Special Series: Basic Science for the Practicing Clinician

Principles and applications of polymerase chain

reaction: basic science for the practicing
physician
Faoud T. Ishmael, MD, PhD, and Cristiana Stellato, MD, PhD

Objective: To review the technology of polymerase chain reaction (PCR) and its use in diagnostic medicine and biomedical
research.
Data Sources: A literature review was performed using the PubMed database for English-language articles published between
January 1, 1985, and November 31, 2007, using the following keywords: polymerase chain reaction, real-time polymerase chain
reaction, and applications of real-time polymerase chain reaction.
Study Selection: Select review articles and primary articles relevant to medical applications of PCR were chosen.
Results: As a revolutionary technique, PCR provides a rapid and accurate means of amplifying DNA. It has enabled the
cloning and manipulation of genes for biomedical research and has facilitated the diagnosis of genetic, infectious, and oncologic
diseases. The use of reverse transcriptases to evaluate RNA levels and the extension of PCR technology to quantify DNA
amplification in real time has brought major advances to the application of PCR. By allowing the determination and
quantification of changes in gene expression, these techniques have provided a greater understanding of disease processes and
now serve as a foundation for diagnostics and basic science research.
Conclusions: Both PCR and real-time PCR have dramatically changed the field of basic science and diagnostic medicine.
These technologies are now a well-established, indispensable part of research and clinical laboratories.
Ann Allergy Asthma Immunol. 2008;101:437–443.

INTRODUCTION vember 31, 2007, using the following keywords: polymerase

Few techniques have revolutionized science and biomedical
research as polymerase chain reaction (PCR). PCR permits
the amplification of specific regions of DNA more than a
billion-fold and allows the manipulation of DNA for tech-
niques such as cloning of genes.1,2 The field of modern
molecular biology owes most of its success to PCR, and large
undertakings, such as the Human Genome Project, were
possible as a result of this technique.3 Furthermore, PCR has
changed the face of medicine and is now routinely used as a
diagnostic tool in the identification of diseases and microbial
infections. The principle of in vitro amplification of DNA
was first described in 1974 by Panet and Khorana.4 As we
know it today, PCR was developed by Mullis and coworkers
in the 1980s, and the significance of this work was recog-
nized with a Nobel Prize in 1993.
To describe in this review the development of PCR and the
current techniques based on this method, a literature review
was performed using the PubMed database for English-lan-
guage articles published between January 1, 1985, and No-
Affiliations: Division of Allergy and Clinical Immunology, Johns Hop-
kins University, Baltimore, Maryland.
Disclosures: Authors have nothing to disclose.
Received for publication February 6, 2008; Received in revised form
March 20, 2008; Accepted for publication March 23, 2008.
chain reaction, real-time polymerase chain reaction, and
applications of real-time polymerase chain reaction. Select
review articles and primary articles relevant to medical ap-
plications of PCR were chosen.
PCR: THE BASICS
One of the key discoveries underlying the PCR technique was
a thermostable DNA polymerase (Taq polymerase) that was
isolated from Thermus aquaticus, a bacterium that grows in
hot springs.2 This polymerase can withstand the heating and
cooling cycles needed for PCR, allowing the efficient ampli-
fication of DNA, as described in the next paragraph.
PCR requires 4 primary components: the thermostable
DNA polymerase, nucleotide triphosphates (which serve as
building blocks for the creation of DNA), sample DNA to be
amplified, and gene-specific primers. The source of sample
DNA can be either genomic DNA, isolated from cells or
tissues, or DNA obtained from RNA samples through reverse
transcription (RT) (see the “RT-PCR” section) (Fig 1). Prim-
ers are short, sequence-specific oligonucleotides that are gen-
erated via chemical synthesis to be complimentary to a cho-
sen DNA sequence of any gene of interest.
PCR is composed of repeating cycles of 3 consecutive
steps that require distinct temperature conditions. Each step is
devoted to a specific process, ultimately leading to the gen-

