INTRODUCTION TO THE ANALYSIS OF DNA

DNA analysis is playing an increasingly important role in refining haematological

diagnosis and determining treatment. Over recent years our understanding of the
molecular basis of both inherited and acquired haematological disorders has improved
considerably. There are several ways in which this knowledge is now being applied in
diagnostic haematology. These include the identification of families at risk for
haemoglobinopathies allowing the provision of early prenatal diagnosis, the
assessment of genetic risk factors in thrombophilia, the diagnosis and characterisation
of leukaemias, the monitoring of minimal residual disease, and the study of host–donor
chimerism following bone marrow transplantation.

EXTRACTION OF DNA

DNA can be extracted from any blood or tissue sample. As a rule, 3 ml of blood in EDTA
will suffice. The DNA is extracted from all nucleated cells and is called genomic DNA. In
the nucleus, the DNA is tightly associated with many different proteins as chromatin. It
is important to remove these as well as other cellular proteins to extract the DNA. This
is achieved through the use of organic solvents or salt precipitation. An aqueous
solution of DNA is obtained, from which the DNA is further purified by ethanol
precipitation.

POLYMERASE CHAIN REACTION

PCR is, fundamentally, a method to amplify genomic or cDNA sequence , development

of the PCR has had a dramatic impact on the study and analysis of nucleic acids.
Through the use of a thermostable DNA polymerase, Taq polymerase extracted from
the bacterium Thermus aquaticus, the PCR results in the amplification of a specific DNA
fragment such that it can be visualised by ethidium bromide staining on an agarose gel.
The procedure takes only a few hours, does not require the use of radionucleotides, and
requires only a very small amount of starting material.

Principle

A DNA polymerase will synthesise the complementary strand of a DNA template in

vitro. A stretch of double-stranded DNA is required for the synthesis to be initiated.
This double-stranded sequence can be generated by annealing an oligonucleotide
(oligo), which is a short, single-stranded DNA molecule usually between 17 and 22
bases in length, to a single-stranded DNA template. These oligos, which are synthesised
in vitro, will prime the DNA synthesis and are therefore referred to as primers.

In the PCR, at least two oligos are used. One primes the synthesis of DNA in the forward
direction, or along the coding strand of the DNA, whereas the other primes DNA
synthesis in the reverse direction, or along the noncoding strand. The other
components of the reaction are the DNA template from which the DNA fragment will be
amplified, the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP)
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required as the building blocks of the DNA that is to be synthesised, the necessary
buffer, and the thermostable DNA polymerase (Taq polymerase).

The first step of the reaction is to denature the template DNA by heating the reaction
mixture to 95°C. The reaction is then cooled to a temperature, usually between 50°C
and 65°C, that permits the annealing of the oligos to the DNA template but only at their
speci ic complementary sequences. The temperature is then raised to 72°C, at which
temperature the Taq polymerase efficiently synthesises DNA, extending from the oligo
in a 5′ to 3′ direction. Cyclical repetition of the denaturing, annealing, and extension
steps, by simply changing the temperature of the reaction in an automated heating
block, results in exponential amplification of the DNA that lies between the two oligos.

The specificity of the DNA fragment that is amplified is therefore determined by the
sequences of the oligos used. A sequence of 17 base pairs (bp) is theoretically unique in
the human genome, and so oligos of this length and longer will anneal at only one
specific place on a template of genomic DNA. One general requirement of the PCR is
therefore some knowledge of the DNA sequence of the gene that is to be amplified. The
relative positioning of the two oligos is another important consideration. They must
prime DNA synthesis in opposite directions but pointing toward one another. There is
also an upper limit to the distance apart that the oligos can be placed; fragments of
several kilobase pairs (kb) in length can be amplified, but the process is most efficient
for fragments of several hundred bp.

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Problems and Interpretation

If the amplification has been successful, a discrete fragment of the expected size is seen
in an ethidium-bromide–stained agarose gel in all samples, except where a blank
control is loaded. If a product is seen in the blank control, then one of the solutions has
been contaminated. In this case, the experiment and all the working solutions must be
discarded and the micropipettes must be cleaned.

