistries are classified into
three categories: -
❖ DNA binding dyes
❖ Probe based chemistries
❖ Quenched dye primers: These combine both primer and probe into one molecule: E.g.
– Amplifluor primers and Lux primers
DNA binding dyes: -These bind to dsDNA in non-specific manner.
• Most commonly used dye is SYBR Green I.
• Other dyes include LC Green I, SYTO 9
Probe based chemistries: -It involves one or more fluorescently labelled oligonucleotides that
hybridize specifically to an internal sequence of the amplified product.
• It includes TaqMan probes, molecular beacons, Scorpion probes and FRET
hybridization probes.
• green and TaqMan hydrolysis probes are the most popular dyes used in real-time PCR.
SYBR
TaqMan probes: -These are also called as hydrolysis probes or dual-labelled probe. These are
linear molecules, usually 18-24 bases in length. These contain fluorescent reporter dye
covalently attached at 5’ end and quencher dyes coupled to 3’ end. While the reporter and
quencher are bound to the probe, the quencher absorbs the fluorescence emitted by the reporter.
During the extension phase of the PCR reaction the probe is degraded, releasing the reporter
and allowing its fluorescence to be detected
• TaqMan probes are sequence-specific oligonucleotide probes carrying a
reporter and a quencher dye.
• The fluorophore is attached at the 5' end of the probe and the quencher dye is
located at the 3' end.
• The TaqMan probe bound to its specific piece of the template DNA during
annealing.
• Taq polymerase then adds nucleotides and removes the TaqMan probe from the
template DNA by hydrolysis.
• This separates the quencher from the reporter, and allows the reporter to give
off its energy as emitted in the form of fluorescence which is then quantified
using a computer.
Commonly used reporters:
1. Fluorescein derivatives like 6-carboxyfluorescein (6-FAM), hexachlorofluorescein
(HEX), tetrachlorofluorescein (TET).
2. Cyanine dye family members – cyanine 3 (Cy3), cyanine 5 (Cy5).
3. Rhodamin based dyes – tetramethylrhodamin (TAMRA), Texas Red.
Commonly used quenchers:
1. Tetramethylrhodamin (TAMRA)
2. 4-(4′-dimethylaminophenylazo) benzoic acid benzoic acid (DABCYL)
3. Black-hole quencher dyes (BHQ-1, -2, -3)
Most common reporter-quencher combination for Taqman probes:
• 5’ reporter dye – 6-FAM or TET
• 3’ quencher - TAMRA
Real-timePCR Vs. Conventional PCR
Real-Time PCR Conventional PCR
Sensitivity High Low
Specificity High Low
-use specific probes -only size discrimination
Quantitative results Yes No
-Specific fluorescence -EtBr staining
Detection method Probe-specific Agarose gel
Fluorescence Electrophoresis
Reaction time 1 hr 3-5 hr
Post-PCR steps No Agarose gel electrophoresis
Cross-contamination No Yes
-Closed system -Open system
- Single step - Multiple steps