PAEDIATRIC RESPIRATORY REVIEWS (2005) 6, 199–208

SERIES: BASIC SCIENCE RESEARCH IN RELATION TO THE LUNG

Basic molecular biology

Albert P. Senft and Ann Marie LeVine*

Divisions of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center,
3333 Burnet Ave.,Cincinnati, OH 45229, USA

KEYWORDS Summary Rapid advances in molecular biology over the past 20 years have and will
Nucleic acids; continue to impact on the practice of medicine. Advances in molecular biology are having
Proteins; an immense impact in determining the underlying aetiology of lung disease and its
PCR; treatment. In this review, basic molecular biology techniques will be discussed with
Immunohistochemistry examples of how these techniques are used in clinical practice.
ß 2005 Elsevier Ltd. All rights reserved.

INTRODUCTION
Molecular biology is a broad area of study aimed at the
common goal of understanding the mechanisms of basic
cellular function. This review has been written as an
introduction to the basic molecular biology techniques
used to study DNA, RNA and proteins. The techniques
utilised in basic and clinical research as well as the strengths
and weaknesses of each technique are described. In addi-
tion, a bibliographical list of reference materials for an in-
depth description of molecular biology techniques has been
included.
NUCLEIC ACIDS
DNA encodes the molecular template for all molecules
necessary for cell function and viability. The nucleotide is
the most basic structural unit of DNA and is comprised of
deoxyribose sugar, a phosphate group and a purine (ade-
nine and guanine) or pyrimidine (thymine and cytosine)
nitrogenous base. DNA is a double helical molecule with
two sugar–phosphate strands serving as the backbone of
the molecule with the nitrogenous bases projecting toward
* Corresponding author. Tel.: +1 513 636 2995;
Fax: +1 513 636 7868.
E-mail address: annmarie.levine@cchmc.org (A.M. LeVine).
1526-0542/$ – see front matter ß 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.prrv.2005.06.006
the centre. The two strands are held together by hydrogen
bonding, a non-covalent interaction, between the purine
and pyrimidine bases. The interaction between the bases
occurs with high fidelity (adenine always pairs with thymine
and guanine with cytosine). Importantly, the two strands of
the DNA molecule can be disassociated and then re-
associated with the addition or removal of energy to break
the forces of hydrogen bonding. Together these two
physical features make DNA a dynamic molecule that
allows for its replication and for transcription of encoded
genes.
The gene is the smallest functional unit of DNA and
encodes for proteins. In general, genes are made up of a
promoter region and a coding region (Fig. 1). The promoter
region of the gene contains short, conserved, defined
nucleotide sequences that positively or negatively regulate
gene transcription. Gene transcription is a tightly regulated
process that is cell-type specific and greatly influenced by
the physiological stress placed upon the cell. Gene tran-
scription produces a ribonucleic acid (RNA) template from
which a protein is produced.
RNA, like DNA, contains a sugar–phosphate backbone
and nitrogenous bases. The major differences are that the
sugar is ribose, the base uracil is incorporated in place of
thymine and the molecule is single-stranded. Total cellular
RNA is a mixture of three types: transfer (tRNA), ribo-
somal (rRNA) and messenger (mRNA), which is the
200
Figure 1 A hypothetical gene, primary transcript and final mRNA.
template for protein production. Since mRNA encodes for
proteins, this will be the focus of the remaining discussion
of RNA. Initial transcription results in the formation of a
primary transcript that includes introns, exons and the 30
untranslated region (30 UTR) (Fig. 1). Exons are segments
that encode the amino acid sequence of the protein.
Introns are intervening sequences that do not encode a
protein but guide splicing of the mature mRNA and allow
alternative combinations of exons to produce variations
of similar proteins. Excision of introns and splicing of
exons must occur before mRNA protein translation can
occur.
Methods for the Analysis of Nucleic Acids
Polymerase Chain Reaction
Polymerase chain reaction (PCR) is used to amplify specific
DNA sequences from limited biological samples and has
revolutionised biomedical research. The technique is based
on the following: DNA can be denatured from a double-
stranded molecule to a single-stranded molecule by heat.
When the single-stranded DNA molecules are cooled they
anneal to a double-strand, the process of two single-
A. P. SENFT AND A. M. LEVINE
stranded DNA molecules annealing occurs with high fide-
lity (i.e. A always pairs with T and C with G). The poly-
merase chain reaction is illustrated in Fig. 2. Sequentially, the
template DNA is denatured by heating. Specific primers
designed for the sequence of interest anneal on the
corresponding DNA sequence of the template DNA when
the reaction is cooled. DNA polymerase extends the DNA
from 50 to 30 building a mirror image of the template strand.
At the end of one cycle, two exact copies of template DNA
