184 Textbook of Biochemistry— Case Studies

MOLECULAR DIAGNOSTICS

Major Topics Covered

1. Nucleic acid electrophoresis
2. In situ hybridization
3. Mutation detection
4. DNA microarray

Electrophoresis of Nucleic Acids

¾¾ In order to analyze nucleic acids by size, the process of electrophoresis in an agarose or polyacrylamide support gel
is usually undertaken.
¾¾ Electrophoresis may be used analytically or preparatively and can be qualitative or quantitative.
¾¾ Large fragments of DNA such as chromosomes may also be separated by a modification of electrophoresis termed
pulsed field gel electrophoresis (PFGE), which uses alternating directions of DNA migration.
¾¾ Schematic illustration of a typical horizontal gel electrophoresis setup for the separation of nucleic acids

Electrophoresis
¾¾ Migration of charged solutes or particles of any size in a liquid medium under the influence of electric field.
¾¾ Developed by Tiselius in 1937.
¾¾ Iontophoresis, isotachophoresis – Electrophoresis of small ions.
¾¾ Zone electrophoresis – Protein electrophoresis when each protein zone is sharply demarcated from neighboring zones
by a protein-free area.
¾¾ Electrophoretogram – Can be scanned and quantified by densitometer.
¾¾ Can be stored and kept as permanent record. Used to separate proteins in serum, urine, CSF, etc.
¾¾ Nucleic acid electrophoresis is widely used as a basic tool in molecular biology (DNA, RNA).
 Supplementary Information to Chapter 49 185

Electrophoretic Apparatus

Starch Gel Electrophoresis (SGE)

¾¾ Prepared from partially hydrolyzed native starch.
¾¾ Used to separate macromolecules on the basis of charge-to-mass ratio and molecule size.
¾¾ Electric field compacts the gel.
¾¾ Crude method, replaced by less cumbersome techniques now.

SGE

Cellulose Acetate Electrophoresis (CAE)

¾¾ Thermoplastic resin – Cellulose treated with acetic anhydride (acetylates hydroxyl groups).
¾¾ Membranes contain 80% air space within interlocking cellulose acetate fibers.
¾¾ Commercially available as dry, opaque, brittle films that can crack easily.
¾¾ Resolution depends on degree of acetylation, prewashing procedure, additives used, pore size and thickness of
membrane.
¾¾ Membranes are soaked in solvent (95/5 methanol/glacial acetic acid) to make it more transparent for analysis, after
run.
186 Textbook of Biochemistry— Case Studies

¾¾ Pre-soaking followed by methanol/acetic acid treatment makes membrane stable for long time.
¾¾ Agarose gel has replaced both methods in many laboratories.

CAE

Agarose Gel Electrophoresis (AGE)

¾¾ Purified, neutral fraction of Agar, by separation of agarose from agaropectin.
¾¾ Used for separation of serum, urine, CSF proteins, LP, Hb, iso-enzymes, etc.
¾¾ Separation based on charge-to-mass ratio only. All proteins can pass through agarose, hence molecular mass does not
act as determinant.
¾¾ Advantages – Lower affinity for proteins, native clarity after drying, little ‘endosmosis’.

AGE
 Supplementary Information to Chapter 49 187

DNA Agarose Gel Electrophoresis

RNA Agarose Gel

Polyacrylamide Gel Electrophoresis (PAGE)

¾¾ Thermo-stable, transparent, strong, relatively chemically inert medium.
¾¾ Depending on concentration can be made into wide range of pore sizes.
¾¾ Average pore size is about 50 A, which allows serum proteins to pass through.
¾¾ Larger proteins like fibrinogen, b2 lipoprotein, a macroglobulin, g globulins are impeded.
¾¾ Separation based on charge-to-mass ratio and molecular size (molecular sieving).
¾¾ Advantages – Better separation, no electroendosmosis.
¾¾ Disadvantages – Acrylamide is neurotoxic.
¾¾ Modified acrylamide compounds which are more hydrophilic which reduce less resistance to passage of larger
molecules (20 kb DNA fragments) and fragments differing in ~2% can be separated.
¾¾ Temperature up to 50–60°C is possible, which enables better separation.
188 Textbook of Biochemistry— Case Studies

PAGE

Disc Electrophoresis
¾¾ Serum protein electrophoresis with AGE yields zones – albumin, a1, a2, b and g globulins.
¾¾ Disc electrophoresis gives 20 different fractions (individual proteins).
¾¾ Used to study individual proteins in serum, esp. genetic variants and iso-enzymes.

