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Polymerase Chain Reaction

Article in Journal of Investigative Dermatology · March 2013

DOI: 10.1038/jid.2013.1 · Source: PubMed

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J Invest Dermatol. 2013 March ; 133(3): e6. doi:10.1038/jid.2013.1.

Research Techniques Made Simple: Polymerase Chain Reaction

(PCR)
Lilit Garibyan1 and Nidhi Avashia2
1Department of Dermatology, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts, USA
2Departmentof Dermatology, Boston University and Boston Medical Center, Boston,
Massachusetts, USA

Introduction
The advent of the polymerase chain reaction (PCR) radically transformed biological science
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from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific
detection and production of large amounts of DNA. PCR-based strategies have propelled
huge scientific endeavors such as the Human Genome Project. The technique is currently
widely used by clinicians and researchers to diagnose diseases, clone and sequence genes,
and carry out sophisticated quantitative and genomic studies in a rapid and very sensitive
manner. One of the most important medical applications of the classical PCR method is the
detection of pathogens. In addition, the PCR assay is used in forensic medicine to identify
criminals. Because of its widespread use, it is important to understand the basic principles of
PCR and how its use can be modified to provide for sophisticated analysis of genes and the
genome.

The PCR Process

PCR is a simple, yet elegant, enzymatic assay, which allows for the amplification of a
specific DNA fragment from a complex pool of DNA. Dr. Kary Mullis, who discovered the
PCR assay, stated it “lets you pick the piece of DNA you’re interested in and have as much
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of it as you want” (Mullis, 1990). PCR can be performed using source DNA from a variety
of tissues and organisms, including peripheral blood, skin, hair, saliva, and microbes. Only
trace amounts of DNA are needed for PCR to generate enough copies to be analyzed using
conventional laboratory methods. For this reason, PCR is a sensitive assay.

Each PCR assay requires the presence of template DNA, primers, nucleotides, and DNA
polymerase. The DNA polymerase is the key enzyme that links individual nucleotides
together to form the PCR product. The nucleotides include the four bases – adenine,
thymine, cytosine, and guanine (A, T, C, G) – that are found in DNA. These act as the
building blocks that are used by the DNA polymerase to create the resultant PCR product.
The primers in the reaction specify the exact DNA product to be amplified. The primers are
short DNA fragments with a defined sequence complementary to the target DNA that is to
be detected and amplified. These serve as an extension point for the DNA polymerase to
build on.
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The above mentioned components are mixed in a test tube or 96-well plate and then placed
in a machine that allows repeated cycles of DNA amplification to occur in three basic steps.
The machine is essentially a thermal cycler. It has a thermal block with holes, into which the
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test tubes or plates holding the PCR reaction mixture are inserted. The machine raises and
lowers the temperature of the block in discrete, precise and pre-programmed steps (Weier &
Gray, 1988). The reaction solution is first heated above the melting point of the two
complementary DNA strands of the target DNA, which allows the strands to separate, a
process called denaturation. The temperature is then lowered to allow the specific primers to
bind to the target DNA segments, a process known as hybridization or annealing. Annealing
between primers and the target DNA occurs only if they are complementary in sequence
(e.g. A binding to G). The temperature is raised again, at which time the DNA polymerase is
able to extend the primers by adding nucleotides to the developing DNA strand (Figure 1).
With each repetition of these three steps, the number of copied DNA molecules doubles.

Analysis of the PCR product

There are two main methods of visualizing the PCR products: (1) staining of the amplified
DNA product with a chemical dye such as ethidium bromide, which intercalates between the
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two strands of the duplex or (2) labeling the PCR primers or nucleotides with fluorescent
dyes (fluorophores) prior to PCR amplification. The latter method allows the labels to be
directly incorporated in the PCR product. The most widely used method for analyzing the
PCR product is the use of agarose gel electrophoresis, which separates DNA products on the
basis of size and charge. Agarose gel electrophoresis is the easiest method of visualizing and
analyzing the PCR product. It allows for the determination of the presence and the size of
the PCR product (Figure 2). A predetermined set of DNA products with known sizes are run
simultaneously on the gel as standardized molecular markers to help determine the size of
the product.

