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BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL
DIAGNOSIS
Dr. Apr. Dieter Deforce
2
1. Introduction
In order to define what “biotechnological techniques used in medical diagnosis”
are lets first take a look at the definition of the word “biotechnology”:
“The means or way of manipulating life forms (organisms) to provide desirable
products for man's use.”
A common misconception is that biotechnology refers only to recombinant DNA
(rDNA) work. However, recombinant DNA is only one of the many techniques used
to derive products from organisms, plants, and parts of both for the biotechnology
industry. A list of areas covered by the term biotechnology would more properly
include: recombinant DNA, plant tissue culture, rDNA or gene splicing, enzyme
systems, plant breeding, mammalian cell culture, immunology, molecular biology,
fermentation, and others.
The biotechnological techniques used in medical diagnosis can be devided into
two big categories: molecular genetic techniques (such as PCR, sequencing, …) and
techniques based on immunology.
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2. Molecular techniques
The development of molecular biological techniques in the diagnosis of viral
infection has been very fast and has led to the discovery of new viruses, the rapid
identification of drug resistance and the active monitoring of the efficacy of therapy.
Some of the technologies described in what follows already have application in
medical diagnosis which are approved by various regulatory organisms such as the
FDA or other national offices. In addition to these validated tests some technologies
are in the process of getting these validations and some technologies already have
applications which are validated and some new applications of the same technology
are now being investigated. Other technologies have no widespread or commercial
use in medical diagnostics yet but the technology is in our opinion very promising
and/or they are being used for research purposes in the field of medical diagnosis.
Some general basic techniques which have applications in the different
technologies described further are:
Base-pairing hybridization assays. Base-pairing is the mechanism with
which complementary DNA strands “hold” together. These strands can be
dissociated from each other at temperatures above their Tm (melting
temperature), and associated at temperatures below their Tm. The Tm is the
temperature at which 50% of the strands are dissociated. In the standard
hybridization assays the target DNA is immobilized on a solid support
such as nitrocellulose or nylon to which single stranded DNA readily
binds. Hybridization of labeled probe to the immobilized target DNA
followed by washing and drying allows for the detection of specific DNA
sequences. The reverse hybridization techniques which have become
popular fix an unlabeled probe to the solid support, while the target DNA
is labeled and allowed to hybridize.
Hybrids
DNA-DNA or DNA-RNA
RNA-RNA
Oligo-DNA or oligo-RNAd
Tm (°C)
81.5 + 16.6(log10[Na+]a) + 0.41(%GCb) – 500/Lc
78 + 16.6(log10([Na+]a/1+0.7[Na+]a)) + 0.7(%GCb) - 500/Lc
For <20 nucleotides:
2(In)
For 20-35 nucleotides: 22 + 1.46(In)
a
Or for another monovalent cation, but only accurate in the 0.01-0.4 M range in M (0.05 M = standard)
Only accurate for %GC in the 30% to 75% range
c
L=length of duplex in base pairs
d
Oligo, oligonucleotide; In, effective length of primer = 2 x (no. of G+C) + (no. of A+T)
b
Note that for each 1% formamide, the T m is reduced by about 0.6°C, while the presence of 6M urea
reduces the Tm by about 30°C.
Nucleic acid hybridization involves mixing single strands of two
sources of nucleic acids, a probe which typically consists of a
homogeneous population of identified molecules (e.g. cloned DNA or
chemically synthesized oligonucleotides) and a target which typically
consists of a complex, heterogeneous population of nucleic acid molecules.
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If either the probe or the target is initially double-stranded, the individual
strands must be separated, generally by heating or by alkaline treatment.
After mixing single strands of probe with single strands of target, strands
with complementary base sequences can be allowed to reassociate.
Complementary probe strands can reanneal to form homoduplexes, as can
complementary target DNA strands. However, it is the annealing of a
probe DNA strand and a complementary target DNA strand to form a
labeled probe-target heteroduplex that defines the usefulness of a nucleic
acid hybridization assay.
Denaturation of double-stranded probe DNA is generally achieved by
heating, a solution of the labeled DNA to a temperature which disrupts the
hydrogen bonds that hold the two complementary DNA strands together.
The energy required to separate two perfectly complementary DNA
strands is dependent on a number of factors, notably:
·
strand length - long homoduplexes contain a large number of
hydrogen bonds and require more energy to separate them; because the
labeling procedure typically results in short DNA probes, this effect is
negligible above an original length (i.e. prior to labeling) of 500 bp;
·
base composition - because GC base pairs have one more
hydrogen bond than AT base pairs, strands with a high % GC composition
are more difficult to separate than those with a low % GC composition;
·
chemical environment - the presence of monovalent cations
(e.g. Na+ ions) stabilizes the duplex, whereas chemical denaturants such as
formamide and urea destabilize the duplex by chemically disrupting the
hydrogen bonds.
A useful measure of the stability of a nucleic acid duplex is the melting
temperature (Tm). This is the temperature corresponding to the midpoint in
the observed transition from double-stranded to single-stranded form.
Conveniently, this transition can be followed by measuring the optical
density of the DNA. The bases of the nucleic acids absorb 260 =
ultraviolet (UV) light strongly. However, the adsorption by doublestranded DNA is considerably less than that of the free nucleotides. This
difference, the so-called hypochromic effect, is due to interactions between
the electron systems of adjacent bases, arising from the way in which
adjacent bases are stacked in parallel in a double helix. If duplex DNA is
gradually heated, therefore, there will be an increase in the light absorbed
at 260 nm (the optical density260 or OD260) towards the value characteristic
of the free bases. The temperature at which there is a midpoint in the
optical density shift is then taken as the Tm.
For mammalian genomes, with a base composition of about 40% GC,
the DNA denatures with a Tm of about 87°C under approximately
physiological conditions. Often, hybridization conditions are chosen so as
to promote heteroduplex formation and the hybridization temperature is
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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often as much as 25°C below the Tm. However, after the hybridization
and removal of excess probe, hybridization washes may be conducted
under more stringent conditions so as to disrupt all duplexes other than
those between very closely related sequences. Probe-target heteroduplexes
are most stable thermodynamically when the region of duplex formation
contains perfect base matching. Mismatches between the two strands of a
heteroduplex reduce the Tm for normal DNA probes, each 1% of
mismatching reduces the Tm by approximately 1°C. Although probe-target
heteroduplexes are usually not as stable as reannealed probe
homoduplexes, a considerable degree of mismatching can be tolerated if
the overall region of base complementarity is long.
Increasing the concentration of NaCl and reducing the temperature
reduces the hybridization stringency, and enhances the stability of
mismatched heteroduplexes. This means that comparatively diverged
members of a multigene family or other repetitive DNA family can be
identified by hybridization using a specific family member as a probe.
Additionally, a gene sequence from one species can be used as a probe to
identify homologs in other comparatively diverged species, provided the
sequence is reasonably conserved during evolution.Conditions can also be
chosen to maximize hybridization stringency (e.g. lowering the
concentration of NaCl and increasing the temperature), so as to encourage
dissociation (denaturation) of mismatched heteroduplexes. If the region of
base complementarity is small, as with oligonucleotide probes (typically
15-20 nucleotides), hybridization conditions can be chosen such that a
single mismatch renders a heteroduplex unstable.
Non-isotopic labeling
Two types of non-radioactive labeling are conducted:
•
Direct nonisotopic labeling, where a nucleotide which
contains the label that will be detected is incorporated. Often such systems
involve incorporation of modified nucleotides containing a fluorophore, a
chemical group which can fluoresce when exposed to light of a certain
wavelength.
