An Introduction to PCR

Article Published: March 17, 2021| By Arianna Ferrini PhD

Contents

What is PCR?

PCR principles and history

Standard PCR experiment overview

PCR variations

- Quantitative real-time PCR (qPCR)

- Reverse transcription-PCR (RT-PCR)

- Reverse transcription-quantitative PCR (RT-qPCR)

 - Digital PCR (dPCR) and digital droplet PCR (ddPCR)

- Microfluidic PCR

PCR troubleshooting

PCR output applications

What is PCR?

Polymerase chain reaction (PCR) is a technique that has revolutionized the world of
molecular biology and beyond, enabling the amplification of nucleotide sequences.
In this article, we will discuss a brief history of PCR and its principles, highlighting
the different types of PCR and the specific purposes to which they are being
applied.

PCR principles and history

In 1983, American biochemist Kary Mullis was driving home late at night when a
flash of inspiration struck him. He wrote on the back of a receipt the idea that
would eventually grant him the Nobel Prize for Chemistry in 1993. The concept was
straightforward: reproducing in a laboratory tube the DNA replication process that
takes place in cells. The outcome is the same: the generation of new
complementary DNA (cDNA) strands based upon the existing ones.

Mullis used the basis of Sanger's DNA sequencing as a starting point for his new
technique. He realized that the repeated use of DNA polymerase triggered a chain
reaction resulting in a specific DNA segment's amplification.

The foundations for his idea were laid by a discovery in 1976 of a thermostable
DNA polymerase, Taq, isolated from the bacterium Thermus aquaticus found in hot
springs.1 Taq DNA polymerase has a temperature optimum of 72 °C and survives
prolonged exposure to temperatures as high as 96 °C, meaning that it can tolerate
several denaturation cycles.

Before the discovery of Taq polymerase, molecular biologists were already trying to
optimize cyclic DNA amplification protocols, but they needed to add fresh
polymerase at each cycle because the enzyme could not withstand the high
temperatures needed for DNA denaturation. Having a thermostable enzyme
meant that they could repeat the amplification process many times over without
the need for fresh polymerase at every cycle, making the whole process scalable,
more efficient and less time-consuming.

The first description of this polymerase chain reaction (PCR) using Taq polymerase
was published in Science in 1985.2

In 1993, the first FDA-approved PCR kit came to market. Since then, PCR has been
steadily and systematically improved. It has become a game-changer in everything
from forensic evidence analysis and diagnostics, to disease monitoring and genetic
engineering. It is undoubtedly considered one of the most important scientific
advances of the 20th century.

Standard PCR experiment overview

The PCR is used to amplify a specific DNA fragment from a complex mixture of
starting material called template DNA. The sample preparation and purification
protocols depend on the starting material, including the sample matrix and
accessibility of target DNA. Often, minimal DNA purification is needed and some
techniques such as direct PCR or extraction-free PCR require no pre-purification of
DNA or RNA. However, PCR does require knowledge of the DNA sequence
information that flanks the DNA fragment to be amplified (called target DNA).
From a practical point of view, a PCR experiment is relatively straightforward and
can be completed in a few hours. In general, a PCR reaction needs five key
reagents:

DNA to be amplified: also called PCR template or template DNA. This DNA can be
of any source, such as genomic DNA (gDNA), cDNA, and plasmid DNA.

DNA polymerase: all PCR reactions require a DNA polymerase that can work at
high temperatures. Taq polymerase is a commonly used one, which can
incorporate nucleotides at a rate of 60 bases/second at 70 °C and can amplify
templates of up to 5 kb, making it suitable for standard PCR without special
requirements. New generations of polymerases are being engineered to improve
reaction performance. For example, some are engineered to be only activated at
high temperatures to reduce non-specific amplification at the beginning of the
reaction. Others incorporate a “proofreading” function, important, for example,
when it is critical that the amplified sequence matches the template sequence
exactly, such as during cloning.

Primers: DNA polymerases require a short sequence of nucleotides to indicate

where they need to initiate amplification. In a PCR, these sequences are called
primers and are short pieces of single-stranded DNA (approximately 15-30 bases).
When designing a PCR experiment, the researcher determines the region of DNA
to be amplified and designs a pair of primers, one on the forward strand and one
on the reverse, that specifically flanks the target region. Primer design is a key
component of a PCR experiment and should be done carefully. Primer sequences
must be chosen to target the unique DNA of interest, avoiding the possibility of
binding to a similar sequence. They should have similar melting temperatures
because the annealing step occurs simultaneously for both strands. The melting
temperature of a primer can be impacted by the percentage of bases that are
guanine (G) or cytosine (C) compared to adenine (A) or thymine (T), with higher GC
contents increasing melting temperatures. Adjusting primer lengths can help to
compensate for this in matching a primer pair. It is also important to avoid
sequences that will tend to form secondary structures or primer dimers, as this will
reduce PCR efficiency. Many free online tools are available to aid in primer design.

