See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/258851372

Molecular Pathology Techniques

Article  in  Clinics in Laboratory Medicine · December 2013

DOI: 10.1016/j.cll.2013.09.004 · Source: PubMed

CITATIONS READS

6 12,513

2 authors:

Mark J Bluth Martin H Bluth

Genome Dynamics International, Southfield, MI Wayne State University
12 PUBLICATIONS   1,371 CITATIONS    160 PUBLICATIONS   1,817 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Pharmacogenetics View project

Toxicology View project

All content following this page was uploaded by Martin H Bluth on 12 January 2018.

The user has requested enhancement of the downloaded file.

M o l e c u l a r P a t h o l o g y Tec h n i q u e s
a, b
Mark J. Bluth, PhD *, Martin H. Bluth, MD, PhD

KEYWORDS
 Molecular pathology  Methodology  RT-PCR  FISH  RFLP  SNP
 Hybrid capture

KEY POINTS
 Polymerase chain reaction (PCR) remains the cornerstone methodology for nucleic acid
amplification.
 Improvements on nucleic acid detection methodologies (ie, PCR) have increased the
detection sensitivity by using fluorescent and bead array–based technologies.
 Single base-pair lesions can be detected via sequencing and related techniques to
discern point mutations in disease pathogenesis.
 Novel technologies such as high-resolution melting analysis provide fast, high-throughput
post-PCR analysis of genetic mutations or variance in nucleic acid sequences.
 Infectious disease can now be detected by fluorophore or chemiluminescent detection
assays, such as Hybrid Capture hybridization technology, allowing for rapid diagnosis.

POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) is a chemical reaction that facilitates the in vitro syn-
thesis of potentially unlimited quantities of a targeted nucleic acid sequence. Basi-
cally, the reaction consists of a target DNA molecule, an excess of the forward and
reverse oligonucleotide primers (typically 15–30 nucleotides long), a thermostable
DNA polymerase (typically Taq or Pfu), an equimolar mixture of deoxyribonucleotide
triphosphates (dATP, dCTP, dGTP, and dTTP), Mg21 or Mn21 (depending on the
type of polymerase used), KCl, and an appropriate Tris-HCl buffer.
The reaction consists of 3 steps: denaturation, annealing, and extension, which
taken together are referred to as a “cycle.” To begin, the reaction mixture is heated
(usually to w95
C) to separate the 2 strands of target DNA (denaturation) and then
cooled to a temperature at which the primers will bind to the target DNA in a
sequence-specific manner (annealing). Immediately after primer annealing, the DNA
polymerase binds (as the temperature is raised to w72
C) and initiates polymerization,
resulting in the extension of each primer at its 30 end (extension). During the following
cycle the primer extension products are subsequently heated to dissociate from the

a
Genome Dynamics International, Uniondale, NY 11556, USA; b Karmanos Cancer Institute,
Detroit Medical Center, Wayne State University School of Medicine, Detroit, Michigan
* Corresponding author.
E-mail address: [email protected]

Clin Lab Med 33 (2013) 753–772

http://dx.doi.org/10.1016/j.cll.2013.09.004 labmed.theclinics.com
0272-2712/13/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved.
754 Bluth & Bluth

target DNA. Each new extension product, as well as the original target, can serve as a
template for subsequent rounds of primer annealing and extension. In doing so, at the
end of each cycle, the PCR products are theoretically doubled.1,2
The whole procedure is carried out in a programmable thermocycler that precisely
controls the temperature at which the steps occur, the length of time that the reaction
is held at the different temperatures, and the number of cycles. Ideally, after 20 cycles
of PCR, a million-fold amplification is achieved and, after 30 cycles, the replicons
approach a billion-fold.

REVERSE TRANSCRIPTION PCR

As described above, PCR is suitable for the amplification of DNA targets because DNA
polymerase does not recognize DNA-primed RNA templates. Reverse transcription
(RT) PCR helps overcome this problem by using the enzyme reverse transcriptase
to first synthesize a strand of complementary DNA (cDNA) using the RNA as a tem-
plate. Because thermolabile RNA is often referred to as the message transcribed
from the DNA template, this process provides a thermostable mirror image of the
RNA transcript. Typically, recombinant reverse transcriptase is added to a reaction
mixture identical to the one for PCR and is incubated at between 37
C and 42
C for
30 minutes during which time the first-strand cDNA synthesis occurs. Subsequently,
the reaction proceeds much like a regular PCR reaction for the appropriate number of
cycles at the appropriate temperatures. This method can, however, present problems
in terms of both the nonspecific primer annealing and inefficient primer extension due
to formation of RNA secondary structures. A secondary RNA structure is a direct
consequence of the low temperature at which the reaction is carried out, due to the
heat labile nature of most RTs. These problems have been largely overcome by the
development of a thermostable DNA polymerase derived from Thermus thermophilus
(ie, Taq polymerase), which, under the proper conditions, can function efficiently as
both an RT and a DNA polymerase.2