•
Indirect nonisotopic labeling, usually featuring the chemical
coupling of a modified reporter molecule to a nucleotide precursor. After
incorporation into DNA, the reporter groups can be specifically bound by
an affinity molecule, a protein or other ligand which has a very high
affinity for the reporter group. Conjugated to the latter is a marker
molecule or group which can be detected in a suitable assay.
The reporter molecules on modified nucleotides need to protrude
sufficiently far from the nucleic acid backbone to facilitate their detection
by the affinity molecule and so a long carbon atom spacer is required to
separate the nucleotide from the reporter group.
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Two indirect nonisotopic labeling systems are widely used:
•
The biotin-streptavidin system utilizes the extremely high
affinity of two ligands: biotin (a naturally occurring vitamin) which acts as
the reporter, and the bacterial protein streptavidin, which is the affinity
molecule. Biotin and streptavidin bind together extemely tightly with an
affinity constant of 1014 one of the strongest known in biology.
Biotinylated probes can be made easily by including a suitable biotinylated
nucleotide in the labeling reaction.
•
Digoxigenin is a plant steroid (obtained from Digitalis plants)
to which a specific antibody has been raised. The digoxigenin-specific
antibody permits detection of nucleic acid molecules which have
incorporated nucleotides containing the digoxigenin reporter group.
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A variety of different marker groups or molecules can be conjugated to
affinity molecules such as streptavidin or the digoxigenin-specific
antibody. They include various fluorophores, or enzymes such as alkaline
phosphatase and peroxidase which can permit detection via colorimetric
assays or chemical luminescence assays, etc.
2.A.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) has become one of the most widely used
techniques in molecular biology. Since the technique is so well known, the basics of
the technique will not be repeated here. It forms the basis for a lot of diagnostics
based on molecular techniques, and a lot of variants have already been developed. We
will discuss some of these variants which have applications in medical diagnostics.
We will also discuss some precautions which should be kept in mind. The last
chapters will discuss some of the specially adopted detection techniques for PCR
products which have applications in medical diagnosis.
An important advantage of PCR in the field of medical diagnosis is that PCR
techniques only require very small amounts of sample. This makes the technique very
useful for prenatal diagnosis, since it requires only such small amounts of sample
chorionic villus samples at about 12 weeks of gestation can be used.
PCR also has its place in bacteriology (see chapter 14, in The science of
Laboratory Diagnosis, John Crocker and David Burnett, ISIS Medical Media, 1999).
2.B.1. Reverse transcriptase PCR (RT-PCR): this technique is used to amplify
RNA from samples. The template for RT-PCR can be either total RNA or
poly(A)+ selected RNA. RT reactions can be primed with random primers,
oligo(dT), or a gene-specific primer. The most commonly used technique
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(which is thechnique used for most applications which will be described in
this course) uses a oligo-dT primer to prime the reverse transcription
process and uses a two step process (see Figure).
5’
mRNA
Oligo(dT) primer
3’
AAAAAAAAAAA(A) A
+
3’
5’
Anneal
3’
5’
AAAAAAAAAAA(A) A
3’
5’
Add reverse transcriptase
3’
5’
AAAAAAAAAAA(A) A
3’
5’
RNase H treatment
3’
5’
single stranded cDNA
ready for PCR
A good overview of the different RT-PCR techniques and things to
consider for optimalisation is given in the manual to be found at:
http://www.lifetech.com/Content/TechOnline/molecular_biology/manuals_pps/pcrtechguide.pdf
The RT-PCR technique is very useful for the detection of viral RNA in
biological samples (eg. Hepatitis C). In the field of virology, the speed of
detection and identification is extremely important and RT-PCR
techniques offer the advantage over more traditional techniques such as
ELISA and viral culture. As an example a protocol for the detection with
RT-PCR
of
foot-and-mouth
disease
is
available
at
http://www.iah.bbsrc.ac.uk/virus/Picornaviridae/Aphthovirus/fmd.htm
2.B.2. Inverse PCR: this is a general method for amplifying DNA flanking a
previously characterized region. It uses primers derived from the
extremities of the known region but instead of pointing towards each other
the 5’ 3’ direction of the primers point away from each other. This
would normally not lead to a PCR product, here however the DNA is first
cut with restriction enzymes such that the known DNA is present in a
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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small restriction fragment (see Firgure). The cut DNA is then circularized
with a ligation enzyme at very low DNA concentrations (low DNA
concentration favors circularization versus ligation to another
fragment).Using conventional PCR the flanking regions can now be
amplified and characterized. This is important in the study of viral
tumourigenesis, when attempting to identify possible insertion sites of
viruses in host DNA and for the assessment of clonality in lymphoid
tumours (Hall et al., Hum. Immunol. 1995, 43, 207-218).
2.B.3. Allele-specific PCR (ARMS test): in this technique primers are
designed to discriminate between target DNA sequences which differ only
a single nucleotide in the region of interest. This is a form of allele-specific
PCR where the primers are designed to differ at the nucleotide(s) that
occur at the 3’ terminus. This technique is based on the fact that in a PCR
reaction it is crucial to have a correct base-pairing at the 3’ end (see
Figure). This method is used to detect specific pathogenic mutations and is
also called the amplification refractory mutation system (ARMS) and some
commercial kits are available for disease diagnosis (such as cystic
fybrosis),
some
can
be
found
on:
http://www.diagnostics.astrazeneca.com/elkits.htm
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2.B.4. Real-time PCR (Quantitative PCR): Several variants already exist of
this technology, but the core idea is the same for all of them. The idea is to
detect how much PCR products are present after each PCR cycle. This
allows for reliable quantitation of PCR products. End-point detections for
the determination of PCR products (where first a normal PCR reaction is
performed and the detection is done at the end) and estimation of the
amount of starting material are not reliable since the exponential
accumulation of PCR products reaches a plateau when a certain amount of
PCR product has been generated. Specificity of the detection of the PCR
products can be based solely on the selection of primers (using SYBR
Green I double stranded DNA binding dye, see Figure) or additional
selectivity can be added by using probes. The latter technique also
commonly known as TaqMan PCR. This TaqMan PCR uses the 5’-3’
nuclease activity of Taq DNA polymerase to detect target sequences
during amplification by PCR (see Figure). In addition to the conventional
primers a probe, (usually 20-30 mer in length) is included in the PCR mix,
designed to hybridize within the target sequence and to be non-extendible
at the 3’ end. The fluorescent emission activity of a fluorescent repoter
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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molecule attached at the 5’ end is neutralized by a quencher molecule at
the 3’ end. When hybridized to its target sequence, the intact probe shows
no signal due to the proximity of the reporter molecule to the quencher
molecule. During amplification Taq DNA polymerase, through its 5’-3’
nucleolytic activity, cleaves the probe into fragments, separating the
reporter from the quencher and allowing for its detection. The level of
fluorescence is directly proportional to the amount of specific
amplification of the target. Real-time PCR as described here is thus the
technique of choice to study the expression level of certain selected genes.
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Using a slight modification this technique can also be used to detect mutations
or determine polymorphisms. In this setup two differently labeled probes are
used. Since the machine can detect multiple colors we are able to determine
which products get formed and thus which sequences are present (see Figure).
The ARMS assay can also be modified to be performed using these real-time
PCR technologies.
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2.B.5. Special precautions to be taken in performing PCR for diagnostic
purposes: Contamination is one of the major reasons for misinterpretation
of results with PCR and occurs when exogenous DNA or RNA, proteases,
nucleases and various inhibitors of Taq polymerase are introduced into the
reaction mix and lead to false positive or negative results. All tubes, tips
and containers must be sterile and disposable. Since PCR is so extremely
sensitive and powerful, extreme care should be taken not to contaminate
the sample with exogenous DNA or previously amplified PCR products.