Deoxynucleotide triphosphates (dNTPs): these serve as the building blocks to

synthesize the new strands of DNA and include the four basic DNA nucleotides
(dATP, dCTP, dGTP, and dTTP). dNTPs are usually added to the PCR reaction in
equimolar amounts for optimal base incorporation.

PCR buffer: the PCR buffer ensures that optimal conditions are maintained
throughout the PCR reaction. The major components of PCR buffers include
magnesium chloride (MgCl2), tris-HCl and potassium chloride (KCl). MgCl2 serves as
a cofactor for the DNA polymerase, while tris-HCl and KCl maintain a stable pH
during the reaction.

The PCR reaction is carried out in a single tube by mixing the reagents mentioned
above and placing the tube in a thermal cycler.

The PCR amplification consists of three defined sets of times and temperatures
termed steps: denaturation, annealing, and extension (Figure 1).
Figure 1: Steps of a single PCR cycle.

Each of these steps, termed cycles, is repeated 30-40 times, , doubling the amount
of DNA at each cycle and obtaining amplification (Figure 2).

Figure 2: The different stages and cycles of DNA molecule amplification by PCR.

Let's take a closer look at each step.

1. Denaturation

The first step of PCR, called denaturation, heats the template DNA up to 95 °C for a
few seconds, separating the two DNA strands as the hydrogen bonds between
them are rapidly broken.

2. Annealing

The reaction mixture is then cooled for 30 seconds to 1 minute. Annealing

temperatures are usually 50 - 65 °C however, the exact optimal temperature
depends on the primers' length and sequence. It must be carefully optimized with
every new set of primers.

The two DNA strands could rejoin at this temperature, but most do not because
the mixture contains a large excess of primers that bind, or anneal, to the template
DNA at specific, complementary positions. Once the annealing step is completed,
hydrogen bonds will form between the template DNA and the primers. At this
point, the polymerase is ready to extend the DNA sequence.

3. Extension

The temperature is then raised to the ideal working temperature for the DNA
polymerase present in the mixture, typically around 72 °C, 74 °C in the case of Taq.

The DNA polymerase attaches to one end of each primer and synthesizes new
strands of DNA, complementary to the template DNA. Now we have four strands
of DNA instead of the two that were present to start with.

The temperature is raised back to 94 °C and the double-stranded DNA molecules –

both the "original" molecules and the newly synthesized ones – denature again
into single strands. This begins the second cycle of denaturation-annealing-
extension. At the end of this second cycle, there are eight molecules of single-
stranded DNA. By repeating the cycle 30 times, the double-stranded DNA
molecules present at the beginning are converted into over 130 million new
double-stranded molecules, each one a copy of the region of the starting molecule
delineated by the annealing sites of the two primers.

To determine if amplification has been successful, PCR products may be visualized

using gel electrophoresis, indicating amplicon presence/absence, size and
approximate abundance. Depending on the application and the research question,
this may be the endpoint of an experiment, for example, if determining whether a
gene is present or not. Otherwise, the PCR product may just be the starting point
for more complex downstream investigations such as sequencing and cloning.

PCR variations

Thanks to their versatility, PCR techniques have evolved over recent years leading
to the development of several different types of PCR technology.

Some of the most widely used ones are:

Quantitative real-time PCR (qPCR)

One of the most useful developments has been quantitative real-time PCR or
qPCR. As the name suggests, qPCR is a quantitative technique that allows real-time
monitoring of the amplification process and detection of PCR products as they are
made.2 It can be used to determine the starting concentration of the target DNA,
negating the need for gel electrophoresis in many cases. This is achieved thanks to
the inclusion of non-specific fluorescent intercalating dyes, such as SYBR® Green,
that fluoresce when bound to double-stranded DNA, or DNA oligonucleotide
sequence-specific fluorescent probes, such as hydrolysis (TaqMan) probes and
molecular beacons. Probes bind specifically to DNA target sequences within the
amplicon and use the principle of Förster Resonance Energy Transfer (FRET) to
generate fluorescence via the coupling of a fluorescent molecule on one end and a
quencher at the other end. For both fluorescent dyes and probes, as the number
of copies of the target DNA increases, the fluorescence level increases
proportionally, allowing real-time quantification of the amplification with reference
to standards containing known copy numbers (Figure 3).

qPCR uses specialized thermal cyclers equipped with fluorescent detection

systems that monitor the fluorescent signal as the amplification occurs.
Figure 3: Example qPCR amplification plot and standard curve used to enable
quantification of copy number in unknown samples.