have been formed. Using this technique, DNA can be
amplified exponentially. This allows minute amounts of
DNA from biological samples to be assayed, making the
technique extremely powerful for basic science and clinical
research.
PCR is a common approach used for the diagnosis of
clinical diseases. For example, PCR can be used for the
detection of herpes simplex virus in cerebral spinal fluid1
and enterovirus in myocardial biopsy samples.2
Northern blot analysis
Northern blot analysis is a sensitive technique for detecting
mRNA expression levels. RNA is separated by agarose gel
electrophoresis. Since, RNA has a negative charge it
BASIC MOLECULAR BIOLOGY
Figure 2 The steps involved in a polymerase chain reaction (PCR).
migrates to the cathode when a current is applied to the gel.
Similar to other forms of electrophoresis, mobility of the
molecule in the gel is directly proportional to its size. After
separation by electrophoresis the RNA is transferred to a
nitrocellulose membrane and cross-linked to the mem-
brane by ultra-violet (UV)-irradiation. To determine mRNA
levels for a specific gene, a short DNA probe is designed for
the specific mRNA of interest. The probe is radiolabelled
for detection and hybridised with a nitrocellulose mem-
brane binding the mRNA molecules with a similar
sequence. The results of hybridisation are detected by
201
autoradiography and reveal the number, size and abun-
dance of transcripts (Fig. 3).
While this technique is time consuming and requires a
significant amount of RNA for analysis, it is still commonly
used because it is extremely quantitative and therefore
provides an accurate assessment of mRNA levels.
Reverse Transcriptase–Polymerase Chain Reaction
Reverse transcriptase–polymerase chain reaction (RT-PCR)
is used to assess the level of mRNA expression. As its
202
Figure 3 The steps involved in a Northern blot analysis.
name indicates, a portion of the assay relies on the
amplification of a DNA template by PCR. Before PCR
amplification, the mRNA is converted into a complimen-
tary DNA (cDNA) molecule. As illustrated in Fig. 4, the
enzyme reverse transcriptase is used to make a DNA
copy of the mRNA. mRNA is selectively copied by using a
primer that binds to the poly A tail of mRNA (not present
in the more abundant rRNA and tRNA). Once the cDNA
is made, the RNA is degraded by RNAse H. DNA
polymerase and T4 DNA polymerase are then used to
build the complementary strand. The result is a collection
of DNA molecules that represents all the specific mRNA
contained in the cell. Similar to the PCR of DNA, primers
are designed for a specific gene and, using these primers,
the product is exponentially amplified by PCR. The
resulting PCR product is subjected to agarose gel
electrophoresis to separate the PCR products by size.
Ethidium bromide intercalates into the DNA and can be
A. P. SENFT AND A. M. LEVINE
visualised using UV light (Fig. 5A). An important advantage
of RT-PCR is that only a small amount of biological
sample is necessary to determine the mRNA level.
The limitation of this method for determining gene
expression is that RT-PCR combined with visualisation
using ethidium bromide is only semi-quantitative due to
the fact that the PCR reaction is linear only within a range
of cycles.
While RT-PCR is not well suited for the quantitative
comparison of mRNA expression, the technique is useful in
screening tissues for the expression of a gene. For example,
RT-PCR based assays are used in the clinical setting for the
detection of RNA viruses, including respiratory syncytial
virus and influenza A virus.3
Real-Time PCR
The technology of real-time PCR developed out of the
necessity for a quantitative technique to determine gene
expression levels in small samples. Measurement of
mRNA for a particular gene by real-time PCR is not
fundamentally different from reverse-transcriptase PCR.
Both techniques require mRNA initially to be reverse
transcribed to a cDNA molecule. The cDNA is then
subjected to PCR. The difference lies in the fact that the
instrument where the PCR reaction is carried out is both
a thermocycler and a detection device. The reaction mix
contains a fluorescent probe that intercalates into the
DNA during synthesis. At the end of each PCR cycle, the
amount of fluorescence is assessed. Based on an algo-
rithm, a threshold level of fluorescence must be reached
for an mRNA to be considered expressed and this is
expressed as a cycle number (Fig. 5B). mRNA expression
is therefore directly related to cycle number; the higher
the relative expression the lower the cycle number
where the curve crossed the threshold. For example,
in Fig. 5B, gene A has a higher expression than gene B
because the cycle number, where it crossed the thresh-
old, is lower.
PROTEINS
Proteins are the molecules that perform all cellular func-
tions. They are translated from the mRNA by ribosomes.
Each amino acid is encoded by at least one triplet nucleo-
tide sequence. Protein translation begins with the codon
AUG, which encodes for methionine and is terminated with
the stop codons UAA, UGA or UAG. Following translation,
proteins are modified (e.g. by glycosylation) and sorted to