VOLUME 101, OCTOBER, 2008 437

 Figure 1. Sources of DNA for polymerase chain reaction (PCR) and reverse transcription (RT)–PCR analysis. A, Genomic DNA isolated from cells or tissues
can be amplified by means of PCR. B, In RT-PCR, messenger RNA (mRNA) can be transcribed by RT into complementary DNA (cDNA), which is then used
as a template for amplification. Exons (ie, regions of DNA that are expressed) are shown in gray.
eration of more copies of the chosen gene (Fig 2). This
reaction is accomplished through the use of a thermocycler,
an apparatus that holds the samples in a heating block, where
rapid and controlled changes in temperatures are performed
in the different phases of the amplification process.
The first PCR step is separation of the double-stranded
DNA (dsDNA) by heating the sample mix to approximately
90°C. In the second step, the sample is cooled, which allows
the annealing— base-specific pairing— of primers to comple-
mentary strands of DNA. The temperature of this step is
determined by using several physicochemical variables of the
chosen primers.5 In the third step, the mixture is heated to
72°C, which is the optimal temperature for the activity of Taq
DNA polymerase. The polymerase catalyzes the synthesis of
new DNA strands using the primers as a starting point and
uses the nucleotide triphosphates present in the mix to gen-
erate the sequence-specific complementary strand, in a pro-
cess called elongation. Repetition of these 3 steps results in
doubling of the copy number with each cycle (copy num-
ber ⫽ 2n, where n is the cycle number). The generation of
PCR products, therefore, follows an exponential pattern and
reaches a plateau after approximately 30 to 40 cycles, when
most reagents have been used and no more PCR product is
generated. Such enormous amplification capability has al-
lowed the detection of DNA from a single cell.6
APPLICATIONS OF PCR
By allowing the amplification and manipulation of DNA,
PCR has greatly expanded the applications of molecular
biology. A gene of interest can be selectively amplified from
genomic DNA using appropriate primers and placed into
other plasmids or vectors for further study. Furthermore,
alteration of the primer sequences can be used to create gene
mutants. Any change in the primer sequence from genomic
DNA will be incorporated into the replicated DNA strand,
allowing researchers to create altered genes for study. These
cloning techniques have been invaluable in studying the
regulation of the expression and the function of genes and
438
their products. The PCR-based techniques are a key tool in
experimental protocols using animal models, where they can
be applied to overexpress or selectively disrupt a gene of
interest, thus providing great insight into the roles of specific
genes in disease pathogenesis.
The power of PCR has also become evident in the field of
diagnostic medicine. The PCR techniques are used to se-
quence genes and can, thus, identify mutations in genetic
diseases. One of the first diagnostic applications of PCR was
the prenatal diagnosis of sickle cell anemia through the de-
tection of a single gene mutation.7 The technique has now
become routine in identifying genetic diseases.8 In addition,
PCR has aided in the diagnosis of cancer by allowing the
detection of mutations in oncogenes and tumor suppressor
genes.9,10
PCR also plays a vital role in forensic medicine. Short
pieces of DNA, termed microsatellite or minisatellite loci
DNA, serve as molecular fingerprints to differentiate individ-
uals.11 Trace amounts of DNA obtained from forensic evi-
dence can be amplified to unambiguously match the sample
to a particular individual. The ability of PCR to amplify and
detect minute amounts of genetic material has tremendously
affected the field of infectious diseases. Physicians can now
rapidly detect the presence of microbes that are difficult to
culture or that require weeks for growth. PCR is commonly
used to identify Mycobacterium tuberculosis, human immu-
nodeficiency virus, herpes simplex virus, syphilis, and count-
less other pathogens.12,13
RT-PCR
Using genomic DNA as starting material for PCR amplifica-
tion provides valuable information, but it cannot indicate
whether the detected gene is actually expressed in a given
cell, tissue, or individual, and it does not allow us to compare
changes in gene expression among different conditions. To
reach these goals, PCR technology has been expanded to use
RT enzymes to amplify messenger RNA (mRNA). This
method is referred to as RT-PCR and is used to convert
ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
 A B
5‘ 3‘
3‘ 3‘ Excitation
5‘ 5‘
5‘ S
3‘ Pol
3‘ 3‘ Pol
5‘ 5‘ 5‘
3‘ Pol 3‘ S S
5‘
N
90 °C 72 °C S
30-40 III. Elongation
I. Melting
cycles Excitation Detection