Iso-electric Focusing (IEF) Electrophoresis

¾¾ IEF separates amphoteric compounds, with increased resolution in a medium possessing a stable pH gradient.
¾¾ Proteins are focused on a particular point on the gel as it migrates to a zone where pH of gel matches protein’s iso-
electric point.
¾¾ Charge becomes zero and migration ceases.
¾¾ Protein zones are very sharp, because pH associated region is very narrow.
¾¾ Normal diffusion is counter-acted by acquisition of charge.
¾¾ Proteins varying in pI by about 0.02 pH units can be separated by IEF.
¾¾ pH gradient is created by carrier ampholytes.
¾¾ Modified version – IPG-IEF, PAGE-IEF, AGE-IEF, CAE-IEF, etc.
 Supplementary Information to Chapter 49 189

IEF Followed by 2nd Dimension (2D gel)

2D (Two-dimensional) Electrophoresis
¾¾ Extensively used proteomics tool; also used in gene mutations, tumor diagnosis.
¾¾ Combines charge-dependent IEF in the first dimension (AGE, large-pore PAGE) with molecular weight dependent
electrophoresis in the second (linear or gradient PAGE).
¾¾ Can resolve 1100 separate protein spots by autoradiography and 400 spots by Coomassie protein staining.

Typical 2D Gel Electrophoretogram

Each protein appears as a characteristic ‘spot’ on 2D gel

Pulsed Field Gel Electrophoresis (PFGE)

¾¾ Power is alternatively applied to different pairs of electrodes or electrode arrays; hence electrophoretic field is cycled
between different directions.
¾¾ Directions vary by 105-180°, and molecules re-orient to new field direction, during each cycle before migration can
continue.
¾¾ Large fragments (50-400 kb) can be resolved using 10 sec pulses.
¾¾ Very large fragments (7 megabases) can be separated by several hours pulses.
190 Textbook of Biochemistry— Case Studies

Pulsed Field Gel Electrophoresis – Current Applied in Diff. Directions

CHEF Electrophoresis
¾¾ It is possible to resolve DNA molecules several million bases in length by periodically changing the angle of the
electric field in the gel.
¾¾ There are a number of variations to the basic setup but all require specially constructed gel boxes, which can be quite
expensive.
¾¾ In addition, some types of alternating-angle elecrophoresis setups dissipate so much power in the gel that special
cooling equipment must be used.

CHEF Electrophoresis Showing Large Fragments Being Separated

 Supplementary Information to Chapter 49 191

Capillary Electrophoresis

Capillary Electrophoresis
¾¾ Separation times are generally only a few minutes.
¾¾ The DNA is detected either by UV absorption or by fluorescent labeling, both of which eliminate the need to use
mutagenic substances (e.g., ethidium bromide) or dispose of radioactive waste.
¾¾ The quantity of DNA required for the separation is in the nanogram range.
¾¾ Single-base resolution can be readily obtained on fragments up to several hundred base pairs in size.

Modes of CE
1. Capillary zone electrophoresis (CZE)
2. Micellar electro-kinetic chromatography (MEKC)
3. Capillary gel electrophoresis (CGE)
4. Capillary IEF, and
5. Capillary ITP (isotachophoresis)
192 Textbook of Biochemistry— Case Studies

Capillary Electrophoresis Unit

Microchip Electrophoresis
¾¾ High-speed separation of proteins and DNA, 4-10 x more than CE.
Simple, capable of chip integration of multiple functions and potential for automation.
¾¾ Newer developments include integrated microchip designs, advanced detection systems, and new applications.