For this reason one should always use positive displacement or barrier
pipettes and there should be separate sets and working areas for samples
pre- and post PCR. There should be a one way traffic of materials and
samples from the pre- to the post-PCR working area. And one should
always use positive and negative controls.
2.B.6. PCR-product carry over prevention (AmpErase): this technology is a
simple yet powerful way to prevent that PCR products would be
reamplified in subsequent PCR amplifications, thereby preventing false
positive results. This method involves substituting dUTP for dTTP in the
PCR mixture, and pretreating all subsequent PCR mixtures with the
enzyme Uracil N-glycosylase (UNG) also calles AmpErase, prior to PCR
amplification (see Figure). Products of the PCR reation will contain
deoxyuridine, and are thus biochemically different from native DNA
molecules which contain thymidine instead of deoxyuridine. UNG
catalyses the cleavage of deoxyuridine containing DNA at deoxyuridine
residues by opening the deoxyribose chain at the C1-position. UNG will
not degrade native DNA (containing thymidine) or RNA because the
uridine in RNA is a ribose sugar and the UNG enzyme only cleaves
deoxyribose sugars. When heated in the first thermal cycling step at the
alkaline pH of the PCR reaction, the contaminating carry-over PCR
product breaks at the positions of the deoxyuridine. Newly synthesized
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PCR products in the PCR reaction will not be degraded because the
temperature is maintained above 55°C, and the UNG enzyme is not active
at these high temperatures. After amplification the UNG is chemically
denatured.
2.B.7. The Line Probe Assay (LiPA): This is a technique for the sequence
specific detection of PCR products. This technique was developed by
Innogenetics. The LiPA tests are based on the reverse hybridization
principle (see Figure, see also chapter 2E on allele specific oligonucleotide
probes). One of the primers used during the PCR reaction is biotinylated at
the 5’ end. Amplified biotinylated PCR-products are then chemically
denatured and the single stranded amplified target DNA is hybridized with
specific oligonucleotide probes which are immobilized as parallel lines on
a nitrocellulose strip. This strip contains several probes with varying
sequences for known mutations or polymorphisms of genes, thus allowing
for mutation analysis or genotyping of the sample. After hybridization,
streptavidin labeled with alkaline phosphatase is added and binds to the
biotinylated bound PCR product. This phosphatase converts the added
chromogen (NBT/BCIP) to a purple precipitate allowing for detection of
the bound DNA. Subsequently the places of hybridization can be read on
the strip. The advantage of this technology is that only one PCR
amplification and one hybrydization step are needed to obtain multiple
answers.
Kits have been developed to perform HLA-typing (Human Leukocyte
Antigens) and for the detection of infectious diseases and prediction of
their drug resistance based on the detection of mutation in the genomic
material of these infectious diseases. A very important feature of this
technique is that the strip can contain many different sequences so that in
one assay a lot of mutations or polymorphisms can be
screened/determined. More information can be found on:
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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http://www.innogenetics.be/Website/Website.nsf/Diagnostic+Products?Op
enView&Start=1&Count=30&Expand=6#6
2.B.8. AMPLICOR technology: this is a technique for detecting specific
PCR products. The technique also gives limited quantitation of the starting
material. This technique was developed by Roche. As with the LiPA test,
the AMPLICOR test is based on the reverse hybridization principle. The
PCR reaction used is based on the AmpErase technique and one of the
primers is biotinylated at the 5’ end. Immediately after amplification, the
strands are chemically denatured by the addition of an alkaline solution.
Denaturation occurs because the hydrogen bonds linking the two strands
are weak and breakdown at high pH. The single strands are then
hybridized onto oligonucleotide capture probes linked to BSA (Bovine
serum albumin) which are bound to a plastic microwell plate. After
hybridization, streptavidin conjugated with horseradish peroxidase is
added and binds to the biotinylated bound PCR product. This peroxidase
oxidizes the tetramethylbenzidine substrate in the presence of hydrogen
peroxide to a blue colored complex, the reaction is stopped after which the
blue turns yellow and the absorbance is measured using a photometer. This
technique allows for the detection of a certain DNA sequence and gives an
idea of the copy number of the target present in the sample. In contrast to
the LiPA technology this technique is not used for detecting mutations or
polymorphisms it is used for detecting the presence or absence of a certain
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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target and to give an idea of the amount of the copies present in the starting
material. Kits have been developed to screen for infectious diseases such
as HIV, HTLV, M. tuberculosis, CMV and others. More information can
be found on: http://us.labsystems.roche.com/ldpage.htm
2.B.
Sequencing
Nowadays almost all sequencing is performed using cycle sequencing and
using fluorescently labeled terminators. This technique which is based on the Sanger
chain termination method will be discussed here in more detail. Cycle sequencing,
also called linear amplification sequencing, uses like the standard PCR reaction
thermostable DNA polymerase and a temperature cycling format of denaturation,
annealing and elongation. The difference is that cycle sequencing employs only one
primer and includes fluorescently labeled ddNTP chain terminators in the reaction.
The ddNTP’s are added in the reaction in much lower amounts than the regular
dNTP’s. Whenever the polymerase adds a fluorescently labeled ddNTP the reaction
stops since further elongation is not possible. The four ddNTP’s used are all four
labeled with a different fluorescent dye (see Figure). The use of only one primer
means that unlike the exponential increase in product during PCR reactions, the
product accumulates linearly. Given the much lower amplification power of cycle
sequencing it is evident that much more starting material is required than for a PCR
reaction. To overcome this problem one can first perform a PCR amplification of the
sequence of interest. After amplification the primers of the PCR reaction first have to
be removed and the purified PCR product is then used as the template in the cycle
sequencing process. If the sequence which is being cycle sequenced is not
polymorphic, this cycle sequencing results in the accumulation of single stranded
products which differ 1 base in length and all products of the same length end with the
same fluorescently labeled ddNTP. When this reaction is then separated on a high
resolution sieving matrix (comparable to polyacrylamide gels) and detection of the
products is being performed with laser fluorescence the sequence can be read as seen
in figure. This is also called automated DNA sequencing. Detection of these
sequences can now be performed by electrophoresis over sieving matrix filled
capillaries. A typical sequence length of 700 bases can be read on one capillary with
an analysis time of 2 hours. Modern systems can contain upto 96 capillaries in one
machine allowing for 96 DNA sequences of 700 bases to be read in 2 hours. More
information on this technique can be found at: http://www.appliedbiosystems.com/ga/
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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1200000
PCR
cycle sequencing
1000000
800000
600000
400000
200000
0
0
5
10
15
20
The very nature of sequencing makes it the most alround technique to search
and diagnose mutations or polymorphisms. Although very versatile and in some cases
the only available technique it is very costly and previously described methods (if
available) might be easier and cheaper to use. Some kits for HLA-typing and for
mutation
screening
in
HIV
are
being
sold
commercially
http://www.appliedbiosystems.com/molecularbiology/md/
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2.C.
DNA microarrays
The DNA microarray technology is a very recent technique in molecular biology,
other terms used to describe this technology are: biochip, DNA chip, gene array,
GeneChip (Affymetrix) and genome chip.
Although this technology is mainly used for exploratory research purposes and
gene-discovery, already it has identified some diagnostic uses (which will be
discussed later on). A major limitation of the technique is the large amount of RNA
required for one array (50-200 µg total RNA or 2-5 µg mRNA). This translates to 107108 cells or 1 to 10 mg of tissue, however as the technique advances the amount of
material required will continue to go down.