45-65 °C
Q R
II. Primer (and Probe) Annealing
P probe
3‘ I.
5‘
3‘ 5‘
P
3‘
Q R
5‘ 5‘
P 3‘ P probe

II.
Pol Data
Analysis

Q R

P
III. Pol

Figure 2. A, Steps in the polymerase chain reaction (PCR): I, double strands of DNA are separated by heating at a high (melting) temperature; II, specific
primers (P) are annealed by bringing the mixture into an optimal temperature range; and III, elevation of temperature to the optimal activity of the heat-stable
Taq polymerase (Pol) results in the elongation phase, in which generation of new strands of DNA occurs through the addition of nucleotides (N) from the primers.
These 3 steps are repeated 30 to 40 times to amplify the gene of interest. B, Detection of PCR products in real-time PCR. Top, DNA binding dyes such as SYBR
Green I (S) fluoresce only when bound to double-stranded DNA. Bottom, Hydrolysis probes are sequence-specific oligonucleotides that contain a reporter
fluorophore (R) that is quenched by an attached molecule (Q). During replication, the Pol II cleaves the oligonucleotide, enabling emission of the reporter’s
fluorescence as it is separated from the quencher. In both types of detection, fluorescence is recorded using the real-time PCR instrument and processed using
software for data analysis.

mRNA into complementary DNA (cDNA) in vitro before
amplification through PCR.
The RTs are RNA-dependent DNA polymerases that are
expressed by RNA-containing retroviruses, which can tran-
scribe DNA using the viral RNA as a template. The enzyme
most widely used for the RT in RT-PCR is the Moloney
murine leukemia virus transcriptase, which synthesizes the
cDNA first strand from a single-stranded RNA template.
RT-PCR can be performed as either a single step, where
mRNA undergoes RT to DNA and then amplification in a
single reaction, or as a 2-step process, where RT and ampli-
fication occur in separate reactions. One-step real-time PCR
may minimize experimental variation because both enzy-
matic reactions occur in a single tube.14 However, RNA is
used as the starting template, which can be prone to degra-
dation across time. Therefore, if the same sample is to be
assayed multiple times across time, a 2-step process may be
desirable. In the latter approach, total mRNA undergoes RT
to cDNA, of which an aliquot is used in subsequent PCRs.
The main disadvantage of this technique is the increased
opportunity for DNA contamination.14 Regardless of the
method, the level of cDNA synthesized is proportional to the
amount of starting mRNA, so this technique can be used to
determine changes in gene expression.
Real-Time PCR
Although PCR represented a tremendous leap in technology,
its uses were mostly qualitative because the ability to quantify
amounts of nucleic acids by conventional means was limited.
Once the PCR is completed, the products are separated based
on size by means of agarose gel electrophoresis and are
detected, in the form of a bright band, with ethidium bromide,
a compound that fluoresces once bound to dsDNA. This
so-called end-point detection is semiquantitative at best for
several reasons: ethidium bromide lacks sensitivity, the end-
point levels of product from the same sample vary from PCR