Principle
¾¾ Similar to CE.
¾¾ Components are fabricated using photolithographic processes, defined by microelectronics industry onto surface of
microchip.
¾¾ Detection with microchip is primarily through LIF, easily implemented with the planar configuration of microchip.
¾¾ Extremely small amounts can be detected in very short time (Typical turnaround time 50–200 sec).

Applications
¾¾ Used in place of conventional slab gel electrophoresis (CE used in HGP).
Detection of HSV DNA in CSF for diagnosis of encephalitis, detection of gene rearrangements correlative with
lymphoproliferative disorders, MTHFR polymorphisms in multiple diseases, fragile X syndrome diagnosis, tetra-
nucleotide repeats in hypercholesterolemia, and diagnosis of muscular dystrophies.
¾¾ Other applications – Electrophoretic microchips for SSCP or heteroduplex analysis of BRCA genes.
¾¾ Genotyping in hereditary hemochromatosis.
 Supplementary Information to Chapter 49 193

In Situ Hybridization
¾¾ In situ hybridization is a method used to localize nucleic acid sequences in cells or tissue sections.
¾¾ The in situ hybridization methodology exploits a fundamental property of nucleic acids: their ability to anneal (bind)
to one another in a sequence-specific manner to form hybrids.
¾¾ Thus, the gene sequence of interest can be detected in situ by labeling a known sequence of DNA or RNA to form a
probe and then introducing the probe to the sample in a single-stranded form.
¾¾ Following annealing, the gene sequence of interest can be visualized via the probe label.

¾¾ It can be done on frozen sections, paraffin tissue sections and formalin-fixed tissues.
There are essentially two types of in situ hybridization (ISH) that can be readily applied to paraffin-embedded tissue
sections: fluorescence in situ hybridization (FISH) using DNA probes and chromogenic in situ hybridization (CISH)
with DNA or RNA probes.
¾¾ One major advantage of fluorescence over nonfluorescent labels to study genetic aberrations is that multiply-labeled
probes can be applied simultaneously to a tissue preparation.
¾¾ Steps involved include –
• Pre-treatment of samples
• Pre-hybridization and hybridization
• Post-hybridization, and
• Detection.
¾¾ In situ hybridization has gained popularity over other molecular biology methods because the DNA/mRNA of interest
can be viewed in the context of the tissue morphology.

Applications of ISH (FISH)

¾¾ ISH is rapidly becoming a technique that can be applied to a wide range of clinical situations.
¾¾ Studies range from the assessment of gene rearrangements in leukemia and lymphoma to the diagnosis of B-cell
lymphoma by demonstration of restriction of light-chain mRNA to the determination of amplification of HER2/neu
in breast cancer.
194 Textbook of Biochemistry— Case Studies

¾¾ Fluorescence in situ hybridization has been used widely in the diagnosis of various types of lymphoma and other
conditions.
¾¾ In research, FISH has been used to determine tumor associated aberrations.
¾¾ Chromogenic in situ hybridization (CISH) is used to diagnose melanoma and lymphoma using mRNA probes for
kappa and lambda chains.
¾¾ In research, CISH has been widely applied to the study of numerical chromosomal aberrations.
¾¾ Utility of FISH/CISH to understand pathological processes of disease and in preneoplasia.
¾¾ Detection of chromosomal aberrations in benign tissue can represent a novel marker of cancer risk.