Base-pairing or hybridization is the underlying principle of DNA microarray
technology. More specifically a reverse nucleic acid hybridization (see also chapter
2E on allele specific oligonucleotide probes) approach is employed: the probes are
unlabeled DNA fixed to a solid support (the arrays of DNA or oligonucleotides) and
the target is labeled and in solution. The DNA microarray is an orderly arrangement
of probes (with known identity, being unlabeled DNA) on a glass or nylon support to
which the target nucleic acids can bind based on complementary base-pairing. The
probes are generally present in spots smaller than 200 microns each enabling the
research on thousands of genes simultaneously.
Two kinds of probes are used, which define two major variants in DNA
microarray technology:
1) cDNA-probes: (500 to 5000 bases long) The cDNA probes are
immobilized to a solid surface such as glass or membranes
using a “spotting” robot. Here the DNA clones for the DNA
probes have been prepared in advance and are then printed onto
the surface of a microscope slide. A good review on how these
cDNA probes are selected and immobilized is given in the
nature
genetics
review
to
be
found
at:
http://www.nature.com/cgitaf/DynaPage.taf?file=/ng/journal/v21/n1s/full/ng0199supp_10.
html
2) oligonucleotide arrays: (20 to 25-mer oligos) These oligos can
be synthesized in-situ (being on the chip) or they can be
synthesized the conventional way followed by on-chip
immobilization. This technology was developed at Affymetrix
(www.affymetrix.com) and involves a combination of
photolithography technology from the semiconductor industry
with the chemistry of oligonucleotide synthesis, a good review
of the production and use of the technology can be found at:
http://www.nature.com/cgitaf/DynaPage.taf?file=/ng/journal/v21/n1s/full/ng0199supp_20.
html
DNA microarrays can be used to determine expression levels of genes, (in this
case usually the gene transcription is compared between two or more different kinds
BIOTECHNOLOGICAL TECHNIQUES IN MEDICAL DIAGNOSIS
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of cell-types (or between treated and non-treated cells)), or to identify sequence (gene
mutation).
1) Expression profiling: In this application RNA is isolated from
the cells to be analyzed. Using an oligo-dT primer, the mRNA
is reverse transcribed into fluorescently labeled cDNA using
the fluorescent labels Cye3-dUTP and Cye5-dUTP. A problem
with this cDNA reverse transcription is the reverse
transcription bias, this is not a problem when comparing the
same mRNA across two cell populations (unless it causes the
mRNA not to be transcribed at all), but it prohibits quantitative
comparisons between different mRNA’s on one array. Since
expression profiling is generally done according to the scheme
depicted in FIGURE ARRAY1, a first cDNA pool (a reference,
or non-stimulated cell pool) is fluorescently labeled with Cye3
and the cDNA pool of the test sample is labeled with Cye5. The
two pools of cDNA are then diluted to have the same overall
fluorescent intensity and after mixing they are hybridized to the
DNA microarray. If a target contains a sequence
complementary to the DNA probe, it will hybridize and be
detectable by fluorescence. After hybridization and washing the
array is scanned by exciting the fluorescent markers with laser
light at their excitation wavelength, and the light emitted at the
specific wavelength of each dye is detected with a chargecoupled device (CCD). Then computer comparisons give the
ratio of expression between both tested cell types. A typical
result
can
be
seen
in:
http://llmpp.nih.gov/lymphoma/figures.shtml
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2) Sequence information: Or screening of DNA variation, has to
be performed by oligonucleotide arrays. This technique offers a
huge potential for assaying for mutations in known disease
genes, as recently shown in the case of the breast cancer
susceptibility gene, BRCA1. In addition, this technique can be
used to identify human single nucleotide polymorphism (SNP)
markers. (see below for both applications). A good review on
this application can be found at: http://www.nature.com/cgitaf/DynaPage.taf?file=/ng/journal/v21/n1s/full/ng0199supp_20.
html
Applications to diagnostics:
I. SNP http://www.affymetrix.com/products/gc_HUSNP1.html
II. Classifying cancers and predicting outcome (see abstract)
Int J Cancer 2001 Feb 15;91(4):474-80
CDNA microarray gene expression analysis of B-cell chronic
lymphocytic leukemia proposes potential new prognostic markers
involved in lymphocyte trafficking.
Stratowa C, Loffler G, Lichter P, Stilgenbauer S, Haberl P, Schweifer
N, Dohner H, Wilgenbus KK
Boehringer Ingelheim Austria, Vienna, Austria.
Human cancer is characterized by complex molecular perturbations
leading to variable clinical behavior, often even in single-disease
entities. We performed a feasibility study systematically comparing
large-scale gene expression profiles with clinical features in human Bcell chronic lymphocytic leukemia (B-CLL). cDNA microarrays were
employed to determine the expression levels of 1,024 selected genes in
54 peripheral blood lymphocyte samples obtained from patients with
B-CLL. Statistical analyses were applied to correlate the expression
profiles with a number of clinical parameters including patient survival
and disease staging. We were able to identify genes whose expression
levels significantly correlated with patient survival and/or with clinical
staging. Most of these genes code either for cell adhesion molecules
(L-selectin, integrin-beta2) or for factors inducing cell adhesion
molecules (IL-1beta, IL-8, EGR1), suggesting that prognosis of this
disease may be related to a defect in lymphocyte trafficking. This
report demonstrates the feasibility of a systematic integration of largescale gene expression profiles with clinical data as a general approach
for dissecting human diseases.
A good overview, with a lot of helpful links can be found at the webpage: www.gene-chips.com.
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It seems likely that eventually oligonucleotide arrays of one sort or
another will replace most other methods for routine mutation screening in
the more common diseases, and that automated DNA sequencing will be
increasingly used for the rarer diseases.
2.D.
Allele specific oligonucleotide hybridization
The allele-specific oligonucleotide (ASO) hybridization is also referred to as
dot-blotting. The general procedure of dot-blotting involves taking an aqueous
solution of target DNA, for example total human genomic DNA, and simply spotting
it on to a nitrocellulose or nylon membrane then allowing it to dry. The variant
technique of slot-blotting involves pipetting the DNA through an individual slot in a
suitable template. The target DNA can also be purified PCR products. In both
methods the target DNA sequences are denatured, either by previously exposing to
heat, or by exposure of the filter containing them to alkali. The denatured target DNA
sequences now immobilized on the membrane are exposed to a solution containing
single stranded labeled probe sequences. After allowing sufficient time for probetarget heteroduplex formation, the probe solution is decanted, and the membrane is
washed to remove excess probe that may have become nonspecifically bound to the
filter. lt is then dried and exposed to an autoradiography film. A useful application of
dot-blotting involves distinguishing between alleles that differ by even a single
nucleotide substitution, mutation detection. This technique has applications in
mutation identification of diseases (eg. Mutations in the Von Willebrand factor). To
do this allele-specific oligonucleotide (ASO) probes are constructed from sequences
spanning the variant nucleotide site. ASO probes are typically 15-20 nucleotides long
and are normally employed under hybridization conditions at which the DNA duplex
between probe and target is stable only if there is perfect base complementarity
between them: a single mismatch between probe and target sequence is sufficient to
render the short heteroduplex unstable (see Figure). Typically, this involves designing
the oligonucleotides so that the single nucleotide difference between alleles occurs in
a central segment of the oligonueleotide sequence, thereby maximizing the
thermodynamic instability of a mismatched duplex. Such discrimination can be
employed for a variety of research and diagnostic purposes. Although ASOs can be
used in conventional Southern blot hybridization (see below), it is more convenient to
use them in dot-blot assays (see Figure). Another method of ASO dot blotting uses a
reverse dot-blotting approach. This means that the oligonucleotide probes are not
labeled and are fixed on a filter or membrane whereas the target DNA is labeled and
provided in solution (examples using this technique are the LiPA and AMPLICOR
technology). Positive binding of labeled target DNA to a specific oligonucleotide on
the membrane is taken to mean that the target has that specific sequence. This
approach, and related DNA microarray methods (already discussed), have many
diagnostic applications.