Reverse transcription-PCR (RT-PCR)

Reverse transcription (RT) -PCR and RT-qPCR are two commonly used PCR variants
enabling gene transcription analysis and quantification of viral RNA, both in clinical
and research settings.

RT is the process of making cDNA from single-stranded template RNA3 and is

consequently also called first-strand cDNA synthesis. The first step of RT-PCR is to
synthesize a DNA/RNA hybrid between the RNA template and a DNA
oligonucleotide primer. The reverse transcriptase enzyme that catalyzes this
reaction has RNase activity that then degrades the RNA portion of the hybrid.
Subsequently, a single-stranded DNA molecule is synthesized by the DNA
polymerase activity of the reverse transcriptase. High purity and quality starting
RNA are essential for a successful RT-PCR.

RT-PCR can be performed following two approaches: one-step RT-PCR and two-
step RT-PCR. In the first case, the RT reaction and the PCR reaction occur in the
same tube, while in the two-step RT-PCR, the two reactions are separate and
performed sequentially.

Reverse transcription-quantitative PCR (RT-qPCR)

The reverse transcription described above often serves as the first step in qPCR
too, quantifying RNA in biological samples (either RNA transcripts or derived from
viral RNA genomes).

As with RT-PCR, there are two approaches for quantifying RNA by RT-qPCR: one-
step RT-qPCR and two-step RT-qPCR. In both cases, RNA is first reverse-transcribed
into cDNA, which is used as the template for qPCR amplification. In the two-step
method, the reverse transcription and the qPCR amplification occur sequentially as
two separate experiments. In the one-step method, RT and qPCR are performed in
the same tube.

Digital PCR (dPCR) and digital droplet PCR (ddPCR)

Digital PCR (dPCR) is another adaptation of the original PCR protocol.4 Like qPCR,
dPCR technology uses DNA polymerase to amplify target DNA from a complex
sample using a primer set and probes. The main difference, though, lies in the
partitioning of the PCR reactions and data acquisition at the end.

dPCR and ddPCR are based on the concept of limiting dilutions. The PCR reaction is
split into large numbers of nanoliter-sized sub-reactions (partitions). The PCR
amplification is carried out within each droplet. Following PCR, each droplet is
analyzed with Poisson statistics to determine the percentage of PCR-positive
droplets in the original sample. Some partitions may contain one or more copies of
the target, while others may contain no target sequences. Therefore, partitions
classify either as positive (target detected) or negative (target not detected),
providing the basis for a digital output format.
ddPCR is a recent technology that became available in 2011.5 ddPCR utilizes a
water-oil emulsion to form the partitions that separate the template DNA
molecules. The droplets essentially serve as individual test tubes in which the PCR
reaction takes place. This technology was put to use in creating sensitive SARS-CoV-
2 tests.

Microfluidic PCR

The recent development of microfluidic handling systems with microchannels and

microchambers has paved the way for a range of practical applications, including
the amplification of DNA via PCR on microfluidic chips.

PCR performed on a chip benefits from microfluidics’ advantages in speed,

sensitivity and low consumption of reagents. These features make microfluidic PCR
particularly appealing for point-of-care testing, for example, for diagnostics
applications. From a practical point of view, the sample flows through a
microfluidic channel, repeatedly passing the three temperature zones reflecting
the different steps of PCR. It takes just 90 seconds for a 10 μL sample to perform
20 PCR cycles.6 The subsequent analysis can then be easily carried out off-chip.

Continue reading below...

Article

A Guide to DNA and RNA Quantification and Quality

Read More

PCR troubleshooting

The different PCR approaches all have advantages and disadvantages that impact
the applications to which they are suited 7. These are summarized in Table 1.

Approach Advantages Limitations

PCR · Easiest PCR to perform · Results are only qualitative

· Low cost of equipment and · Requires post-amplification

reagents analyses that increase time
and risk of error
· Several downstream
 applications (e.g., cloning) · Products may need to be
confirmed by sequencing

qPCR · Produces quantitative results · Requires more expensive

reagents and equipment
· Probe use can ensure high
specificity · Less flexibility in primer and
probe selection
· High analytical sensitivity
· Less amenable to other
· Low turnaround time
downstream product
· Eliminates requirements for confirmation analyses (such
post-amplification analysis as sequencing) due to the
small length of the amplicon

· Not suitable for some

downstream applications
such as cloning

RT-PCR · Can be used with all RNA · RNA is prone to degradation

and RT- types
· The RT step may increase
qPCR
the time and potential for
contamination

dPCR and · Fast · Costly

ddPCR
· No DNA purification step · Based on several statistical
assumptions
· Provides absolute
quantification

· Increased sensitivity for

detecting the target in limited
clinical samples

· Highly scalable
 Microfluid · Accelerated PCR process · Still very new technology
ic PCR
· Reduced reagent · Requires extensive sample
consumption preparation to remove debris
and unwanted compounds
· Can be adapted for high
throughput · Restricted choice of
materials for the microfluidic
· Portable device for point-of-
device due to high
care applications
temperatures
· Allows single-cell analysis

Table 1: Key advantages and disadvantages of different PCR approaches.