VOLUME 101, OCTOBER, 2008 439

run to run, and there is a limited dynamic range for precise
quantification of the product based on densitometric analysis
of the brightness of the band.14,15
Other methods in molecular biology were available for
measuring quantities of specific nucleic acid sequences, such
as Northern and Southern blots, in situ hybridization, and
RNase protection assays. However, each of these methods
had shortcomings, including lack of sensitivity, propensity to
be labor intensive and time-consuming, the need for radioac-
tivity, and limited ranges of detection.14,15 A more sensitive,
accurate, and simple method to quantify nucleic acids was
needed, and it became evident that PCR could become quan-
titative by measuring the amount of amplified material as it is
generated at each cycle. This process, termed real-time PCR,
became a reality as a result of the development of fluorescent
chemistries and instrumentation that allows detection and
quantitation of DNA not at the end of the amplification
process but while it is synthesized. This yields results in
numerical values that provide true quantitative capabilities.
Both PCR and RT-PCR can be performed using this technol-
ogy.
Real-time PCR instruments combine the ability to excite,
detect, and record fluorescence using a regular thermocycler.
The fluorescent dyes or probes used in the PCR mix require
an energy source for excitation, and real-time instruments use
a variety of energy sources, including lamps (ie, tungsten
halogen), lasers, and light-emitting diodes.15 Most instru-
ments incorporate lamps that can be combined with filters to
excite dyes at specific wavelengths. Fluoresce emission is
then measured using a photodetector, such as a camera or
photomultiplier tubes. Multiple wavelengths can be measured
at once, which confers the ability to detect multiple targets in
a single reaction tube (also known as multiplex analysis).
Specific software is used to collect and elaborate the data in
graphic and numeric form for end-user analysis.
A variety of fluorescence-based real-time PCR assays are
currently available.14 Dyes that emit fluorescence only after
binding to dsDNA are commonly used, and SYBR Green I
has proved to be the most sensitive agent. Once bound to the
minor groove of dsDNA, SYBR Green fluoresces 1,000-fold
more than when it is free in solution (Fig 2A).5 The fluores-
cence intensity increases proportionally to the amount of
dsDNA, enabling quantitation of PCR products. Similar to
the ethidium bromide dye used in end-point PCR, binding of
SYBR Green is not sequence specific—this characteristic
being given by the use of gene-specific primers— but detects
all newly generated cDNA.
Another layer of specificity can be uniquely added in
real-time PCR by using an approach alternative to the use of
nonspecific dyes such as SYBR Green. This is accomplished
through the addition to the mix of a probe that is a fluores-
cent-labeled, sequence-specific synthetic oligonucleotide
working together with the primers to provide higher gene-
specific detection. Widely used are hydrolysis probes, which
contain a reporter dye at the 5⬘ end and a quencher molecule
at the 3⬘ end of the molecule.16 This PCR assay is based on
440
the detection of the fluorescence that is generated on cleavage
of the probe as the PCR product is synthesized (Fig 2B).
When the probe is intact, the quencher prevents the reporter
from fluorescing through a process known as fluorescence
resonance energy transfer.17 As the new DNA strand is
synthesized, the 5⬘ nuclease activity of the DNA polymerase
cleaves the probe, separating the quencher and the reporter
dye. Because fluorescence resonance energy transfer requires
the close proximity of the reporter and quencher molecules,
increased fluorescence emission occurs as the dye and the
quencher are separated, and the signal intensity is propor-
tional to the amount of PCR product generated. This method
requires a different probe for each gene to be assayed and is
more expensive than using DNA binding dyes.15 However, it
provides greater specificity because only sequence-specific
amplification is measured, and it is particularly useful in
detecting DNA mutations and single-nucleotide polymor-
phisms through the use of a melting curve analysis.15 In this
important application, fluorescently labeled oligonucleotides
are hybridized to an amplified PCR product in tandem. Heat-
ing of the sample results in dissociation of the probe and a
change in fluorescence. If a mutation is present, a mismatch
between the DNA and the probe occurs, and dissociation will
occur at a lower temperature. This method holds excellent
sensitivity, and even single-base mutations are easily detect-
able.15 This technology has been applied to a variety of
diagnostic tests, including the detection of mutations in
pathogenic bacteria.
For SYBR- and probe-based approaches, visualization of
PCR products can be monitored as the reaction proceeds
using the fluorescent chemistries. PCR is a dynamic process
that can be divided into 4 phases: a linear ground phase, an
exponential phase, a linear phase, and a plateau phase (Fig
3A).14,18 The linear ground phase represents the first 10 to 15
cycles, in which the fluorescence signal has not yet risen
above the background. In the exponential phase, doubling of
product occurs at every cycle, and the level of PCR product
is accurately measured by the fluorescence emission. As the
components of the sample mix are consumed, the PCR begins
to slow and enters the linear phase, which is characterized by
a high level of variability between runs. Finally, when all of
the reactants are consumed, the process enters the plateau
phase, where no more products are synthesized.
The PCR products are quantified by the cycle number,
referred to as the cycle threshold (CT), at which, in the
exponential phase, the fluorescent intensity becomes detect-
able above background. The more target DNA present in the
starting material, the faster an increase in fluorescent signal
will occur and the lower the CT value will be. Therefore,
results obtained using real-time PCR can be used to quantify
absolute amounts of DNA and to compare relative amounts of
specific nucleic acid sequences between various samples.
Quantitation in Real-Time PCR
A variety of different methods and algorithms have been
developed for quantification of changes in gene expression,
ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY
 plateau
A
100