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2.E.
Oligonucleotide ligation assay
The oligonucleotide ligation assay (OLA) tests for base substitution
mutations. The test is based on the principle that two oligonucleotides
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constructed to hybridize to adjacent sequences in the target, with the join sited
at the position of the mutation will only be covalently joined by DNA ligase if
they are perfectly hybridized. Various formats of the technique exist but the
most recent and advanced technique uses modified tails on one probe and
fluorescent tags (FAM, HEX or TET) on the other probe with the ligation
products being electrophoresed and analysed on an automated sequencer.
These modifications have allowed the number of known point mutation sites
which can be screened at any one time to be increased and Applied
Biosystems now have kits capable of screening for 31 known CF mutations in
a single reaction tube and single lane of an electrophoresis run. In this test a
cocktail of fifteen pairs of primers is used to amplify regions of the CFTR
gene where common mutations are located. Each amplified segment (or
amplicon) is probed with three oligonucleotide probes. The common probe
hybridises to the amplicon at a sequence that is present in both normal and
mutant alleles and is labeled with one of three fluorescent tags. The mutant
and normal probes are allelic and can be distinguished by their length since
they have varying numbers of mobility modifying tails attached. These tails
are composed of Pentaethylene oxide (PEO) units (Figure). At any one locus,
if the mutation is not present only the normal and common probe will form a
ligation product, if a homozygous mutation is present only the mutant and
common probes will ligate and in heterozygous samples both normal and
mutant probes will ligate to the common probe in equal amounts but giving
products of different mobility. The rTth DNA ligase facilitates ligation by
catalysing the formation of a phosphodiester bond between the 5' phosphate of
the common probe and the 3' hydroxyl of either the normal or mutant probe.
This reaction will ONLY occur when both probes are hybridised and perfectly
matched to the complementary target amplicon. Following ligation, the
product for each amplicon has a unique combination of electrophoretic
mobility and fluorescence which allows identification of the sample phenotype
by size separation of the ligation product in each of the three colours (the CF
kit is designed to have 10-12 blue, 10-12 green and 9-11 yellow peaks). A red
labelled internal size standard allows Genescan analysis software to size
products precisely, label fragment data and display fragments as labelled peaks
in plot displays (Figure). This technique is suitable to mutation detection in
any gene where a number of known mutations or common polymorphisms
occur.
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2.F.
In Situ amplification
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Also called in situ PCR represents for most histopathologists the marriage of
standard histopathology and molecular biology. In situ PCR is able to amplify a single
copy target nucleic acid sequence so that it can be detected in fixed tissues and cells
and aims to correlate PCR results with morphology. Although in situ hybridization
(ISH) has traditionally been used to localize nucleic acid sequences within the cell,
this method cannot detect low copy numbers of target sequences (1-20 copies). This
technique holds great potential for the field of diagnostic histopathology. Sample
preparation is of great importance for these techniques. The objective of sample
preparation is to provide within single cells a discrete reaction chamber to contain the
PCR reaction. Cells, either in suspension or fixed to some form of solid phase support,
are rendered sufficiently permeable so that oligonucleotide primers and other reaction
components may diffuse into the cell. The permeation is controlled so that the
products of amplification are retained within the cells upon thermal amplification and
tissue morphology is retained so that meaningful histopathological interpretations can
be made. Typical pretreatments are to fix cells or tissue in formalin or
paraformaldehyde and affix the cells or tissue to glass slides using (for example)
Denhardt's solution (dewaxing and rehydration is, necessary for paraffin waxembedded tissues). Tissues or cells are then digested with a carefully optimized low
concentration of proteinase-K over 60 minutes to prepare for in situ PCR. A number
of alternatives have been described including detergent, chemical denaturant, or
microwave pre-treatments (see Figure). The PCR reagents are then added directly
onto the cells or tissue and the reaction is sealed of with a cover slide which is sealed
to the microscope slide to avoid evaporation of the reaction during the thermal cycling
process. The PCR reaction itself is analogous to a traditional PCR reaction and
involves the same reagents. After amplification the PCR products are detected by
different techniques.
A more detailed description of the technique and its many possible variations
(analogous to the variations described in the previous chapter on PCR) can be found
in chapter 52 “In Situ Amplification” of the book “The Science of Laboratory
Diagnosis” by Crocker and Burnett (ISIS Medical Media, 1999).
When applied to cancer studies in situ PCR can allow researchers to observe
the expression of genes and the presence of mutations in the abnormal cells of tissue
sections. The technique an also be used to locate cells with gene rearrangements and
chromosomal translocations, to pinpoint unique gene alterations associated with
neoplastic disorders, to detect latent viral infections and many more. In contrast to the
situation in acute virus infection, in latent or persistent infection virus-infected cells
comprise only a small fraction of the total number of cells in the tissue or tissue fluids
under examination. Thus conventional in situ hybridization methods may not reveal
infection. The techniques provide important tools for the study of viral pathogenesis.
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2.G.
Southern blotting
In this procedure, the target DNA is digested with one or more restriction
endonucleases, size-fractionated by agarose gel electrophoresis, denatured and
transferred to a nitrocellulose or nylon membrane for hybridization (Figure).
Fragments are fractionated by size in a conventional agarose gel electrophoresis
system. Following electrophoresis, the test DNA fragments are denatured in strong
alkali. As agarose gels are fragile, and the DNA in them can diffuse within the gel, it
is usual to transfer the denatured DNA fragments by blotting on to a durable
nitrocellulose or nylon membrane, to which single-stranded DNA binds readily. The
individual DNA fragments become immobilized on the membrane at positions which
are a faithful record of the size separation achieved by agarose gel electrophoresis.
Subsequently, the immobilized single-stranded target DNA sequences are allowed to
associate with labeled single-stranded probe DNA. The probe will bind only to
related DNA sequences in the target DNA, and their position on the membrane can be
related back to the original gel in order to estimate their size.
More detailed information on the technique
http://www.protocol-online.net/molbio/DNA/southern.htm
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2.G.1. Restriction fragment length polymorphisms: this technique uses the
Southern blotting technique. Because the genomic DNA samples are
fractionated by separation of restriction fragments according to size, mutations
which alter a restriction site, and significantly large insertions or deletions
occurring between neighboring restriction sites, can be typed. Such mutations
will result in altered restriction fragment lengths, that is restriction fragment
length polymorphisms (RFLPs). Direct detection of pathogenic point
mutations by restriction mapping is an application of RFLP technology to
diagnostics. Very occasionally, a pathogenic mutation directly abolishes or
creates a restriction site, enabling direct screening for the pathogenic mutation.
For example, the sickle cell mutation is a single nucleotide substitution (A 
T) at codon 6 in the -globin gene, which causes a missense mutation (Glu 
Val), and at the same time abolishes an MstII restriction site which spans
codons 5 to 7. The nearest flanking restriction sites for MstII, located 1.2 kb
upstream in the 5'-flanking region and 0.2 kb downstream at the 3' end of the
first intron, are well conserved. Consequently, a -globin DNA probe can
differentiate the normal A -globin and the mutant S-globin alleles in MstIIdigested human DNA: the former exhibits 1.2 kb and 0.2 kb MstII fragments,
whereas the sickle cell allele exhibits a 1.4 kb MstII fragment (Figure). RFLP
is also a powerful tool for the study of viral pathogenesis and for the genotypic
classification of viruses.