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The absence of a fragment in all tracks indicates that the PCR has failed. This could
occur for a number of reasons, the most obvious being the poor quality or omission of
one of the essential reagents. The reaction may also fail if the magnesium concentration
is too low or if the annealing temperature is too high. DNA quality is often one of the
major reasons for failure. If one particular DNA sample repeatedly fails to amplify, then
the sample should be reextracted with phenol and chloroform and reprecipitated in
one-tenth volume of 5 mol/l ammonium acetate and 2.5 volumes of ethanol. Another
problem is the presence of nonspecific fragments or just a smear of amplified product.
This can occur if the magnesium concentration is too high or if the annealing
temperature is too low.

Analysis of Polymerase Chain Reaction Products

Presence or Absence of a Polymerase Chain Reaction Product

PCR products are most commonly and conveniently visualised by agarose gel
electrophoresis.

Amplification Refractory Mutation System

Principle

Point mutations and small insertions or deletions can be identified directly by the
presence or absence of a PCR product using allele-specific primers. Two different oligos
are used that differ only at the site of the mutation (the amplification refractory
mutation system, or ARMS, primers) with the mismatch distinguishing the normal and
mutant base located at the 3′ end of the oligo. In a PCR, an oligo with a mismatch at its 3′
end will fail to prime the extension step of the reaction. Each test sample is amplified in
two separate reactions containing either a mutant ARMS primer or a normal ARMS
primer. The mutant primer will prime amplification together with one common primer
from DNA with this mutation but not from a normal DNA. A normal primer will do the
opposite. A second pair of unrelated primers at distance from the ARMS primers is
included in each reaction as internal control to demonstrate that efficient amplification
has occurred. This is essential because a failure of the ARMS primer to amplify is
interpreted as a significant result and must not be the result of suboptimal reaction
conditions.

Interpretation

In all the samples, apart from the blank control, the fragment produced by amplification
with the internal control primers must be seen. If this is the case, then the presence or
absence of a mutation is simply determined by the presence or absence of the expected
fragment produced by amplification with the mutant ARMS primer and the common
primer. The presence or absence of the normal allele is determined in the same way in
the reaction that includes the normal ARMS primer. In this way, heterozygous,
homozygous normal, and homozygous mutant genes can be distinguished.
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Gap-PCR
Large deletions can be detected by Gap-PCR. Primers located 5′ and 3′ to the
breakpoints of a deletion will be too far apart on the normal chromosome to generate a
fragment in a standard PCR. When the deletion is present, these primers will be
brought together, enabling them to give rise to a product.

By the same principle, primers can be brought together by chromosomal translocation,

giving rise to a diagnostic product. Breakpoints may be clustered over too large a
region for genomic DNA to be used in these instances. However, leukaemic
translocations can also give rise to transcribed fusion genes. Primers from different
genes are then juxtaposed in a hybrid messenger RNA (mRNA) molecule and can give
rise to a reverse transcription-PCR (RT-PCR) product.

Size of the PCR Product

Principle

Deletions and insertions can be identified simply, after agarose gel electrophoresis,
when their size significantly alters that of the PCR product. A higher resolution of
fragment sizes is obtained after polyacrylamide gel electrophoresis.

Restriction Enzyme Digestion

Principle

Restriction enzymes (RE) cleave DNA at short specific sequences. Because many RE are
available, it is not uncommon for a single point mutation to coincidentally create or
destroy an RE recognition sequence. If this is the case, digestion of the appropriate PCR
product prior to agarose gel electrophoresis enables the mutation to be identified. A
difference in the size of the restriction fragments seen in normal and mutant samples
can be predicted from a restriction map of the amplified fragment and the site of the
mutation that changes a restriction site. The observed fragments should be consistent
with either the mutant or the normal pattern.