FISH

CISH

Outline of Common Mutation Detection Techniques

Technique Advantages Disadvantages
Protein-truncation test Cheap; rapid; allows detection of Mutations can be missed when gene product is very short; does not
genomic deletions detect missense mutations; RNA required to examine small exons
Single-strand-conformation Simple, well-established technique Low sensitivity; labour-intensive; does not detect exon deletions*
analysis of genomic DNA
Denaturing high-performance Detects almost all intra-exonic and Expensive equipment required; does not detect exon deletions*
liquid chromatography splice-site mutations; rapid
DNA chips Can potentially identify all sequence Expensive equipment required; high cost per chip
variants; very rapid
Direct sequencing Identifies most intra-exonic and Expensive; exon deletions can be missed if detailed single-nucleotide-
splice-site mutations polymorphism analysis is not carried out
Multiplex ligation-dependent Detects all exon deletions Cannot detect intra-exonic mutations
probe amplification
This type of deletion is thought to be rare in most populations.
 Supplementary Information to Chapter 49 195

Single Strand Conformation Polymorphism (SSCP)/Heteroduplex analysis

¾¾ Single-strand conformation polymorphism (SSCP) and heteroduplex analysis are separate mutation scanning methods
in their own right.
¾¾ They are, however, unusual in that they can be carried out simultaneously on a single gel.
¾¾ The technique of SSCP analysis was originally described in 1989.
¾¾ It involves the heat denaturation of polymerase chain reaction (PCR) amplified DNA followed by electrophoresis
under non-denaturing conditions.
¾¾ The fragments in the original protocol were visualized by radiolabeling and autoradiography, although a variety of
non-isotopic methods are now available, including silver staining, fluorescent labels, and ethidium bromide staining.
¾¾ The SSCP technique relies on the propensity for single-stranded DNA (ssDNA) in nondenaturing conditions to take
on a three-dimensional, or secondary structure that is highly sequence dependent.
¾¾ Consequently, sequence differences can cause alterations to the DNA’s secondary structure.
¾¾ Because the electrophoretic mobility of DNA under nondenaturing conditions is dependent on its shape as well as
other factors like charge, point mutations can give rise to mobility shifts.

Schematic of the principle of SSCP analysis. A single nucleotide polymorphism of either thymine or cytosine leads to different single-stranded conformations
of the DNA molecule. Under non-denaturing gel electrophoresis, this SMP results in different mobilities of the DNA fragments.
196 Textbook of Biochemistry— Case Studies

¾¾ Heteroduplexes are hybrid DNA molecules that, although largely matched, have one or more mismatched base pairs.
¾¾ Heteroduplexes have been used as a tool to scan for point mutations since 1992.
¾¾ They typically appear on native polyacrylamide gels as one or two bands of reduced mobility relative to the
homoduplex DNA.
¾¾ The mismatched bases present in heteroduplexes are thought to affect electrophoretic mobility by inducing bends in
the DNA.
¾¾ Because two different DNA sequence variants must be present to form heteroduplexes, they may need to be created
for some types of analysis.
¾¾ For example, in order to analyze male samples for loci on the X chromosome by heteroduplex analysis, heteroduplexes
can be created by mixing, denaturing, and annealing the test sample PCR amplification with a known normal control
amplification.
¾¾ In heterozygotes, however, heteroduplexes form as a natural byproduct of PCR reactions.
¾¾ During the latter stages of PCR amplification, when the polymerase activity is limiting, some of the denatured ssDNA
can spontaneously reanneal without primer extension with an opposite strand from the other allele, thus creating
heteroduplex DNA.
¾¾ Combined SSCP/heteroduplex analysis exploits the tendency for a proportion of the DNA denatured during sample
preparation for SSCP to spontaneously reanneal to form dsDNA and, hence, heteroduplexes when there are sequence
differences in the sample.
¾¾ The gel conditions for both SSCP and heteroduplex analysis are compatible and so it is possible to get “two techniques
for the price of one.”