Typically, when PCR is used to detect the presence or absence of a specific DNA product, it
is called qualitative PCR. Qualitative PCR is a good technique to use when PCR is
performed for cloning purposes or to identify a pathogen. For example, in the report by
Dworkin et al, qualitative PCR was used to detect the presence of Merkel cell polyomavirus
in cutaneous squamous cell carcinoma (SCC) in immunocompetent individuals (2009).
Using genomic DNA isolated from SCCs excised from immunocompetent individuals and
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primers specific to virus genes, the investigators were able to demonstrate the presence of a
351base pair (bp) viral gene in 6 out of 16 samples tested, by the presence of a PCR-product
band about 351 bp long, as seen on a 2% agarose gel with ethidium bromide (Figure 3). The
experiment also included template DNA from a polyomavirus containing plasmid as a
positive control (P) and a negative water control (W). The first lane marked by (M) is the
molecular marker, which is used to identify the size of the detected PCR product. The
presence of a viral specific gene detected by PCR is marked by (+); absence of viral gene is
marked by (−).

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Quantitative PCR
Quantitative real-time or qRT-PCR provides information beyond mere detection of DNA. It
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indicates how much of a specific DNA or gene is present in the sample. qRT-PCR allows for
both detection and quantification of the PCR product in real-time, while it is being
synthesized (Van Guilder et al, 2008). The two common methods used to detect and
quantify the product include (1) fluorescent dyes that non-specifically intercalate with
double-stranded DNA and (2) sequence-specific DNA probes consisting of fluorescently
labeled reports. These permit detection only after hybridization of the probe with its
complementary DNA target. Real-time PCR can be combined with reverse transcription,
which allows messenger RNA to be converted into cDNA (i.e., reverse transcription), after
which quantification of the cDNA is performed with qPCR (Valasek & Ripa, 2005).
Quantification of the desired gene during the exponential amplification avoids problems that
are associated with end-point PCR, which is analyzation after completion of the final PCR
cycle.

Analysis of tumors is an ideal example of the use of PCR. It can be used to isolate and
amplify DNA of tumor suppressor genes or proto-oncogenes. In turn, quantitative PCR can
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be utilized to quantify the amount of the particular gene isolated. On the other hand,
quantitative PCR can be used to analyze single cells and quantify any combination of DNA,
mRNAs, and proteins. (Stahlberg et al, 2012)

Advantages and limitations of PCR

There are multiple advantages to PCR. First, it is a simple technique to understand and to
use, and it produces results rapidly (Bolognia et al, 2008). It is a highly sensitive technique
with the potential to produce millions to billions of copies of a specific product for
sequencing, cloning, and analysis. This is true of qRT-PCR as well, but qRT-PCR has the
advantage of quantification of the synthesized product. Thus, it can be used to analyze
alterations of gene expression levels in tumors, microbes, or other disease states.

Although PCR is a valuable technique, it does have limitations. Because PCR is a highly
sensitive technique, any form of contamination of the sample by even trace amounts of DNA
can produce misleading results (Bolognia et al, 2008; Smith & Osborn, 2009). In addition, in
order to design primers for PCR, some prior sequence data is needed. Therefore, PCR can
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only be used to identify the presence or absence of a known pathogen or gene. Another
limitation is that the primers used for PCR can anneal non-specifically to sequences that are
similar, but not completely identical to target DNA. In addition, incorrect nucleotides can be
incorporated into the PCR sequence by the DNA polymerase, albeit at a very low rate.

SUMMARY AND FUTURE DIRECTION

PCR is a simple and widely used process in which minute amounts of DNA can be
amplified into multiple copies. In addition to the rapidity with which this assay works, it is
able to quantitatively demonstrate how much of a particular sequence is present. As with all
methods, the validity of the results should be compared with the specificity associated with
the method. The future of PCR is promising, combining various assays and approaches to

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produce greater insight into various gene combinations (Botes et al, 2011). For example, in a
study to link distinct taxa within the microbial community to specific metabolic processes,
stable isotope probing was combined with qPCR (Postoller et al, 2011; Smith & Osborn,
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2009). Microarray experiments can be validated by qPCR approaches due to its rapidity.