Allele-Specific Oligonucleotide Hybridization

Principle

Under appropriate conditions, short oligonucleotide probes will hybridize to their exact
complementary sequence but not to a sequence where there is even a single base
mismatch. A pair of oligos is therefore used to test for the presence of a point mutation:
a mutant oligo complementary to the mutant sequence and a normal oligo
complementary to the normal sequence, with the sequence difference placed near the
centre of each oligo.

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Interpretation

The oligos should hybridise to their exact DNA sequence, such that the mutant oligo
gives a signal with a homozygous mutant control but not with a normal control. When
this is the case, the interpretation of the result is straightforward; a positive signal from
a particular oligo indicates the presence of that allele in the test sample. Heterozygotes
and homozygotes are distinguished by using the mutant and normal oligos in tandem.
Nonradioactive probes, with detection systems involving horseradish peroxidase, are
now quite widely used in this procedure.

INVESTIGATION OF HAEMOGLOBINOPATHIES

Sickle Cell Disease

There are occasions when it is beneficial to make this diagnosis by DNA analysis :

1. prenatal diagnosis, which can be performed at 10 weeks of pregnancy

2. in distinguishing HbS/S from HbS/β thalassaemia
3. in confirming the diagnosis of sickle cell anaemia in a neonate).

The sickle cell mutation in codon 6 of the β globin gene (GAG ➙ GTG) results in the loss
of a Bsu36 I (or Mst II, Sau I, OxaN I, or Dde I) restriction enzyme site that is present in
the normal gene. It is therefore possible to detect the mutation directly by restriction
enzyme analysis of a DNA fragment generated by the PCR. A pair of primers are used to
amplify exons 1 and 2 of the β globin gene, and the products of the PCR are digested
with Bsu36 I. The loss of a Bsu36 I site in the sickle cell gene gives rise to an abnormally
large restriction fragment that is not seen in normal individuals .

β Thalassaemia

Although more than 100 β thalassaemia mutations are known, each of these groups has
its own subset of mutations, so that as few as 5 different mutations may account for
more than 90% of the affected individuals in a population. This makes the direct
detection of β thalassaemia mutations a reasonable possibility, and it has become the
method of choice where it is most important—in prenatal diagnosis.

The majority of mutations causing β thalassaemia are point mutations affecting the
coding sequence, splice sites, or promoter of the β globin gene. Favoured methods for
their detection are either ARMS or reverse dot blot analysis. Larger deletions can be
identified directly from the size of the amplified product.

α Thalassaemia

In contrast to the β thalassaemias, the most common α thalassaemia mutations are

deletions. Two categories exist: those that remove only one of the two alpha globin
genes on any one chromosome (α+ thalassaemia) and those that remove both of the
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alpha genes from one chromosome (α0 thalassaemia), the common deletions can now
be identified by a reasonably robust Gap-PCR.

DISORDERS OF COAGULATION

Thrombophilia

Considerable advances have been made in our understanding of the genetic risk factors
found in patients with venous thromboembolism (VTE). Among these are the diverse
mutations causing protein C, protein S, and antithrombin deficiency. The most common
of the known genetic risk factors for VTE is a resistance to the anticoagulant effect of
activated protein C caused by the Arg506Gln substitution in factor V (FVL); around
20% of subjects of north European origin presenting for the irst time with
thromboembolism are heterozygous for this mutation. Because of their prevalence, and
because the tests have become relatively simple, there is a tendency toward
indiscriminate testing for these genetic risk factors in thrombophilia, but without
careful and informed counselling this may often be inappropriate