Conformation Sensitive Gel Electrophoresis (CSGE)

¾¾ There are two major principles on which CSGE works.
¾¾ Single base mismatches produce conformational changes in double-
stranded DNA, which leads to differential migration of heteroduplexes
and homoduplexes.
¾¾ The gel contains partially denaturing agents, in the form of formaldehyde
and ethylene glycol, the conformational changes produced in the first
instance get increased further, leading to a clear separation of the
heteroduplex bands.
¾¾ Therefore, a normal sequence will give a homoduplex bandcorresponding
to normal DNA (as shown in the figure), whereas mutant DNA can give
theoretically up to 4 bands.
 Supplementary Information to Chapter 49 197

Protein Truncation Test (PTT)

¾¾ The protein truncation test (PTT), occasionally referred to as the in vitro synthesized-protein (IVSP) assay, is a
method for screening the coding region of a gene for mutations that result in the premature termination of mRNA
translation.
¾¾ The techniques involved in performing PTT are relatively straightforward and begin with the isolation of genomic
DNA or RNA.
¾¾ The polymerase chain reaction (PCR) is used to amplify a DNA template, usually of 1–3 kb in size, that is tested in
an in vitro transcription and translation assay.
198 Textbook of Biochemistry— Case Studies

¾¾ Truncated proteins are identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography or fluorography.
¾¾ A single, large exon of a gene can be amplified directly from genomic DNA in several overlapping fragments.
¾¾ The complete coding sequence of a large gene, with many small exons, can be amplified in several overlapping
fragments by reverse-transcription PCR (RT-PCR) starting from RNA.
¾¾ There are two main advantages of PTT compared to most other mutation detection methods.
¾¾ Several kilobase segments of a gene can be rapidly screened in a single reaction, and PTT only identifies those
mutations that have a clear pathological effect on protein function (i.e., those that result in a truncated protein and are
likely to result in loss of function).
¾¾ Missense mutations and neutral polymorphisms are not identified.
¾¾ If it is important to identify missense mutations, another mutation detection method, such as single-strand conformation
polymorphism (SSCP), would need to be used in conjunction with PTT.

Denaturing High Performance Liquid Chromatography (DHPLC)

¾¾ Denaturing high-performance liquid chromatography (DHPLC) is one of many techniques that have been developed
to screen genes for sequence variations.
¾¾ Depending on the mode of operation, DHPLC can be used to detect single-nucleotide substitutions or small insertions
or deletions in double-stranded DNA fragments as well as analyze and purify single-stranded nucleic acids.
¾¾ Although DHPLC can identify DNA fragments that contain sequence variations, additional methods (usually DNA
sequencing) are required to confirm the precise nature of the mutation.
¾¾ Denaturing HPLC is a form of ion-pair reversed-phased chromatography in which nucleic acids can be bound to a
hydrophobic column [usually poly-(styrene-divinylbenzene particles)] via an intermediary such as triethylammonium
acetate (TEAA).
¾¾ The affinity of this interaction is dependent on size, nucleotide composition, and column temperature.
¾¾ An increasing gradient of an organic solvent, usually acetonitrile, is passed over the column and gradually removes
the TEAA, leading to the release of bound DNA (or RNA) from the column.
¾¾ After being eluted, the DNA can be detected via ultraviolet (UV) absorbance or, if the DNA has been labeled,
fluorescence.
¾¾ Under nondenaturing conditions (i.e., column temperatures at which double-stranded DNA remains fully paired), the
interaction is almost completely dependent on fragment size.
¾¾ Working in this mode, it is possible to accurately size and quantify PCR fragments.
 Supplementary Information to Chapter 49 199

¾¾ At high column temperatures, double-stranded DNA is completely denatured into single strands and elution from the
column is dependent on both size and sequence.
¾¾ It has been demonstrated that for short fragments (< 30 nucleotides), it is possible to separate all oligonucleotides
differing by a single base and that for larger fragments (up to at least 60 nucleotides), all possible substitutions can be
detected with the exception of a C to G transversion.
¾¾ For most purposes, DHPLC is performed under partially denaturing conditions.
¾¾ DNA containing a mismatch has a slightly lower affinity for the column because it makes fewer ion-paring bonds
with the hydrophobic column and will elute before the fully paired DNA.
¾¾ Typically, single-nucleotide mismatches can be reliably detected in DNA fragments of 300–400 nucleotides, although
much higher detection limits (up to 1500 bp) have been reported.