How To Guide

PCR Troubleshooting
READ MORE
PCR output applications

PCR has become an indispensable tool in modern molecular biology and has
completely transformed scientific research. The technique has also opened up the
investigation of cellular and molecular processes to those outside the field of
molecular biology and consequently also finds utility by scientists in many
disciplines.

Whilst PCR is itself a powerful standalone technique, it has also been incorporated
into wider techniques, such as cloning and sequencing, as one small but important
part of these workflows.

Research applications of PCR include:

Gene transcription - PCR can examine variations in gene transcription among cell
types, tissues and organisms at a specific time point. In this process, RNA is
isolated from samples of interest, and reverse-transcribed into cDNA. The original
levels of RNA for a specific gene can then be quantified from the amount of cDNA
amplified in PCR.

Genotyping - PCR can detect sequence variations in alleles of specific cells or

organisms. A common example is the genotyping of transgenic organisms, such as
knock-out and knock-in mice. In this application, primers are designed to amplify
either a transgene portion (in a transgenic animal) or the mutation (in a mutant
animal).

Cloning and mutagenesis - PCR cloning is a widely used technique where double-
stranded DNA fragments amplified by PCR are inserted into vectors (e.g., gDNA,
cDNA, plasmid DNA). This for example, enables the creation of bacterial strains
from which genetic material has been deleted or inserted. Site-directed
mutagenesis can also be used to introduce point mutations via cloning. This often
employs a technique known as recombinant PCR, in which overlapping primers are
specifically designed to incorporate base substitutions (Figure 4). This technique
can also be used to create novel gene fusions.

Figure 4: Diagram depicting an example of recombinant PCR.

Sequencing - PCR can be used to enrich template DNA for sequencing. The type of
PCR recommended for the preparation of sequencing templates is called high-
fidelity PCR and is able to maintain DNA sequence accuracy. In Sanger sequencing,
PCR-amplified fragments are then purified and run in a sequencing reaction. In
next-generation sequencing (NGS), PCR is used at the library preparation stage,
where DNA samples are enriched by PCR to increase the starting quantity and
tagged with sequencing adaptors to allow multiplexing. Bridge PCR is also an
important part of the second-generation NGS sequencing process.

Both as an independent technique and as a workhorse within other methods, PCR

has transformed a range of disciplines. These include:
Genetic research - PCR is used in most laboratories worldwide. One of the most
common applications is gene transcription analysis9, aimed at evaluating the
presence or abundance of particular gene transcripts. It is a powerful technique in
manipulating the genetic sequence of organisms – animal, plant and microbe -
through cloning. This enables genes or sections of genes to be inserted, deleted or
mutated to engineer in genetic markers alter phenotypes, elucidate gene functions
and develop vaccines to name but a few. In genotyping, PCR can be used to detect
sequence variations in alleles in specific cells or organisms. Its use isn’t restricted
to humans either. Genotyping plants in agriculture assists plant breeders in
selecting, refining, and improving their breeding stock. PCR is also the first step to
enrich sequencing samples, as discussed above. For example, most mapping
techniques in the Human Genome Project (HGP) relied on PCR.

Medicine and biomedical research - PCR is used in a host of medical

applications, from diagnostic testing for disease-associated genetic mutations, to
the identification of infectious agents. Another great example of PCR use in the
medical realm is prenatal genetic testing. Prenatal genetic testing through PCR can
identify chromosome abnormalities and genetic mutations in the fetus, giving
parents-to-be important information about whether their baby has certain genetic
disorders. PCR can also be used as a preimplantation genetic diagnosis tool to
screen embryos for in vitro fertilization (IVF) procedures.

Forensic science - Our unique genetic fingerprints mean that PCR can be
instrumental in both paternity testing and forensic investigations to pinpoint
samples' sources. Small DNA samples isolated from a crime scene can be
compared with a DNA database or with suspects' DNA, for example. These
procedures have really changed the way police investigations are carried out.
Authenticity testing also makes use of PCR genetic markers, for example, to
determine the species from which meat is derived. Molecular archaeology too
utilizes PCR to amplify DNA from archaeological remains.

Environmental microbiology and food safety - Detection of pathogens by PCR,

not only in patients' samples but also in matrices like food or water, can be vital in
diagnosing and preventing infectious disease.
PCR is the benchmark technology for detecting nucleic acids in every area, from
biomedical research to forensic applications. Kary Mullis's idea, written on the back
of a receipt on the side of the road, turned out to be a revolutionary one.

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