Fluorescence 80
linear
60

40
exponential
20
Threshold
0
0 10 20 30 40
ground
Cycle Number

B 100 Test gene - Sample 1

Test gene - Sample 2
Housekeeping gene - Sample 1
Log Fluorescence

10
Housekeeping gene - Sample 2
Threshold

1 ∆CT
∆CT
0.1
∆∆CT

0.01
0 5 10 15 20 25 30 35 40
Cycle Number
C 100
Known copy number
Unknown
80
Fluorescence

101
60 102
CT Value

103 4
10
40 105
106
CT 107
20
Threshold
Log Copy Number
0
0 10 20 30 40
Cycle Number
Figure 3. A, Phases of polymerase chain reaction (PCR) as detected by means of real-time technology. In the initial linear ground phase, PCR products are
not yet detectable. In the exponential phase, products are detectable and the reaction is proceeding optimally. The threshold for detection of the cycle threshold
(CT) value (see the “Real-Time PCR” section) is set in this phase (dashed line). In the linear phase, reactants are being consumed, and by the time the reaction
reaches the plateau phase, there is no further DNA synthesis. B, Method of relative quantitation for changes in gene expression. Log-scale transformation of the
amplification plot shown in part A allows easier visualization and determination of the CT value. Changes in expression of a particular gene between 2 conditions
(test gene samples 1 and 2) can be quantitated by comparing the difference in CT values (⌬⌬CT) after normalization, for each sample, with expression of a
housekeeping gene (⌬CT) (see the “Quantitation in Real-Time PCR” section). C, Absolute quantitation of a nucleic acid can be accomplished by generating a
standard curve from varying dilutions of a known amount of the chosen DNA. The copy number of the unknown can be determined by fitting the CT value to
the standard curve.

and more detailed descriptions can be found in a recent
review.14 One method frequently used for this analysis is
comparing the difference in CT values (⌬⌬CT) (Fig 3B). First,
transformation on a logarithmic scale of the amplification
curve allows easier visualization and determination of the CT
value and quantitation of the sample. Relative changes in
gene expression can then be determined by first normalizing
data to a control gene, whose expression does not change as
experimental conditions vary. A variety of reference or
“housekeeping” genes have been used as such internal stan-