REAL-TIME PCR

Real-time (also called “quantitative” [qPCR, qRT-PCR] or “kinetic” [kPCR, kRT-PCR])

is a closed-system assay that can be used to determine the relative quantity of gene
expression as well as genotyping by detection of single-nucleotide polymorphisms
(SNP).
In principle, the method works much like the PCR mentioned above; however, real-
time PCR also uses an additional oligonucleotide probe. This probe is target message-
specific and contains a fluorochrome at one end and a quencher molecule at the other.
When unhybridized, the probe forms a hairpin structure that brings the fluorochrome in
proximity with and binds the quencher, effectively muting its fluorescence. However,
when hybridized, the quencher molecule is cleaved, and the bound fluorochrome
is now unencumbered and can be detected by a fluorescence absorption assay.
Single-nucleotide differences like SNPs can be detected in PCR products by the
sequence-specific hybridization of the probe. Because it is possible to have different
colored fluorochromes, the probes can be differentially labeled, allowing both alleles
of an SNP to be typed in the same tube. These molecules can be used in a closed sys-
tem for allelic discrimination of PCR products. Both assays can be read in real time or
end-point formats, using a fluorescent thermocycler or LightCycler. This PCR-based
assay can be consolidated by combining an amplification primer and the fluorescent
detection component in the same molecule to enable real-time genotyping.
 Molecular Pathology Techniques 755

Once suitable oligonucleotides are designed, the genotyping of a sample is straight-

forward. The instrument is programed to amplify the DNA and to perform a melting
curve analysis. A perfect match has a higher melting temperature than a mismatch.
In this way, the LightCycler directly genotypes a sample after amplification with no
additional handling. With dual-color detection, it is possible to simultaneously geno-
type 2 different mutations in one PCR run.3–5

MULTIPLEX PCR

Multiplex PCR (mpPCR) consists of multiple primer sets within a single PCR mixture to
produce amplicons of differing sizes that specifically identify different DNA se-
quences. Primer sets are designed so that their annealing temperatures are optimized
to work correctly within a single reaction. The resultant amplicons are different enough
in size to form distinct bands when visualized by gel electrophoresis. By its original
design, this assay is typically efficient for elucidating the presence and relative con-
centrations of from 2 to 20 distinct messages and is limited by the resolution capacity
of electophoretic gel separation.6

XTAG TECHNOLOGY

xTAG technology (Luminex Corp, Austin, TX, USA) is a next-generation form of multi-
plexing that overcomes the resolution limits of mpPCR by combining the methods of
multiplex amplification with particle-based flow cytometry. Like mpPCR, multiple re-
actions can be carried out in a single reaction; however, because of the added flow
component, many more tests can be run and resolved at the same time.
Using a viral panel as an example, after obtaining a biologic sample, the mRNA is
reverse transcribed to cDNA. The cDNA is then amplified using a panel of primers
that can specifically amplify many different pathologic/pathogenic nucleic acid se-
quences at the same time. Each pathogen-specific primer used is tagged with a
unique oligonucleotide sequence (called the tag) as well as a fluorophore. After the
multiplex amplification step is completed, the reaction is mixed with microscopic
beads that are internally tagged with varying amounts of fluorescent molecules at
the time of production. Each different type of bead is also labeled with a unique oligo-
nucleotide sequence that is complementary to the unique tag on the pathogen-
specific primer (called the anti-tag). If both the tag and the anti-tag are present,
then hybridization occurs, binding the fluorophore-labeled amplicon to its appropriate
fluorophore-labeled bead. The beads are then processed and placed in a special flow-
enabled luminometer equipped with 2 lasers for reading. The first of the 2 lasers iden-
tifies the bead based on its internal dye content and the second laser detects how
much, if any, tagged amplicon is bound to its surface.7
This technology allows for the resolution of 100 or more tests from one sample at
one time in one tube. It is adaptable to perform tests on nucleic acids, peptides,
and proteins in a variety of sample matrixes.

STRAND DISPLACEMENT AMPLIFICATION

Strand displacement amplification method allows for rapid isothermal amplification of

target nucleic acid molecules using a series of primers, DNA polymerase (exo-Bst),
and a restriction endonuclease (BsoBI), to amplify a unique nucleic acid sequence
exponentially.8 BsoBI recognizes the nucleic acid sequence: 50 C-(C or T)-C-G-(A or
G)-G 30 . One of the primers, commonly called the “bump” primer, contains a sequence
756 Bluth & Bluth

complementary to a unique target sequence, but also contains a 50 linker with a built-in
BsoBI restriction site.
Strand displacement amplification can be thought of as occurring in 2 segments: a
target generation phase and an exponential amplification phase. In the target gener-
ation phase after the heat denaturation of the native nucleic acid, primers anneal
(Fig. 1) and the polymerization reaction occurs in both directions in the presence of
modified dCTPaS (Fig. 2), which results in the production of a double-stranded prod-
uct with a BsoBI restriction site 50 to the target sequence. Next, the BsoBI restriction
site within the primer is digested with BsoBI. However, because the newly polymerized
DNA strands (the strands complementary to and extended from the “bump” primer)
were synthesized with dCTPaS, only one side of the BsoBI site is sensitive to diges-
tion, resulting in the production of one nicked strand (Fig. 3). Next, the exo-Bst poly-
merase binds to the nicked strand, on the 50 side of the nick, and polymerizes a new
strand extending from the nick site, displacing the previous strand in the process. The
strand is nicked again with BsoBI and the process repeats (Fig. 4). The newly dis-
placed strand will serve as a template in following rounds of amplification. The expo-
nential amplification phase describes the continuous repetition of this process and
can produce copies in excess of a million-fold within 2 hours.9,10
The reaction can be performed with real-time analysis if coupled with a fluorescent
probe and multiplexed via an initial purifying step in which the “bumper” primer is
covalently linked to magnetic or fluorescently labeled beads.