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2.G.2. Restriction mapping: this technique, which also uses the Southern blotting
technique is used to detect gene deletions. Certain diseases are associated with
a high frequency of deletion of all or part of a gene. If a partial restriction map
has been established for the gene under investigation, deletions can be
screened by Southern blot hybridization using an appropriate intragenic DNA
probe. If the deletion is a small one, for example a few hundred base pairs, it
is often apparent as a consistent reduction in size of normal restriction
fragments in the gene. An individual who is homozygous for this mutation, or
is a heterozygote with one normal allele and another with a small deletion, can
easily be identified by detecting the aberrant size restriction fragments. Large
deletions will lead to absence of specific restriction fragments. Homozygous
deletion of large DNA segments can easily be detected as complete absence of
appropriate restriction fragments associated with the gene. If, however, an
individual is heterozygous for a relatively large gene deletion, the deletion
may still be detected by demonstrating comparatively reduced intensity of
specific gene fragments.
For example, patients with 21-hydroxylase
deficiency often have deletions of about 30 kb of the 21-hydroxylase/C4 gene
cluster. Such pathological deletions eliminate the functional 21-hydroxylase
gene, CYP21, and an adjacent C4B gene, leaving the related CYP21P
pseudogene and C4A genes. Patients with homozygous deletions will show
absence of diagnostic restriction fragments associated with CYP21 and C4B,
while carriers of the deletion will show a 2:1 ratio of CYP21P:CYP21 and of
C4A:C4B (Collier et al., 1989; Figure).
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2.G.3. Northern blotting: Northern blot hybridization is a variant of Southern
blotting in which the target nucleic acid is RNA instead of DNA. A principal
use of this method is to obtain information on the expression patterns of
specific genes. Once a gene has been cloned, it can be used as a probe and
hybridized against a Northern blot containing, in different lanes, samples of
RNA isolated from a variety of different tissues (see Figure). The data
obtained can provide information on the range of cell types in which the gene
is expressed, and the relative abundance of transcripts. Additionally, by
revealing transcripts of different sizes, it may provide evidence for the use of
alternative promoters, splice sites or polyadenylation sites.
2.H.
Chromosome banding
Chromosome banding is the technique used in cytogenetics. Cytogenetic
analysis is a highly skilled laboratory discipline and it takes several years of training.
We will restrict this to a basic explanation of the technique.
Chromosomes can only be seen in dividing cells, and obtaining dividing cells
directly from the human body is difficult. Bone marrow is a possible source, but it is
much easier all round to take an accessible source of nondividing cells and culture
them in the laboratory. Blood is the material of choice - most people don't mind
giving a few millilitres, and the T lymphocytes in blood can be easily induced to
divide by treatment with lectins such as phytohemagglutinin. Other common sources
include fibroblasts grown from skin biopsies, and (for prenatal diagnosis) chorionic
villi or fetal cells shed into the amniotic fluid. Although chromosomes were described
accurately in some organisms as early as the 1880s, for many decades all attempts to
prepare spreads of human chromosomes produced a tangle that defied analysis. The
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key to getting analyzable spreads was a new technique, growing cells in liquid
suspension and treating them with hypotonic saline to make them swell. This allowed
the first good quality preparations to be made in 1956. White cells from blood are put
into a rich culture medium laced with phytohemagglutinin and allowed to grow for
48-72 hours, by which time they should be dividing freely. Nevertheless, because M
phase (Figure) occupies only a small part of the cell cycle, few cells will be actually
dividing at any one time.
The mitotic index (proportion of cells in mitosis) is increased by treating the
culture with a spindle disrupting agent such as colcemid. Cells reach M phase of the
cycle, but are unable to leave it, and so cells accumulate in metaphase of mitosis.
Often it is preferable to study prometaphase chromosomes, which are less contracted
and so show more detail. Cell cultures can be prevented from cycling by thymidine
starvation; when the block is released the cells progress through the cycle
synchronously. By trial and error, the time after release can be determined when a
good proportion of cells are in the desired prometaphase stage. Meiosis can only be
studied in testicular or ovarian samples. Female meiosis is especially difficult, as it is
active only in fetal ovaries, whereas male meiosis can be studied in a testicular biopsy
from any postpubertal male who is willing to give one. The results of meiosis can be
studied by analyzing chromosomes from sperm, although the methodology for this is
cumbersome. Meiotic analysis is used for some investigations of male infertility.
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Until the 1970s chromosomes were identified on the basis of their size and the
position of the centromeres. This allowed chromosomes to be classified into groups
but not unambiguously identified. The introduction of banding techniques finally
allowed each chromosome to be identified, as well as permitting more accurate
definition of translocation breakpoints, subchromosomal deletions, etc. Banding
resolution can be increased by using more elongated chromosomes, for example
chromosomes from prometaphase or earlier, rather than metaphase. Typical highresolution banding procedures for human chromosomes can resolve a total of 400, 550
or 850 bands (Figure).
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Chromosome Banding techniques
G-banding - the chromosomes are subjected to controlled digestion
with trypsin before staining with Giemsa, a DNA-binding chemical
dye. Dark bands are known as G bands. Pale bands are G negative
(Figure).
Q-banding - the chromosomes are stained with a fluorescent dye
which binds preferentially to AT-rich DNA, such as Quinacrine, DAPI
(4',6-diamidino-2-phenylindole) or Hoechst 33258, and viewed by UV
fluorescence. Fluorescing bands are called Q bands and mark the same
chromosomal segments as G bands.
R-banding - is essentially the reverse of the G-banding pattern. The
chromosomes are heat-denatured in saline before being stained with
Giemsa. The heat treatment denatures AT-rich DNA, and R bands are
Q negative. The same pattern can be produced by binding GC-specific
dyes such as chromomycin A3, olivomycin or mithramycin.
T-banding - identifies a subset of the R bands which are especially
concentrated at the telomeres. The T bands are the most intensely
staining of the R bands and are visualized by employing either a
particularly severe heat treatment of the chromosomes prior to staining
with Giemsa, or a combination of dyes and fluorochromes.
C-banding - is thought to demonstrate constitutive heterochromatin,
mainly at the centromeres. The chromosomes are typically exposed to
denaturation with a saturated solution of barium hydroxide, prior to
Giemsa staining.
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The chromosome constitution is described by a karyotype that states the total
number of chromosomes and the sex chromosome constitution. Human females and
males are 46,XX and 46,XY respectively. When there is a chromosomal abnormality
the karyotype also describes the type of abnormality and the chromosome bands or
subbands affected. Chromosomes are displayed as a karyogram (often loosely
described as a karyotype). Karyograms such as shown in the Figure are prepared by
cutting up a photograph of the spread, matching up homologous chromosomes and
sticking them back down on a card – or nowadays more often by getting an image
analysis computer to do the job. Chromosome banding picks out structural
organization on a 1-10 Mb scale. Various treatments involving denaturation and/or
enzymatic digestion, followed by incorporation of a DNA-specific dye, can cause
human and other mitotic chromosomes to stain as a series of light and dark bands.