Interpretation

The factor V primer pair gives rise to a fragment of 181 bp; after Taq I cleavage, the
normal gene (1691G) gives rise to fragments of 157 bp and 24 bp, whereas the mutant
gene (1691A) remains uncut at 181 bp. Although the 24 bp fragment is not easily
detected, the 157 bp and 181 bp fragments are clearly resolved on a 3% agarose gel
When only the smaller fragment is seen, the sample is normal (1691 G/G); when both
the smaller and larger fragments are seen, the sample is heterozygous for FVL (1691
A/G); when only the larger fragment is seen, the sample is homozygous for FVL (1691
A/A). The same principle applies for the prothrombin fragment, with the normal gene
(20210G) being cut by Taq I to 98 bp and the mutant gene (20210A) remaining uncut at
118 bp. The Taq I enzyme is relatively robust, and partial digestion—the only potential
pitfall in this analysis—is avoided by using a significant excess of enzyme. The presence
of both the factor V and prothrombin fragments in the same tube controls for the
efficacy of the restriction enzyme because one of these is usually normal and therefore
shows complete digestion. The analysis only requires confirmation when an individual
appears to have both the prothrombin and factor V mutations.

Clotting Disorders

Diverse mutations underlie haemophilia A and haemophilia B, and these are usually
identified by screening exons for mutation by single-strand conformation
polymorphism (SSCP) technique, denaturing high-performance liquid chromatography,
or direct DNA sequence analysis. It may still be relevant to determine carrier status and
offer prenatal diagnosis through genetic linkage analysis.

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LEUKAEMIA AND LYMPHOMA

Translocations, Molecular Analysis, and Minimal Residual Disease

Frequently, the breakpoints within the translocation are too widely distributed to allow
direct amplification of DNA by PCR. In such cases, the mRNA from the fusion gene can
be reverse transcribed using RT to yield cDNA, which can than be amplified by PCR. In
addition, RT-PCR is an exquisitely sensitive tool that has been exploited in the detection
of residual disease.

BCR–ABL Reverse Transcriptase-Polymerase Chain Reaction

Principle and Interpretation

The BCR–ABL analysis is performed by two-stage RT-PCR. The RNA extracted from
nucleated cells is reverse transcribed by RT to generate coding or cDNA using random
primers 6 bp long, or hexamers. Following the RT step, the samples are subjected to
multiplex PCR, to test for the presence or absence of BCR–ABL. Multiplex PCR is similar
to conventional PCR but includes more than one pair of primers in a single PCR test.
This strategy enables the detection of the vast majority of the BCR–ABL transcripts. The
most commonly observed transcripts are b3a2, b2a2, and e1a2, giving rise to 385, 310,
and 481 bp amplicons, respectively

If BCR–ABL is undetectable by multiplex PCR in follow-up samples from patients

undergoing therapy, the cDNA is tested at higher level of sensitivity by nested PCR.
Nested PCR enables the detection of one leukaemic cell in a background of 105 to 106
normal cells. The choice of primers for nested PCR is dependent on the type of
transcript detected by multiplex PCR at presentation. The primers for b3a2 and b2a2
are the same; however, for e1a2 transcript a different set of primers is used. Nested
PCR is not indicated for testing cDNA from a patient suspected of having CML.
Diagnosis is made by expression of BCR-ABL by multiplex PCR. Furthermore, in post-
therapy samples this will indicate molecular relapse.

Monitoring Minimal Residual Disease

Effective clinical management of haematological malignancies depends on accurate and

precise measurement of a patient's response to therapeutic agents. This includes
examination of cellular morphology in peripheral blood and marrow specimens.
Although these studies are essential, they lack sensitivity; therefore the malignant clone
has frequently advanced to levels at which it is refractory to further treatment before
relapse is recognised. The last 3 decades have seen a remarkable advance in the
development of technology to monitor patients' response to therapy to a sensitivity of 1
× 10-5 (i.e., the detection of one malignant cell in a background of 100,000 normal cells).
To enable this, a disease-specific marker is essential, as illustrated by targeting the
novel fusion gene BCR–ABL, which maps to the Philadelphia chromosome associated
with CML. The principle aim is to detect and measure minimal residual disease (MRD)
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using the most sensitive techniques available to a clinical laboratory with accuracy and
precision and thus recognise early signs of relapse. The clinical utility of such studies
has been amply confirmed by close and regular measurement of MRD in patients with
CML. This is increasingly being shown to apply to other adult and childhood leukaemias
using disease-specific markers.