TRANSCRIPTION-MEDIATED AMPLIFICATION

Transcription-mediated amplification (TMA) is an isothermal nucleic acid-based

method that can amplify RNA or DNA targets a billion-fold in less than 1 hour
(Fig. 5). This system is useful for detecting the presence of Mycobacterium tubercu-
losis and Chlamydia trachomatis.
Developed at Gen-Probe (Hologic Gen-Probe, San Diego, CA, USA), TMA technol-
ogy uses 2 primers and 2 enzymes: RNA polymerase and reverse transcriptase. One
primer contains a promoter sequence for RNA polymerase. In the first step of ampli-
fication, this primer hybridizes to the target rRNA at a defined site. Reverse transcrip-
tase creates a DNA copy of the target rRNA by extension from the 3’ end of the
promoter primer. The RNA in the resulting RNA:DNA duplex is degraded by the RNase
activity of the reverse transcriptase. Next, a second primer binds to the DNA copy. A
new strand of DNA is synthesized from the end of this primer by reverse transcriptase,
creating a double-stranded DNA (dsRNA) molecule. RNA polymerase recognizes the

Fig. 1. Primer hybridization. (Courtesy and Ó Becton, Dickinson and Company. Reprinted
with permission.)
 Molecular Pathology Techniques 757

Fig. 2. Primer extension. (Courtesy and Ó Becton, Dickinson and Company. Reprinted with
permission.)

promoter sequence in the DNA template and initiates transcription. Each of the newly
synthesized RNA amplicons reenters the TMA process and serves as a template for a
new round of replication. The amplicons produced in these reactions are detected by
a specific gene probe via hybridization protection assay followed by a chemilumines-
cence detection protocol.11,12

DNA SEQUENCING

DNA sequencing using the enzymatic extension reaction makes use of the difference
between normal deoxyribonucleotides and dideoxyribonucleotides. Deoxyribonucle-
otides contain a hydroxyl group at position 3 on the pentose sugar ring, allowing
DNA polymerase to join it with the phosphate group of the next nucleotide. A dideox-
ynucleotide can be incorporated into a growing chain, but because it does not contain
a hydroxyl group at position 3, no additional nucleotides can be added, effectively ter-
minating polymerization of that chain at that point.
The reaction is the same as a standard PCR with the exception of the supplementa-
tion of a small concentration of a labeled dideoxynucleotide to the reaction mix in

Fig. 3. Single-strand digestion. (Courtesy and Ó Becton, Dickinson and Company. Reprinted
with permission.)
758 Bluth & Bluth

Fig. 4. Nicked-strand displacement. (Courtesy and Ó Becton, Dickinson and Company.

Reprinted with permission.)

addition to the regular quantities of the normal deoxynucleotide triphosphates (dATP,

dTTP, dCTP, and dGTP). Using dideoxythymidine triphosphate (ddTTP) as an
example, the DNA to be sequenced is denatured, complementary primers anneal,
and primer extension occurs as normal. However, when ddTTP is incorporated instead
of dTTP, DNA polymerization on that molecule terminates. When the reaction is carried

Promoter-primer Binds to rRNA Target

Reverse Transcriptese (RT) Creates DNA Copy

RNAse H Activities of RT Degrades the rRNA

Second Primer Binds to DNA

RT Creates DNA Copy

RNA Polymerase RT Creates DNA Copy

Transcribes RNA Cycle Repeats

100-1000 Copies of RNA Promoter Primer

Amplicon are Produced Binds to DNA

RNAse H Activities of RT
Degrades the RNA
Second Primer Binds
to RNA Amplicon RT Creates DNA Copy

Fig. 5. Illustration of transcription mediated amplification. (Courtesy of Gen-Probe, Inc., San

Diego, CA; with permission.)
 Molecular Pathology Techniques 759

out in the presence of the optimal concentrations of both dTTP and ddTTP, there will be
molecules synthesized that stop at each of the thymidine nucleotides in the sequence.
The fragments generated can then be separated out by size via gel electrophoresis and
the tag on the ddNTP allows the fragment to be visualized. In the beginning, the reac-
tion was carried out using 4 different tubes, each one containing a different ddNTP. The
reaction uses radiolabeled nucleotides (ie, S35), and when completed, the reactions
were separated in parallel lanes of a gel (one lane per ddNTP), which was then exposed
to film, yielding a staggered pattern of bands each with a one nucleotide base differ-
ence in size from the next. When the order of the bands from all 4 lanes is read in
size order from bottom to top, it would correspond to the sequence of the DNA frag-
ment that was amplified from the primer onward (Fig. 6).
This method is excellent for sequencing fragments up to 600 base pairs. As a result,
the method could get costly for the time and reagents necessary to perform multiple
reactions required to verify the sequence of longer DNA fragments. Today, however,
the same reaction can be performed using ddNTPs labeled with different fluorophores
in one tube, run in one lane of a capillary gel, and read with a laser detector. This
advance allows for more accurate sequence reading while using less time and re-
agents (Fig. 7).