their appropriate location (e.g. soluble or membranous) by
a highly ordered process.
With the advent of high-throughput techniques (e.g.
microarrays that will be discussed in a future review),
examining changes in gene expression under various
experimental and pathophysiological conditions has pro-
vided a wealth of information and a basis for exploring the
BASIC MOLECULAR BIOLOGY
Figure 4 The generation of cDNA from mRNA by the reverse transcriptase reaction.
underlying mechanisms of disease processes. However,
changes in gene expression only represent potential
changes in protein levels and/or cellular function. Basic
approaches to protein analysis are therefore important
to understand whether changes in gene expression corre-
late to changes in protein expression and altered cellular
function.
Methods for the Analysis of Proteins
Sodium dodecyl sulphate polyacrylamide gel
electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophor-
esis (SDS-PAGE) is a technique that is used to separate
203
proteins based on their molecular weight. The detergent
SDS has a negative charge and when added to the sample it
binds hydrophobic amino acid residues. Proteins bind
proportionally to the same amount of SDS. Therefore,
the charge to mass of all proteins is equivalent and all SDS–
protein complexes are negative. Protein samples are pre-
pared either under reducing conditions where b-mercap-
toethanol is added and the sample is boiled, or under non-
reducing conditions where b-mercaptoethanol and heat
are omitted. The specific sample preparation conditions are
determined by experimental necessity. Once the samples
have been prepared for electrophoresis, the sample is
loaded on the gel and an electric field is applied. The rate
of migration of the protein is inversely proportional to the
logarithm of its molecular weight, meaning larger proteins
204
Figure 5 Results from polymerase chain reaction (PCR) and
real-time PCR analysis. A, this is representative of an ethidium
bromide stained agarose gel of PCR products. In the example
more product is detected in lane A than lane B. B, this
represents a threshold curve of PCR product formation from
real-time PCR analysis. In this example the PCR product
of sample A is more abundant since it reaches the critical
threshold level of fluorescence at an earlier cycle number than
sample B.
move more slowly through the gel because they encounter
more resistance. Protein bands that result from differential
migration are commonly stained for visualisation with
Comassie blue or silver and are compared against refer-
ence proteins with known molecular weights run on the
same gel.
Western immunoblot
SDS-PAGE analysis is a powerful technique that allows
the examination of global changes in protein levels from
biological samples. However, it lacks specificity. Western
immunoblot analysis utilises the power of antibody spe-
cificity to ascertain information about the levels of a
particular protein in biological samples. In this technique,
proteins are first separated by SDS-PAGE analysis. Pro-
teins are then transferred to a membrane (nitrocellulose
or polyvinylidenefluoride (PVDF)) by applying a current
across the gel and membrane in such a way that the
proteins migrate from the gel to the membrane (Fig. 6).
After the proteins have been transferred, the membrane
is incubated with an antibody (primary antibody) raised
against the protein of interest. Following incubation with
A. P. SENFT AND A. M. LEVINE
the primary antibody, the membrane is incubated with an
antibody that is conjugated to horseradish peroxidase and
is capable of recognising the primary antibody. For exam-
ple if the antibody against protein X was raised in a rabbit,
the second antibody used would be an anti-rabbit horse-
radish peroxidase conjugated antibody. The highly specific
antibody to protein binding is detected using a perox-
idase-dependent luminescence generating system fol-
lowed by exposure to X-ray film. The bands that
appear reflect the size and relative amount of the studied
protein (Fig. 6).
Western blotting is an extremely powerful tool for
examining the presence or absence of proteins in a sample
and for comparing samples ascertained under different
conditions. The major limitation of Western immunoblot-
ting is that it is semi-quantitative.
Enzyme Linked Immunosorbent Assay
This technology for analysing proteins also exploits the
specificity of antibodies to single proteins. Enzyme Linked
Immunosorbent Assay (ELISA) systems use two antibodies,
the first captures the protein and the second is used for
detection. The first antibody is bound to the bottom of a
well (Fig. 7). The protein sample (serum, bronchoalveolar
lavage fluid, etc) is added to the well and incubated to allow
the specific protein to bind to the antibody. The well is
washed extensively so that only the protein that specifically
interacts with the antibody is retained. A second antibody
that recognises the specific protein and is conjugated with
alkaline phosphatase is added to the well. This is also
washed extensively and what is retained in the well is
an antibody–protein–antibody sandwich. Protein quanti-