The introduction of quantitative real-time PCR (QR-PCR) at the end of the last
millennium made quantification of MRD more widely accessible.

Principle

QR-PCR permits quantification of number of transcripts of gene of interest at high

levels of sensitivity. This is achieved by developing the technology that permits the
detection of PCR products as they accumulate. Furthermore, the rate of accumulation is
proportional to the number of mRNA molecules of the target gene in the starting
material during the exponential phase of the PCR. The accumulation of amplicons is
detected by including a sequence-specific specific probe labelled with fluorochromes in
addition to the primers as in conventional PCR. Since the advent of QR-PCR several
types of probes have been developed, although all are dependent on the fluorescence
resonance energy transfer (FRET) principle. The two commonly used systems involve
hybridisation or hydrolysis of the probe. A widely used methodology is TaqMan, which
involves hydrolysis of the probe. This technology is based on the 5′exonuclease assay,
which exploits the inherent 5′ to 3′ exonuclease activity of the Taq DNA polymerase.
The Taq DNA polymerase cleaves a dual-labelled probe annealed to the target sequence
during PCR amplification . Briefly, the cDNA synthesised from total RNA is added to the
PCR reaction containing standard PCR components plus a probe that anneals to the
template between the two primers as per conventional PCR. The probe has a
fluorescent reporter dye, and quencher dye. While the probe is intact, the proximity of
the quencher greatly reduces the fluorescence emitted by the reporter dye by Forster
resonance energy transfer (FRET) through space. Adequate quenching is observed for
probes with the reporter dye at the 5′ end and the quencher at the 3′ end.

The number of cycles taken to achieve this is called the cycle threshold (Ct), and it is
inversely proportional to the starting material of the target. The number of transcripts
of the target is read off a standard curve. The target gene and endogenous control gene
transcripts are measured off the respective standard curves. The level of expression of
the target gene is reported as a percentage ratio to obtain a normalised value for the
gene of interest independent of the integrity of the RNA and efficiency of the reverse
transcription reaction.

Interpretation

Patients who achieve complete cytogenetic remission may harbour up to 1 × 1010

leukaemic cells. The kinetics of the leukaemic load in these patients and their response
to therapeutic agents can only be monitored by measuring MRD. Although it should be
noted that patients in whom QR-PCR fails to detect BCR–ABL may still harbour up to 1 ×
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106 leukaemic cells. Investigators have also reported that patients who achieve BCR–
ABL:ABL ratio <0.045% while receiving α-interferon therapy have a considerably
reduced risk of cytogenetic relapse, and therefore it might be possible to reduce or
withdraw chemotherapy from these individuals. Thus with close regular monitoring of
MRD it is possible to reduce or avoid exposing patients to toxic drugs unnecessarily.
More recently, Q-PCR has been used to monitor patients being treated with imatinib
mesylate. Early results from an international multicentre study indicate that a 3 log
reduction in BCR–ABL copies is consistent with a good prognosis. Quantification of
BCR–ABL also helps to identify patients at risk of relapse, and therefore provides a
window for early clinical intervention with the aim of reversing disease progression.
For patients who have undergone stem cell transplantation (SCT) this invariably means
infusion of lymphocytes isolated from the original donor (i.e., adoptive
immunotherapy). there is little doubt that a con irmed 1 log increase in BCR–ABL
transcripts is clinically signi icant (i.e., from 0.002% to 0.02%). Patients who achieve a
0.02% BCR–ABL:ABL ratio on three consecutive occasions are said to be in molecular
relapse.