PYROSEQUENCING

Pyrosequencing is a method of DNA sequencing based on sequencing by the principle

of synthesis. First, a sequencing primer is hybridized to a single-stranded DNA template
in the presence of the enzymes, DNA polymerase, ATP sulfurylase, luciferase, and
apyrase, and the substrates, adenosine 50 phosphosulfate (APS) and luciferin.13–15
The first of 4 dNTPs are then added to the reaction and DNA polymerase incorpo-
rates it only if it is complementary to the base in the template strand. Each incorpora-
tion event is accompanied by the release of pyrophosphate (PPi) in a quantity
equimolar to the amount of incorporated nucleotide (Fig. 8, step 2).
ATP sulfurylase quantitatively converts PPi to ATP in the presence of APS. This ATP
drives the luciferase-mediated conversion of luciferin to oxyluciferin, which generates
visible light in amounts proportional to the ATP generated. The light produced in the
luciferase-catalyzed reaction is detected by a charge-coupled device camera and
seen as a peak in a Pyrogram. The height of each peak is proportional to the number
of a specific nucleotide incorporated (Fig. 8, step 3).
Apyrase, a nucleotide degrading enzyme, continuously degrades ATP and unincor-
porated dNTPs. Apyrase switches off the light-promoting reaction and regenerates
the reaction solution. The next dNTP is then added (Fig. 8, step 4).

Fig. 6. Classic Sanger sequence film with representative sample sequence. (Adapted from
Freeman S. Biological science. 2nd edition. Upper Saddle River, NJ: Pearson Education,
Inc.; 2005. p. 410; with permission.)
 760
Bluth & Bluth
Fig. 7. Next Gen Sanger sequencing using only one tube and fluorescent-labeled ddNTPs.
 Molecular Pathology Techniques 761

Fig. 8. Stepwise illustration of the pyrosequencing method. (Image kindly provided by Ó

QIAGEN all rights reserved.)

The addition of dNTPs is performed one at a time (G then C then T then A then G then
C then T, etc). As the process continues, the cDNA strand is built up and the nucleotide
sequence is determined from the signal peaks in the Pyrogram (Fig. 8, Step 5).
Deoxyadenosine alpha-thiotriphosphate is used as a substitute for dATP because it
is used efficiently by DNA polymerase, but is not recognized by luciferase (all figures
762 Bluth & Bluth

from describing pyrosequencing adapted from http://www.adelaide.edu.au/saef/

new/whatis/).

DENATURING GRADIENT GEL ELECTROPHORESIS

Denaturing gradient gel electrophoresis is a widespread technique that can be used to

separate similar sized fragments of DNA or RNA based on the composition of the
double-stranded fragments. The melting temperature (ie, the temperature at which
base pairs in a dsDNA fragment lose their bond) depends on the base-pair composi-
tion of a fragment. Even in the case of a one base-pair substitution, the fragment will
melt at a different temperature.16
By adding a GC-rich tail (called a GC clamp) to one of the primers for the amplifica-
tion, a fragment is produced that will only partially melt when it is run into a denaturing
gradient gel. The GC clamp will remain double stranded, which causes the fragment to
stop migrating when it reaches a certain point in the gel. This process will occur at a
different position in the gel when one or more base pairs are substituted, deleted, or
inserted. Therefore, allelic variation or mutation can be detected (Fig. 9) using this
method.17 Clinically, Denaturing Gradient Gel Electrophoresis (DGGE) could be
used to confirm the presence of and distinguish between which types of mycobacteria
are present in a sample.18 It can also be used to determine what mutations exist in
BRCA1 and BRCA2 genes of an individual.19
In this method, the gradient is typically a urea and formamide (UF) gradient. The use
of the UF gradient results in the ability to run the gel at a much lower temperature than

Fig. 9. Illustration of DGGE. Lane 1: homozygous GC. Lane 2: heterozygous sample. Lane 3:
homozygous AT. (Courtesy of Dr R.W.M. Hofstra, Department of Medical Genetics, Univer-
sity of Groningen, The Netherlands.)
 Molecular Pathology Techniques 763

without, using the gradient. A 10% increase in UF concentration has the same effect
as a 3.2
C increase in temperature.