References
1. Mullis KB. The unusual origin of the polymerase chain reaction. Scientific American. 1990; 262(4):
56–61. 64–5. [PubMed: 2315679]
2. Weier HU, Gray JW. A programmable system to perform the polymerase chain reaction. DNA.
1988; 7(6):441–7. [PubMed: 3203600]
3. Dworkin AM, et al. Merkel cell polyomavirus in cutaneous squamous cell carcinoma of
immunocompetent individuals. J Invest Derm. 2009; 129(12):2868–74. [PubMed: 19554019]
4. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene
expression analysis. BioTechniques. 2008; 44(5):619–26. [PubMed: 18474036]
5. Valasek MA, Repa JJ. The power of real-time PCR. Advances in physiology education. 2005; 29(3):
151–9. [PubMed: 16109794]
6. Stahlberg A, Thomsen C, Ruff D, et al. Quantitative PCR analysis of DNA, RNAs, and Proteins in
the same single cell. Clin Chem. 2012; 58:1682–1691. [PubMed: 23014600]
7. Botes M, de Kwaadsteniet M, Cloete TE. Application of quantitative PCR for the detection of
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microorganisms in water. Food Microbiol. 2011 Aug; 28(5):848–61. [PubMed: 21569926]

8. AUTHOR/S. TITLE OF CHAPTER. In: Bolognia, J.; Jorizzo, J.; Rapini, R., editors. Dermatology.
Vol. 2008. Elsevier; Spain: 2008. PAGE RANGE
9. Postollec F, Falentin H, Pavan D, et al. Recent advances in quantitative PCR (qPCR) applications in
food microbiology. Food Microbiol. 2011 Aug; 28(5):848–61. [PubMed: 21569926]
10. Smith C, Osborn M. Advantages and limitations of quantitative PCR (qPCR)-based approaches in
microbial ecology. FEMS Microbiol Ecol. 2009 Jan; 67(1):6–20. [PubMed: 19120456]
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WHAT PCR DOES

• PCR is a very sensitive technique that allows rapid amplification of a specific
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segment of DNA.

• PCR makes billions of copies of a specific DNA fragment or gene, which allows
detection and identification of gene sequences using visual techniques based on
size and charge.

• Modified versions of PCR have allowed quantitative measurements of gene

expression with techniques called real-time PCR

LIMITATIONS

• The DNA polymerase used in the PCR reaction is prone to errors and can lead
to mutations in the fragment that is generated

• The specificity of the generated PCR product may be altered by nonspecific

binding of the primers to other similar sequences on the template DNA

• In order to design primers to generate a PCR product, some prior sequence

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information is usually necessary.

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Figure 1.
Schematic presentation of the PCR principle

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Figure 2. Visualization of the PCR products on an agarose gel

From Riedl et al, 2004: Identification of an alternatively spliced mouse Langerin transcript.
(a) Ethidium bromide-stained agarose gel showing PCR products from full-length mouse
Langerin obtained from C57BL/6 fresh LC (fLC) and from fetal skin-derived dendritic cells
(FSDDC).
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Figure 3. Qualitative PCR detects the presence of Merkel cell polyomavirus in cutaneous
squamous cell carcinoma (SCC) in immunocompetent individuals
From Drowkin et al, 2009: MCPyV detection. (a) The presence of MCPyV in SCCs,
genomic normal DNA, and adjacent skin DNA was determined by PCR using VP1 primers.
A representational result is shown with 6 of 16 samples tested showing a PCR product at
351 bp. All experiments included DNA from an MCPyV plasmid as a positive control (P)
and a negative water control (W). M, molecular weight marker; +, positive for virus; −,
negative for virus.
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