Banding patterns are interesting (as well as being useful to cytogeneticists) because
they provide evidence of some sort of structure over 1-10 Mb regions. The banding
patterns correlate with other properties. Regions that stain as dark G bands replicate
late in S phase of the cell cycle and contain more condensed chromatin, while R bands
(light G bands) generally replicate early in S phase, and have less condensed
chromatin. Genes are mostly concentrated in the R bands, while the later replicating,
more condensed G-band DNA is less active transcriptionally. There are also
differences in the types of dispersed repeat elements found in G and R bands. Bands
similar to G bands can be produced by staining with quinacrine, which preferentially
binds to AT-rich DNA, while the R-banding pattern can be elicited using
chromomycin, which preferentially binds GC-rich DNA. However, the AT content of
human G band DNA is only a few per cent higher than R band DNA. The differences
depend on the differences in the scaffold-loop structure. Chromatin loops are thought
to attach to the chromosome scaffold at special scaffold attachment regions (SARs).
There are more SARs per unit length of DNA in G bands than in R bands. G bands
have smaller loops and a tighter 'queue' of SARs along the scaffold, so that there are
more SARs per unit length of chromosome, leading to stronger staining with ATselective stains like Giemsa.
Clinical cytogenetics is now established as a vital medical technique enabling
the clinical diagnosis of many genetic conditions (such as the Down syndrome). More
information in the analysis of the chromosome can be found in “Chromosome
Analysis” of the book “The Science of Laboratory Diagnosis” by Crocker and Burnett
(ISIS Medical Media, 1999).
2.I.
In situ hybridization (ISH)
The ISH technique is used to localize specific nucleic acid sequences in the cell
(compare to in situ amplification).
2.J.1. Chromosome in situ hybridization: A simple procedure for mapping genes
and other DNA sequences is to hybridize a suitable labeled DNA probe
against chromosomal DNA that has been denatured in situ. To do this, an air-
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dried microscope slide preparation of metaphase or prometaphase
chromosomes is made, usually from peripheral blood lymphocytes or
lymphoblastoid cell lines. Treatment with RNase and proteinase K results in
partially purified chromosomal DNA, which is denatured by exposure to
formamide. The denatured DNA is then available for in situ hybridization
with an added solution containing a labeled nucleic acid probe, overlaid with a
coverslip. Depending on the particular technique that is used, chromosome
banding of the chromosomes can be arranged either before or after the
hybridization step. As a result, the signal obtained after removal of excess
probe can be correlated with the chromosome band pattern in order to identify
a map location for the DNA sequences recognized by the probe. Chromosome
in situ hybridization has been revolutionized by the use of fluorescence in situ
hybridization (FISH) techniques. The sensitivity and resolution of in situ
hybridization has significantly increased by the use of FISH. In this technique,
the DNA probe is either labeled directly by incorporation of a fluorescentlabeled nucleotide precursor, or indirectly by incorporation of a nucleotide
containing a reporter molecule (such as biotin or digoxigenin) which after
incorporation into the DNA is then bound by fluorescently labeled affinity
molecule. To increase the intensity of the hybridization signal, large DNA
probes are preferred, usually cosmid clones containing around 40 kb of insert.
Because such large sequences will contain many interspersed repetitive DNA
sequences, it is necessary to use chromosome in situ suppression
hybridization. Essentially, this is a form of competition hybridization: before
the main hybridization, the probe is mixed with a large excess of unlabeled
total genomic DNA and denatured, thereby saturating the repetitive elements
in the probe,' so that they no longer mask the signal generated by the unique
sequences. FISH has the advantage of providing rapid results which can be
scored conveniently by eye using a fluorescence microscope. In metaphase
spreads, positive signals show as double spots, corresponding to probe
hybridized to both sister chromatids (Figure). Using sophisticated image
processing equipment and reporter-binding molecules carrying different
fluorophores, it is possible to map and order several DNA clones
simultaneously. The maximum resolution of conventional FISH on metaphase
chromosomes is several megabases. The use of the more extended
prometaphase chromosomes can permit 1 Mb resolution but, because of
problems with chromatin folding, two differentially labeled probe signals may
appear to be side-by-side, unless they are separated by distances greater than 2
Mb. Recently, however, new variations have been developed, permitting very
high resolution. There are numerous applications for chromosome FISH.
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Tissue in situ hybridization: In this procedure, a labeled probe is hybridized against
RNA in tissue sections. Tissue sections are made from either paraffin-embedded or
frozen tissue using a cryostat, and then mounted on to glass slides. A hybridization
mix including the probe is applied to the section on the slide and covered with a glass
coverslip. Typically, the hybridization mix has formamide at a concentration of 50%
in order to reduce the hybridization temperature and minimize evaporation problems.
Although double-stranded cDNAs have been used as probes, single-stranded
complementary RNA probes (riboprobes) are preferred: the sensitivity of initially
single-stranded probes is generally higher than that of double-stranded probes,
presumably because a proportion of the denatured double-stranded probe renatures to
form probe homoduplexes. cRNA riboprobes that are complementary to the mRNA
of a gene are known as antisense riboprobes and can be obtained by cloning a gene in
the reverse orientation in a suitable vector such as pSP64. In such cases, the phage
polymerase will synthesize labeled transcripts from the opposite DNA strand to that
which is normally transcribed in vivo. Useful controls for such reactions include
sense riboprobes which should not hybridize to mRNA except in rare occurrences
where both DNA strands of a gene are transcribed. Labeling of probes is performed
using either selected radioisotopes, notably 35S, or by nonisotopic labeling (Figure).
In the former case, the hybridized probe is visualized using autoradiographic
procedures. The localization of the silver grains is often visualized using only darkfield microscopy (direct light is not allowed to reach the objective; instead, the
illuminating rays of light are directed from the side so that only scattered light enters
the microscopic lenses and the signal appears as an illuminated object against a black
background). However, bright-field microscopy (where the image is obtained by
direct transmission of light through the sample) provides better signal detection.
Fluorescence labeling is a popular nonisotopic labeling approach and detection is
accomplished by fluorescence microscopy.
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A more detailed description of the ISH technique can be found in
chapter 51 “In Situ Hybridization” of the book “The Science of Laboratory
Diagnosis” by Crocker and Burnett (ISIS Medical Media, 1999).
Some commercial kits are available for diagnosis of diseases such as
cytomegalovirus, Epstein-Barr virus (more information can be found on
http://www.novocastra.co.uk/ish.htm ).
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3. Immunological techniques
A broad range of immunological techniques have well established applications in
the field of medical diagnosis. A good overview of these techniques and some
necessary background information can be found in chapter 2 in the book:
Immunobiology, Janeway and Travers, Garland Publishing Inc., 1997.
3.A.
Serological techniques
Essentially serological techniques include all techniques based on the use of
antibodies. The term derives from the fact that originally these assays were conducted
using the sera from immunized individuals. The use of antibodies is often called
serology. Since this description includes almost the entire field of immunological
techniques used in medical diagnosis, we will describe in this chapter some
“serological techniques” which cannot be classified in one of the chapters below
although these chapter also could be classified by the definition under the term
serological techniques.
3.A.1 Blood grouping: A commonly used technique for blood grouping,
which is very easy in its use is based on a gel system. Gel phase
systems rely on trapping the reacted cells in a solid or semi-solid
matrix, with negative results falling through the gel. In this system
monoclonal antibodies directed against the A, B and D (rhesus system)
antigens are chemically bonded to the matrix. Cells containing the
corresponding are trapped in the matrix when they are pulled through
the gel by centrifugation (see figure).
3.A.2 Latex agglutination: Polysterene latex micro-particles coated with
viral (or other antigens) agglutinate when mixed with patient serum
containing specific antibody. When a patient has antibodies to a viral
antigen this implies that he/she has been infected with the virus. The
antigen-coated latex particles are mixed with patient’s serum on a glass
slide or in a microtiter well. A visible agglutination pattern appears if
specific antibody is present. These tests are in wide use in virus
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laboratories because of their speed, simplicity and ease of use.