HIGH-RESOLUTION MELTING ANALYSIS

High-resolution melting analysis (HRM) is a technique for fast, high-throughput post-

PCR analysis of genetic mutations or variance in nucleic acid sequences. It enables
researchers to detect and categorize genetic mutations rapidly (eg, SNPs), identify
new genetic variants without sequencing (gene scanning), or determine the genetic
variation in a population (eg, viral diversity) before sequencing.
The first step of the HRM protocol is the amplification of the region of interest, using
standard PCR techniques, in the presence of a specialized dsDNA binding dye (such
as SYBR Green). This specialized dye is highly fluorescent when bound to dsDNA and
poorly fluorescent in the unbound state. This change allows the user to monitor the
DNA amplification during PCR (as in real-time or quantitative PCR).
After completion of the PCR step, a high-resolution melt curve is produced by
increasing the temperature of the PCR product, typically in increments of 0.008 to
0.2
C, thereby gradually denaturing an amplified DNA target. Because SYBR Green
is only fluorescent when bound to dsDNA, fluorescence decreases as duplex DNA
is denatured, which produces a characteristic melting profile; this is termed melting
analysis. The melting profile depends on the length, GC content, sequence, and het-
erozygosity of the amplified target. When set up correctly, HRM is sensitive enough to
allow the detection of a single base change between otherwise identical nucleotide
sequences.20,21

SOUTHERN AND NORTHERN HYBRIDIZATIONS

Both Southern and Northern hybridizations combine electrophoretic separation of test

nucleic acid with transfer to a solid support and subsequent hybridization. These as-
says, therefore, not only give information about the presence of hybridization but also
permit determination of the molecular weight of the hybridizing species.
The original procedure was termed Southern blot hybridization or Southern blotting,
after its inventor, E. M. Southern. In this assay, the sample is DNA. Northern blotting
was named by analogy for the technique using RNA samples. (Extending the analogy
even further, the Western blot is a similar procedure in which proteins are subjected to
electrophoresis and transfer; a Southwestern blot has been described for a technique
separating and blotting DNA followed by incubation with protein solutions to permit
evaluation of specific DNA-binding proteins.)
Sample preparation is time-consuming and labor intensive for both of these tech-
niques. Degradation of sample nucleic acids is not tolerated by the assays, and a rela-
tively large amount of starting material is required. For Southern hybridizations, the DNA
must be purified with minimal shearing because sizing of the DNA fragments is achieved
through digestion with one or more restriction enzymes. Shearing and degradation intro-
duce random breaks in the sample, reducing the quantity available to be cut specifically
at appropriate recognition sequences. Impurities in the sample may interfere with the
activity and sequence specificity of the restriction enzyme. Partially or improperly
digested samples can produce spurious band sizes or result in such a reduced concen-
tration of the specific band that it is no longer detected during hybridization. For North-
ern hybridizations, the starting material is RNA, and extreme care must be taken to avoid
degradation during sample collection and preparation because of the ubiquitous nature
of RNases. RNA is composed of fragment sizes determined by transcription and
764 Bluth & Bluth

processing of message and ribosomal RNA. It is not digested before electrophoresis,

but is separated under denaturing conditions to remove secondary structure.
The size-separated fragments in the agarose gel are then transferred to a nylon or
nitrocellulose membrane. As originally designed, the transfer occurred passively
through capillary action. Most current applications use vacuum or pressure to speed
the transfer. After transfer, baking or ultraviolet cross-linking immobilizes the nucleic
acids and the entire membrane is then hybridized with labeled probe under stringent
conditions.
Hybridization is followed by autoradiographic, colorimetric, or chemiluminescent
detection of bands that are bound to the probe. Interpretation involves both detection
of a hybridizing species and determination of the molecular weight of the molecule.
These technically demanding assays require several days to perform but may be
required in clinical applications in which the information cannot be obtained in any other
format. The presence of bands at molecular weights different from normal or germline
(developmentally unaltered) samples can indicate a change in the genetic material.22–25

RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS

Restriction fragment length polymorphism (RFLP) analysis is a method that uses re-
striction endonuclease digestion of DNA and gel electrophoretics separation of the
resulting fragments (Fig. 10). This technique allows the study of small variances called
polymorphisms that occur in the DNA sequences between individuals of the same
species. These variances occur in the form of differing numbers of small sequence re-
peats of DNA—called tandem repeats, minisatellites, and microsatellites—that are
normally found in the noncoding regions of DNA. Polymorphisms can occur as a result
of mutations—deletions, inversions, additions, substitutions, and translocations—to
the DNA sequence.
Briefly, DNA is carefully isolated (so as to cause as little degradation and mechan-
ical fragmentation as possible) and then digested with a particular endonuclease.
The digested DNA is electrophoretically separated in an agarose gel. At this point
the separated DNA samples can be viewed using DNA binding dyes, such as
ethidium bromide, to compare the banding patterns. Alternatively, the DNA frag-
ments can be Southern blotted (see above), hybridized with labeled probes, and
then analyzed.
Therefore, if one were to digest DNA from 2 individuals (excluding identical twins and
clones) and separate the resulting fragments by gel electrophoresis, some differences
may be found in the 2 resulting banding patterns because of the differing number of
tandem repeats between restriction sites from one individual to another. Analysis of
the banding of an individual yields what is commonly known as a “genetic fingerprint.”
Using RFLP analysis, one may also observe a difference in the RFLP patterns be-
tween normal and diseased tissue (ie, tumor) from the same individual. If a tumor re-
sults from a genetic alteration (mutation), that alteration may result in a change in the
size of one or more bands as a result of additions or deletions of DNA. In addition,
single-nucleotide alterations may be observable via RFLP analysis if the alteration oc-
curs within the sequence of just one of the endonuclease restriction sites, rendering
that particular site unrecognizable to and uncut by the enzyme, which ultimately
changes the banding pattern.26