VOLUME 101, OCTOBER, 2008 441

dards, including glyceraldehyde-3-phosphate dehydrogenase,
␤-actin, 18S ribosomal RNA, ribosomal protein 36B4, and
cyclophilin. To normalize the expression of a specific gene to
a housekeeping gene, the difference in CT between the target
and the reference gene under 1 treatment condition is calcu-
lated (⌬CT) (Fig 3B). This value can be then used to compare
different conditions, such as the expression of a gene in cells
under cytokine challenge vs unchallenged cells. The relative
changes in gene expression brought by the treatment are
determined by subtracting the ⌬CT value of the challenged
condition from the ⌬CT value of the resting condition
(⌬⌬CT). Because the absolute number of mRNA copies in the
sample chosen as the reference (in the case of the example,
the level of expression of a certain gene in unchallenged
cells) is unknown, only relative changes can be calculated in
this approach. In a PCR in which all the components are used
at 100% efficiency, the amount of amplified product doubles
with each cycle, so a ⌬⌬CT value of 1 within 2 samples
corresponds to a 2-fold difference from the amount of nucleic
acid in the reference sample. When expression of the studied
gene is increased with treatment, the ⌬⌬CT value is negative
because the CT number is smaller in the sample in which gene
expression is greater. Once normalized over a housekeeping
gene, a change in CT of 2 between 2 samples corresponds to
a 4-fold difference in gene expression; a change in CT of 3 is
an 8-fold difference, and so forth. Fold change over the
reference sample is then expressed as a positive number after
logarithmic transformation of the ⌬⌬CT value, as 2⫺⌬⌬CT.
The method of reporting differences in gene expression
based on normalization toward reference genes is based on
the assumption that the expression of the latter genes does not
change with the experimental condition. Because this is not
always true, it is important to use more than 1 reference gene
or to test the validity as true housekeeping of the chosen
reference gene in each specific experimental setting.
Absolute quantitation of the PCR product can also be
performed and is preferentially indicated for pathogen quan-
tification, gene therapy, basic transcription studies, and qual-
ity control (Fig 3C). The actual nucleic acid copy number can
be measured if a known quantity of the gene of interest is
available. The known sample can be diluted to create a
standard curve, and unknown samples of interest can be
compared for absolute quantitation.19 The main limitation of
this quantitation method is the availability of known stan-
dards and the need to run standard curves for each gene of
interest with each assay.
APPLICATIONS OF QUANTITATIVE RT-PCR
Quantitative RT-PCR, based on real-time technology, has
drastically improved the PCR method, providing extremely
high sensitivity, reproducibility, and specificity. Changes in
gene expression can be precisely compared between patients
and controls or among patient groups under different treat-
ments or vs placebo. Because very small amounts of mRNA
can easily be amplified, this process can be performed on
442
very small samples of blood or other bodily fluids and from
tissue from biopsies or brushings.
The RT technology can be used to determine global
changes in gene expression through array studies. RNA sam-
ples taken from a cell, tissue, or a patient can undergo RT into
cDNA and hybridization to a DNA microarray containing
thousands of genes. As the amount of cDNA is proportional
to mRNA expressed in any given tissue, the microarray can
be used to determine the differential gene expression between
2 or more conditions. This is a powerful approach that has
been used to characterize a variety of diseases. Because it
may be difficult in array studies to precisely quantify levels of
changes in gene expression, real-time PCR has been an
invaluable tool in validating the array results by accurately
measuring and confirming results for selected candidate
genes.
Real-time PCR has extended the value of PCR in medical
diagnostics not only to detect the presence of microbes but
also to actually quantify bacterial, viral, and fungal loads.15
The best example of determining the quantity of pathogen-
specific product is using this technique to obtain viral loads in
human immunodeficiency virus–infected patients. Clinically,
this has been used as a key measurement to determine when
treatment should be started and as a means of assessing the
efficacy of treatment after initiation.20
In conclusion, PCR and real-time PCR techniques have
revolutionized basic science research and diagnostic medi-
cine. They have enabled the understanding of countless phys-
iologic and pathologic mechanisms and are invaluable tools
routinely used in diagnosing multiple diseases. As PCR-
based technology continues to grow and new applications are
developed, this approach will play an even larger and more
central role in biomedical research and clinical medicine.
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Requests for reprints should be addressed to:
Cristiana Stellato, MD, PhD
Johns Hopkins Asthma and Allergy Center
5501 Hopkins Bayview Cir
Room 1A.12A
Baltimore, MD 21224
E-mail: stellato@jhmi.edu
443