fication is determined by the relative amount of colour
liberated by alkaline phosphatase acting on the substrate.
The sample with an unknown concentration of protein is
then compared to a standard curve generated from
serial dilutions of a known concentration of the protein.
ELISA is a quantitative technique commonly employed
to analyse cytokine levels in various biological samples
including serum, bronchoalveolar lavage and cerebrospinal
fluid.
Methods for Analysing Whole Lung
The focus of this review has been to discuss molecular
biology techniques that are applicable to a whole tissue, a
cell or a subcellular compartment but require a homo-
genate of the sample for analysis. The lung is a complex
organ with a host of cell types and an elaborate, physiolo-
gically significant architecture. Examining a lung homogenate
may not be informative with regard to a disease process that
is limited to a small portion of the lung. Therefore, the
following molecular biology techniques are useful for exam-
ining mRNA and protein expression in the whole lung on
histological sections.
BASIC MOLECULAR BIOLOGY
Figure 6 The steps involved in a Western immunoblot analysis.
In situ hybridisation
The principles underlying this are the same as those
described for Northern blotting, above. In the case of in
situ hybridisation, a radiolabelled oligonucleotide probe is
generated to the mRNA of interest. The probe is hybri-
dised against the histological tissue section. The power of
this technique is that one can visualise the location of
mRNA expression in the context of the whole tissue. In situ
hybridisation allows for an estimation of the level and
location of expression of mRNA. Since the lung is extre-
mely heterogeneous, in situ hybridisation is a powerful
technique since it allows gene regulation to be evaluated
over the entire lung. An example of the technique is shown
in Fig. 8.
205
Immunohistochemistry
Immunohistochemistry is used to assess the location
of specific proteins on a histological tissue section.
The technique is similar to Western immunoblot ana-
lysis where detection of the protein is determined by
using specific antibodies against that protein. The differ-
ence is that instead of the protein binding to a mem-
brane, the protein is retained in the cellular architecture
found in the histological tissue section. Similar to in situ
hybridisation, this technique allows not only the deter-
mination of relative protein expression levels but also
the distribution of the protein expression. A re-
presentative example of immunohistochemistry is shown
in Fig. 9.
206
Figure 7 The steps involved in an Enzyme Linked Immunosorbent Assay (ELISA) analysis.
SUMMARY
Molecular biology techniques have become powerful tools in
the practice of medicine. The most immediate examples are
the use of ELISA and PCR in routine diagnostic tests. A more
interesting and more powerful use of these basic techniques
is their use in biological samples from patients. Obtaining in-
formation about the underlying mechanisms of human dis-
eases is vital. Information obtained from patients allows the
basic research models to be validated as acceptable models
for studying specific disease processes. Ultimately, results
from translational research may enhance disease prevention
and lead to more effective and specific medical treatments.
A. P. SENFT AND A. M. LEVINE
FURTHER READING: HANDBOOKS
OF MOLECULAR BIOLOGY
TECHNIQUES
The following books are excellent, practical, in-depth
resources for protocols to perform the techniques
described in this review article.
Ausubel F M, Kingston R E, Moore D D, Seidman J G,
Smith J A, Struhl K (eds). Current Protocols in Molecular
Biology. New York: John Wiley and Sons, 2005.
Bonifacino J S, Dasso M, Harford J B, Lippincott-Schwartz
J, Yamada K M (eds). Current Protocols in Cell Biology. New
York: John Wiley and Sons, 2005.
BASIC MOLECULAR BIOLOGY 207

Figure 8 In situ hybridisation for surfactant protein-C (SP-C) mRNA in mouse lung. The micrographs depict in situ hybridisation for SP-
C in adult FVB/N mouse lung (magnification 10X). A depicts the dark-field image to visualise the probe and B depicts the bright-field
image of the lung section stained with toludine blue to show the morphological structure. Cellular sites of SP-C expression determined by
hybridisation with the anti-sense probe that is complimentary to SP-C mRNA appear as focal clusters (white) throughout the lung
parenchyma consistent with the location of alveolar Type II cells (A). In contrast, the bronchiolar epithelium (arrows) is negative for SP-C
expression (A and B). The image shown is by courtesy of Dr Stephan Glasser, Cincinnati Children’s Hospital Medical Center.

Figure 9 Immunohistochemistry for pro surfactant protein-C (SP-C) protein in mouse lung. The micrograph shown has
been immunohistochemically stained for pro SP-C in 6 week old FVB/N mouse lung (magnification 20X). The arrows point to
alveolar Type II cells that stain positive for pro SP-C. The image shown is by courtesy of Dr Susan Wert, Cincinnati Children’s Hospital
Medical Center.
208
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