REVERSE LINE-BLOT HYBRIDIZATION

Reverse line-blot hybridization, also called “spacer oligonucleotide typing” (spoligo-

typing),27 is a method that can detect and identify pathogens based on the presence
 Molecular Pathology Techniques 765

Fig. 10. Process illustrating RFLP Analysis. (Courtesy of Santa Monica College, Santa Monica,
CA; with permission.)

and comparison of pathogen-specific genes and is useful for confirming the pres-
ence of specific pathogens and a proper course of treatment based on possible
multidrug resistance. For example, wild-type M tuberculosis is a slow-growing bac-
terium, requiring 2 to 6 weeks to culture, and is sensitive to treatment with rifampicin
(RIF). Mutations in the rpoB gene can render it resistant to RIF. Using this informa-
tion, the mutation hot-spot region of the rpoB gene is first amplified by mpPCR us-
ing as many as 20 different biotinylated primers, which yield labeled amplified
products.28 The PCR products are hybridized to a set of wild-type and mutant oligo-
nucleotide probes, which are covalently bound to a membrane, by reverse line blot-
ting (Fig. 11). It is called “reverse” because, in contrast to Southern or Northern
blotting where the sample is transferred onto a membrane and then probed, the
probe is first systematically bound to the membrane and the sample is then hybrid-
ized to it.
766 Bluth & Bluth

Fig. 11. Preparation of the probe-labeled reverse line-blot hybridization membrane.

Positive hybridization is detected on film after streptavidin-peroxidase incubation

and enhanced chemiluminescence. RIF-sensitive strains only hybridize with the
wild-type probes, whereas resistant strains will fail to hybridize with one or more
wild-type probes and show additional hybridization signals for the mutant probes
(Fig. 12). The resultant hybridization pattern elucidates the genotype of the M tuber-
culosis and thus a proper treatment protocol.

HYBRID CAPTURE

Hybrid capture is a nucleic acid hybridization technology that can precede signal
amplification and often uses fluorophore or chemiluminescent detection. To date,
human papillomavirus (HPV) cannot be cultured in vitro, and immunologic tests
are inadequate to determine the presence of HPV cervical infection. Indirect evi-
dence of anogenital HPV infection can be obtained through physical examination
and by the presence of characteristic cellular changes associated with viral replica-
tion in Papanicolaou smear or biopsy specimens. Alternately, biopsies can be
analyzed by nucleic acid hybridization to detect the presence of HPV DNA directly.
In the case of modern molecular-based HPV tests, specimens containing the target
DNA hybridize with a specific HPV RNA probe cocktail. The resultant RNA:DNA
 Molecular Pathology Techniques 767

Fig. 12. Results of a reverse line-blot hybridization assay.

hybrids are captured onto the surface of a solid media (ie, a microplate well coated
with antibodies specific for RNA:DNA hybrids or covalently linked to beads). Immo-
bilized hybrids are then reacted with alkaline phosphatase–conjugated antibodies
specific for the RNA:DNA hybrids and detected with a chemiluminescent substrate.
Several alkaline phosphatase molecules are conjugated to each antibody. Multiple
conjugated antibodies bind to each captured hybrid, resulting in substantial signal
amplification. As the substrate is cleaved by the bound alkaline phosphatase, light
is emitted that is measured as relative light units on a luminometer (Fig. 13). The inten-
sity of the light emitted denotes the presence or absence of target DNA in the spec-
imen.29 In the case of HPV, although this technique can elucidate the presence and
load of the virus, it cannot determine which specific types of HPV are present. For
the identification of the HPV strains present in the sample, PCR with HPV strain–
specific primers would be required.30

BRANCHED DNA ASSAYS

In contrast with techniques that rely on PCR, the sensitivity of branched DNA (bDNA)
methods is achieved by signal amplification on the bDNA probe after direct binding of
768 Bluth & Bluth

Fig. 13. Hybrid capture test principle. (Image kindly provided by Ó QIAGEN all rights
reserved.)