Currently good latex agglutination tests are available for viral antigens
such as rubella, toxoplasmose and cytomegalovirus. This test is also
used for the diagnose of bacterial infections such as Staphylococcus
aureus http://www.bindingsite.com/micro.html .
Staphylococcus aureus Rapid Latex test
This kit incorporates a slide agglutination procedure for use in the
differentiation of Staph. aureus from other staphylococci on the basis of
protein A / coagulase production.
Polystyrene latex particles are coated with fibrinogen and IgG, and will
readily agglutinate in the presence of a suspension of Staph. aureus
carrying clumping factor (coagulase) and/or protein A. The kit is available
in 100 or 300 test packs which include latex reagent, control reagent,
reaction cards and mixing sticks.
3.A.3 Hemagglutination This technique is in fact a variant of the latex
agglutination. Here red blood cells are used as the “particle”. The
antigens already present on the red blood cell can be used as in the
blood grouping test explained on p 2:11 and 2:12 of the chapter in
“Immunobiology”. Or the red blood cells can be coated with antigens,
an example is the THPA test for Syphilis.
3.A.4 Complement based techniques These techniques use the
complement system. This is a very complex system of proteins in our
body. A short description of the essentials will be given here.
Complement was discovered as a heat-labile component of normal
plasma that allows some antibodies to kill bacteria. The complement
system is made up of a large number of distinct plasma proteins; one is
activated directly by bound antibody to trigger a cascade of reactions
each of which results in the activation of another complement
component. Some components of this complement system, especially
the terminal complement components are able to directly kill cells by
creating pores in their membrane. Especially bacteria and red blood
cells are sensitive to complement killing. However under some
conditions also nucleated cells get killed by the complement. A widely
used example of the use of this technique is the traditional serological
HLA-typing. Until recently, the 'traditional' method used to distinguish
HLA non-identical individuals employed antibody. HLA-A and -B
locus antigens were the first to be defined in this manner, using
alloantisera obtained from subjects immunized by blood transfusions
pregnancy or renal allograft rejection. Using this technique, called a
microcytotoxicity assay, peripheral blood T lymphocytes (HLA class I
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antigens) and B lymphocytes (HLA-DR, -DQ) from potential allograft
recipients of unknown HLA type are incubated with antiserum of
characterized HLA specificity. These cells are then exposed to
complement. If antibody reacts with HLA proteins expressed on the
cell surface, complement is activated and the cells are killedcomplement mediated lysis. In the absence of reaction no lysis occurs.
Killing is visualized usually by a two-colour fluorescent dye system,
one of which labels all viable cells green. The other can only penetrate
killed cells, labelling them red, therefore killing is assessed semiquantitatively by loss of green cells and is usually expressed as a
percentage. Thus, where lysis occurs, the specificity of the antibody
identifies which HLA proteins are expressed. Several types of error are
possible with serological typing. A failure to identify a rare or crossreactive antigen is a frequent problem. This is principally because the
relevant antisera either have not been used, or are not available.
Linkage disequilibrium, geographical origins and racial breeding
patterns are contributing factors, moreover some antigens are
expressed at a lower frequency or not at all in certain populations.
Such errors are most likely to occur in laboratories using limited
numbers of antisera. In addition, a misinterpretation of antiserum
reactions can also produce errors. Antiserum may contain more than
one antibody, each with differing HLA specificities and even
monospecific HLA antibodies frequently cross-react with other HLA
antigens. Furthermore HLA typing of DR antigens using serology is
particularly susceptible to misinterpretation since B lymphocytes are
both more cross-reactive to a wide range of DR antigens and more
susceptible to non-specific complement mediated lysis, giving rise to
'falsepositive' results. Technical variations also account for a 15-30%
inter-laboratory error in serological typing. Variation in complement
activity between batches and quality of antiserum are the main
contributory factors. Serotyping is a rapid method for HLA
genotyping, however, the reagents used are not specific enough to
determine the precise structural identity of MHC molecules in
genetically non-identical individuals. This can only be achieved by
direct analysis of the MHC genes themselves.
3.A.5 Line Immunoassay (LIA) This provides a highly specific and
sensitive tool for detecting and characterizing specific antibodies to
microorganisms or other antigens by virtue of their binding to antigens
that have been affixed to nitrocellulose membranes. The antigens are
produced by recombinant molecular techniques or artificially
synthesized (synthetic peptides) and are then directly attached to a
nitrocellulose membrane. The nitrocellulose membrane is then
incubated with patient’s serum to allow antibody to bind to the
antigens fixed to the membrane. Enzyme-labeled anti-human
immunoglobulin is then added on a principle similar to that of ELISA.
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Appropriate washing steps are included. On addition of substrate,
antigen bands, to which specific antibody is present, become visible.
The technique is unique in that antibody to single viral or bacterial
proteins or other antigenic epitopes can be detected, hence making the
test very specific. Commercial tests are available for the detection of
HIV, Hepatitis C and human T-cell lymphotropic virus antibodies. LIA
tests are also available for the detection of auto-antibodies implicated
in an important group of autoimmune diseases: Sjogren's syndrome,
systemic lupus erythematosus, drug induced lupus, diffuse
scleroderma,
mixed
connective
tissue
disease,
CREST,
polymyositis/dermatomyositis.
http://www.innogenetics.be/Website/Website.nsf/7df3b6bb9c0862e8c1
2567380052687f/e1e06bbd2ad052aec12569a700578ce8?OpenDocume
nt
3.A.6 Western blotting This technique is described on p 2:23 of the
chapter in “Immunobiology”. Western blotting is applied to the field of
virology for the detection of the presence of antibodies specific for
certain viruses (much as in the LIA assays). Western blotting can also
be used to detect in stead of the presence of antibodies in a sample, the
presence of certain antigens in a sample. It the latter setting the
membrane is screened with specific monoclonal antibodies. In this
setting it is also used for the diagnosis of prion infection.
3.B.
ELISA
The basics of the ELISA technique are explained on pages 2:9 to 2:10 of the
chapter in “Immunobiology”. ELISA is such a basic technique that we will not further
discuss the principles of the technique in this course. ELISA is probably also the most
widely used technique in medical diagnosis. ELISA kits are commercially available
for the detection and quantification of so much targets that giving a list is useless and
impossible. Just as an example we would like to refer to an ELISA test for the
detection of EPO in patient’s serum http://biochem.roche.com/packinsert/1693417A.pdf . A bit more explanation on variants of the ELISA technique
used in diagnostics can be found on pages 218-219 in The science of Laboratory
Diagnosis, John Crocker and David Burnett, ISIS Medical Media, 1999.
3.C.
Flowcytometry
Flowcytometry is another widely used technique in medical diagnostics,
especially in the field of haematology. The background information on the technique
and the machinery has been touched in the course “Biologisch-farmaceutische
analyse”. For the application of flowcytometry to medical diagnosis monoclonal
antibodies are used to study the presence of specific markers on cells, which can be
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indicative of disease processes or of specific cells. These antibodies are labeled with
fluorescent markers which can be detected by the flowcytometer. See also p 2:27 to
2:30 of the chapter in “Immunobiology”.
Some applications of flowcytometry to haematology are discussed in Chapter
32 of The science of Laboratory Diagnosis, John Crocker and David Burnett, ISIS
Medical Media, 1999.
An immense array of antibodies with different fluorescent labels specially
designed for flowcytometry are commercially available. One of the big suppliers is
Pharmingen http://www.pharmingen.com .
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4. European legislation
http://europa.eu.int/eur-lex/en/lif/dat/1998/en_398L0079.html
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