Advantages of QR-PCR

The major advantage of QR-PCR is the ability to detect accumulation of amplicons

during the exponential phase of PCR. This permits quantification of the DNA of interest
in the starting material. This is not possible with conventional PCR because samples are
analysed at the end of the PCR run and therefore any differences in the copy number
between samples in the starting material are generally not discernable. Post-PCR
handling is eliminated, on completion of an assay, the sealed micro-titre plate is
discarded and thereby minimising risk of contamination. This also eliminates the need
to handle carcinogenic ethidium–bromide–stained gel on completion of QR-PCR assay.
The QR-PCR offers higher level of specificity because, in addition to primers annealing
to DNA sequence of interest, a third oligonucleotide (the probe) anneals to the region
between the primers at a higher temperature. Conventional PCR is also less amenable
to automation. More importantly, because QR-PCR can be automated, interlaboratory
standardisation becomes feasible when measuring patients' response to therapy. This
permits rapid evaluation of new therapeutic modalities because methodology and
protocols can be standardised, permitting international interlaboratory studies.

The Lymphoproliferative Disorders

DNA analysis useful in LPD in:

1. monitoring residual disease

2. certain cases in which the diagnosis is ambiguous, genetic techniques may be
useful. Examples include cases of controversial lineage, lymphomas in which the
histology is ambiguous, and occult lymphomas.
3. DNA analysis may also help in determining whether a lymphocytosis is
monoclonal, oligoclonal, or polyclonal.

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Translocations do occur in these disorders and may be used in monitoring disease, as
described for CML earlier. However, the most commonly used markers, because they
are more universally applicable, are the rearranged immunoglobulin (Ig) and TCR
genes.

Principle

This analysis is possible because the Ig and TCR genes undergo a rearrangement during
the normal differentiation of B and T lymphocytes, respectively, but not during
differentiation of other cells. This rearrangement results in a unique fusion of variable
diversity and joining (VDJ) segments, interdigitated by random nucleotide (N region)
insertion or deletion. The sequence and length of the DNA at these sites of
recombination are therefore characteristic of any particular lymphocyte clone.

More recently, because of its simplicity, the small amount of DNA required, and
potential sensitivity, PCR has been used to detect rearrangement of the Ig and TCR
genes. Because of the N region diversity, a polyclonal population of cells will give rise to
a ladder of various fragment sizes; however, if one clone becomes abnormally large, a
discrete fragment size will begin to dominate the products of the PCR—the basis of the
so-called “fingerprinting” method for the diagnosis of lymphoproliferative disorders. To
gain further sensitivity in following disease, the product of a “clonal” amplification can
be sequenced to derive a clone-specific sequence at the site of rearrangement. This
sequence can then be used for the design of clone-specific oligonucleotide probes or
primers that can be used in ASOH, ARMS, or real-time PCR. This methodology has been
used to monitor MRD in lymphoproliferative disorders.

HOST-DONOR CHIMERISM STUDIES

Following allogeneic stem cell transplantation it is important to monitor the

engraftment of donor cells in the host. This can be achieved in a number of ways, one of
which is the use of DNA markers. The method of choice is the PCR amplification of STR
loci, which, because of their highly polymorphic nature, are likely to give informative
differences between any host-donor pair.

Principle

Provided the ampli ication cycle number is kept reasonably low (25 cycles) the PCR
reaction is semi-quantitative, and the amount of product will reflect the amount of
starting material. Therefore, once an informative difference is established, the amount
of PCR product of the different host and donor alleles will reflect the proportion host
and donor DNA in a sample.

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GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Most of the polymorphic variants of glucose-6-phosphate dehydrogenase (G6PD) that

are associated with acute haemolysis have been defined at the molecular level, and it
may be important to distinguish these from a G6PD-deficient variant that is the cause of
chronic haemolysis. Because it is an X-linked disorder, it may be difficult to identify
heterozygous females, in whom levels of enzyme activity may vary from reduced to
normal. This is particularly important in female relatives of men with chronic
haemolytic anaemia caused by G6PD de iciency. Furthermore, G6PD de iciency can be
masked by reticulocytosis in individuals with acute haemolysis (particularly in
individuals with an A-variant) and in neonates.

In such cases, it may be bene icial to test for a G6PD-deficient variant by DNA analysis.
Although more than 100 G6PD mutations have now been described, some of the most
common deficient variants are readily diagnosed by restriction enzyme digestion of the
appropriate PCR products.

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