a large hybridization complex to the RNA target sequence. This series of hybridization
steps results in a “sandwich” complex of probes and target sequence. These unusual
synthetic oligonucleotides are composed of a primary sequence and secondary se-
quences that result in a branched structure extending from the primary sequence.
There is no synthesis reaction taking place. However, there are 2 steps to this assay:
the capturing step and the signal amplification step. In the capturing step there are 2
capturing oligonucleotide probes: the capture probe and the capture extender probe.
The capture probe is linked to the bottom of a microwell plate much like the one used
in hybrid capture. The capture extender probe will hybridize to both the capture probe
and a specific sequence on the target RNA, effectively anchoring it to the solid
medium (bottom of the microwell plate). The assay then continues to the signal ampli-
fication step whereby label extender probes are hybridized to specific sequences at
precise distances from each other on the target RNA. The label extender probes are
designed to serve as platforms for hybridization to preamplifier probes, which only
remain attached if hybridized to 2 adjacent label extender probes. Next, amplifier
probes, oligonucleotides labeled with alkaline phosphatase, are hybridized to the pre-
amplifier probes. Finally, the assay is treated with a chemoreactive substrate that fa-
cilitates the chemiluminescent reaction and is read by a luminometer. This assay can
be multiplexed by linking the capture probe to beads instead of the surface of a micro-
well (Fig. 14).
The signal in the bDNA assay is proportional to the number of alkaline phosphatase–
labeled probes that hybridize to bDNA secondary sequences. As such, the target RNA
can be blocked with blocking probes to increase the stringency of the primary probes
bound to it. As with all chemiluminescent-based assays of this type, quantification is
achieved by establishing a standard curve as well as negative and positive controls for
each run.31
 Molecular Pathology Techniques
Fig. 14. Illustration of virus detection using the Qauntiplex 3.0 Assay via bDNA technology developed by the Bayer Corporation. (From Campas M,
Katakis I. DNA biochip arraying, detection and amplification strategies. Trends Analyt Chem 2004;23(1):49–62; with permission.)

769
770 Bluth & Bluth

IN SITU HYBRIDIZATION

In situ hybridization is simply the detection of specific genetic information within a

morphologic context.32 In situ hybridization facilitates simultaneous detection, locali-
zation, and quantification of individual DNA or RNA molecules at the cellular level in a
fixed sample using labeled oligonucleotides or peptides as probes. This specialized
type of solid-support assay involves taking morphologically intact tissue, cells, or
chromosomes affixed to a glass microscope slide through the hybridization process.
Briefly, after a tissue sample is fixed, it is permeabilized and labeled probes are added
and allowed to hybridize to target molecules. The samples are then washed and
viewed using bright-field or fluorescent microscopy.
Autoradiographic, chromogenic33 (CISH), and fluorescent (FISH) methods of detec-
tion have been applied. Evaluation of the final product is analogous to evaluation of
immunohistochemistry and requires experience in histopathology. The strength of
the method lies in linking microscopic morphologic evaluation with detection via
hybridization.
The method also has applications in cytogenetic analysis of metaphase chromo-
some spreads or of interphase nuclei. In this context, the detection is usually accom-
plished via FISH. Detecting numerical aberrations or translocations of chromosomes
can be achieved rapidly using probes for specific targets. FISH avoids some of the dif-
ficulties of conventional cytogenetics and may have greater sensitivity for some tar-
gets. Although FISH cannot completely replace karyotyping, it can complement and
reduce the need for the frequency of cytogenetic analysis.34
Newer variations of FISH and CISH are exploiting the possibilities of automation
(fast-FISH) and expanding the information that can be obtained in a single assay. FIC-
TION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for
Investigation Of Neoplasms) combines immunophenotyping with FISH; GOLDFISH
(Gold-Facilitated In Situ Hybridization) is a gold-enhanced bright field chromogenic
in situ gene amplification assay,35 and fiber FISH makes it possible to detect and
map chromosomal break points simultaneously. In situ hybridization can be very labor
intensive and tedious and, because of the extremely labile nature of mRNA, gives
inconsistent results for molecular diagnostic purposes. However, recent improve-
ments resulting in automated processing of slides through the assay hold much prom-
ise for more widespread adoption of this technique.

Other Technologies
Microarray-based technologies have the capacity to interrogate tens of thousands of
genes at one time. As such, although array-based algorithms have been FDA
approved in selective cases (p450 cytochrome oxidase), the application of the clinical
marketplace is not well defined. Array-based methods can be found elsewhere36;
however, time will determine how such methods are to be interpreted, reported to
the physician, and ultimately, used for effective patient management.

REFERENCES

1. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of

DNA with a thermostable DNA polymerase. Science 1988;239:487–91.
2. Myers TW, Gelfand DH. Reverse transcription and DNA amplification by a Ther-
mus thermophilus DNA polymerase. Biochemistry 1991;30:7661–6.
3. Heid CA, Stevens J, Livak KJ, et al. Real time quantitativePCR. Genome Res
1996;6:986–94.
 Molecular Pathology Techniques 771

4. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for
gene expression analysis. Biotechniques 2008;44(5):619–26.
5. Spackman E, Suarez DL. Type A influenza virus detection and quantitation by
real-time RT-PCR. Methods Mol Biol 2008;436:19–26.
6. Chamberlain JS, Gibbs RA, Ranier JE, et al. Deletion screening of the Duchenne
muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res
1988;16(23):11141–56.
7. Merante F, Yaghoubian S, Janeczko R. Principles of the xTAG respiratory viral
panel assay. J Clin Virol 2007;40:S31–5.
8. Walker GT, Little M, Nadeau J, et al. Strand displacement amplification—an
isothermal, in vitro DNA amplification technique. Nucleic Acids Res 1992;20:
1691–6.
9. Walker GT, Little MC, Nadeau JG, et al. Isothermal in vitro amplification of DNA by
a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A 1992;89:
392–6.
10. Walker GT. Empirical aspects of strand displacement amplification [review]. PCR
Methods Appl 1993;3(1):1–6.
11. Down JA, O’Connell MA, Dey MS, et al. Detection of Mycobacterium tuberculosis
in respiratory specimens by strand displacement amplification of DNA. J Clin Mi-
crobiol 1996;34(4):860–5.
12. Available at: http://www.gen-probe.com/science/#technologies-0.
13. Ronaghi M, Uhlén M, Nyrén P. A sequencing method based on real-time pyro-
phosphate. Science 1998;281(5375):363.
14. Nyrén P. The history of pyrosequencing. Methods Mol Biol 2007;373:1–14.
15. Nordstrom T, Ronaghi M, Forsberg L, et al. Direct analysis of single-nucleotide
polymorphism on double-stranded DNA by pyrosequencing. Biotechnol Appl
Biochem 2000;31:107–12.
16. Fischer SG, Lerman LS. DNA fragments differing by single base-pair substitu-
tions are separated in denaturing gradient gels: correspondence with melting
theory. Proc Natl Acad Sci U S A 1983;80(6):1579–83.
17. Available at: http://www.ingeny.com/htdocs/DGGE.html.
18. McAuliffe L, Ellis Richard J, Lawes Jo R, et al. 16S rDNA PCR and denaturing
gradient gel electrophoresis; a single generic test for detecting and differenti-
ating Mycoplasma species. J Med Microbiol 2005;54:731–9.
19. van der Hout Annemarie H, van den Ouweland Ans MW, van der Luijt Rob B, et al.
A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2:
application in a Dutch cancer clinic setting. Hum Mutat 2006;27(7):654–66.
20. Ririe K, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA
melting curves during the polymerase chain reaction. Anal Biochem 1997;245:
154–60.
21. Available at: http://www.kapabiosystems.com/public/pdfs/kapa-hrm-fast-pcrkits/
Introduction_to_High_Resolution_Melt _Analysis_Guide.pdf.
22. Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. 5th edition.
New York: Garland Science, Taylor & Francis Group; 2008. p. 538–9.
23. Southern EM. Detection of specific sequences among DNA fragments separated
by gel electrophoresis. J Mol Biol 1975;98(3):503–17.
24. Alwine JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose
gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA
probes. Proc Natl Acad Sci U S A 1977;74(12):5350–4.
25. Kevil CG, Walsh L, Laroux FS, et al. An improved, rapid northern protocol. Bio-
chem Biophys Res Commun 1997;238:277–9.
772 Bluth & Bluth

26. Rosetti S, Englisch S, Bresin E, et al. Detection of mutations in human genes by a

new rapid method: cleavage fragment length polymorphism analysis (CFLPA).
Mol Cell Probes 1997;11:155–60.
27. Molhuizen HO, Bunschoten AE, Schouls LM, et al. Rapid detection and simulta-
neous strain differentiation of Mycobacterium tuberculosis complex bacteria by
spoligotyping. Methods Mol Biol 1998;101:381–94.
28. Cowan LS, Diem L, Brake MC, et al. Transfer of a Mycobacterium tuberculosis
genotyping method, spoligotyping, from a reverse line-blot hybridization,
membrane-based assay to the luminex multianalyte profiling system. J Clin Mi-
crobiol 2004;42(1):474–7.
29. Poljak M, Brenc  ic
 A, Seme K, et al. Comparative evaluation of first- and
second-generation digene hybrid capture assays for detection of human pap-
illomaviruses associated with high or intermediate risk for cervical cancer.
J Clin Microbiol 1999;37(3):796–7.
30. Available at: http://www.thehpvtest.com/w/media/5C4BD0982BED4E3788F65B
36AF829AAD.ashx.
31. Collins ML, Zayati C, Detmer JJ, et al. Preparation and characterization of RNA
standards for use in quantitative branched-DNA hybridization assays. Anal Bio-
chem 1995;226:120–9.
32. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid molecules in
cytological preparations. Proc Natl Acad Sci U S A 1969;63(2):378–83.
33. Tautz D, Pfeifle C. A non-radioactive in situ hybridization method for the localiza-
tion of specific RNAs in Drosophila embryos reveals translational control of the
segmentation gene hunchback. Chromosoma 1989;98(2):81–5.
34. Jin L, Lloyd RV. In situ hybridization: methods and applications. J Clin Lab Anal
1997;11(1):2–9.
35. Tubbs R, Pettay J, Skacel M, et al. Gold-facilitated in situ hybridization: a bright-
field autometallographic alternative to fluorescence in situ hybridization for detec-
tion of HER-2/neu gene amplification. Am J Pathol 2002;160:1589–95.
36. Bluth MH. Hybridization array technologies in McPherson and Pincus: Henry’s
clinical diagnosis and management by laboratory methods. 22nd edition.
New York: Saunders Publishing; 2011. p. 1282–9.

View publication stats