DNA Sequencing
¾¾ DNA sequencing is the determination of the order of nucleotides in a DNA molecule.
¾¾ The most popular method for doing this is called the Sanger sequencing method or chain-termination method.
¾¾ This method uses synthetic nucleotides that lack the 3' hydroxyl group and are unable to form the 3'–5' phosphodiester
bond necessary for chain elongation. These nucleotides are called dideoxynucleotides (ddNTPs).
¾¾ The sequencing reaction consists in a singlestranded template DNA to which a short complementary primer is
annealed and extended by a DNA polymerase.
¾¾ The sequencing reaction contains a low concentration of ddNTPs, each labeled with a different fluorochrome, in
addition to the normal deoxynucleotides.
¾¾ Once a ddNTP is incorporated in the elongating chain, it blocks further chain extension; as a result, a mixture of
chains of lengths determined by the template sequence is accumulated.
200 Textbook of Biochemistry— Case Studies

¾¾ DNA sequencing is considered the standard for identification of nucleic acids and detection of mutations, and CGE
is routinely used in research and clinical situations in which panels of mutations or large numbers of samples are
analyzed and when turnaround time is critical.
¾¾ Direct sequencing of amplified fragment-length polymorphism bands also has been used as a polymorphism isolation
strategy of population genetic parameters in genomic DNA of non-genomic model species.

Automated Sequencing

Other Techniques for Mutation Detection

1. Denaturing gradient gel electrophoresis (DGGE) – 99% sensitivity.
2. Allele specific oligonucleotide (ASO) analysis and its variants (AMRS, amplification refractory mutation system).
3. Chemical cleavage of mismatch (CCM) and enzyme mismatch cleavage (EMC) – Large fragments can be analyzed.

Labeling Technique for DNA Arrays

 Supplementary Information to Chapter 49 201

RNA samples are labeled using fluorescent nucleotides (left) or radioactive nucleotides (right), and hybridized to
arrays. For fluorescent labeling, two or more samples labeled with differently colored fluorescent markers are hybridized
to an array. Level of RNA for each gene in the sample is measured as intensity of fluorescence or radioactivity binding to
the specific spot. With fluorescence labeling, relative levels of expressed genes in two samples can be directly compared
with a single array.

DNA Arrays—Technical Foundations

¾¾ An array works by exploiting the ability of a given mRNA molecule to hybridize to the DNA template.
¾¾ Using an array containing many DNA samples in an experiment, the expression levels of hundreds or thousands genes
within a cell by measuring the amount of mRNA bound to each site on the array.
¾¾ With the aid of a computer, the amount of mRNA bound to the spots on the microarray is precisely measured,
generating a profile of gene expression in the cell.

An Experiment on a Microarray
In this schematic diagram:
GREEN represents Control DNA
RED represents Sample DNA
YELLOW represents a combination of Control and Sample DNA
BLACK represents areas where neither the Control nor Sample DNA
Each color in an array represents either healthy (control) or diseased (sample)
tissue. The location and intensity of a color tell us whether the gene, or mutation,
is present in the control and/or sample DNA.

DNA Microarray
202 Textbook of Biochemistry— Case Studies

Microarray is a tool for analyzing gene expression

that consists of a glass slide.

Each blue spot indicates the location of a PCR product. On a real

microarray, each spot is about 100µm in diameter.

Photolithography

¾¾ Light directed oligonucleotide synthesis.

¾¾ A solid support is derivatized with a covalent linker molecule terminated with a photolabile protecting group.
¾¾ Light is directed through a mask to deprotect and activate selected sites, and protected nucleotides couple to the
activated sites.
¾¾ The process is repeated, activating different set of sites and coupling different based allowing arbitrary DNA probes
to be constructed at each site.
 Supplementary Information to Chapter 49 203

Affymetrix GeneChip® Arrays

A combination of photolithography and combinatorial chemistry to manufacture GeneChip® Arrays. With a minimum number of steps,
Affymetrix produces arrays with thousands of different probes packed at extremely high density. Enable to obtain high quality, genome-wide
data using small sample volumes.

Data from an experiment showing the expression of thousands

of genes on a single GeneChip® probe array.