With the huge increase in knowledge of the human genome
and its DNA sequence, growing numbers of disease genes can
now be examined using DNA analysis. Few laboratory tests at
the disposal of the modern clinician have the potential
specificity and information content of these techniques. Only a
few years ago, DNA analysis was mainly applicable to
presymptomatic diagnosis of inherited conditions and the
detection of carriers following initial diagnosis of the patient by
more conventional laboratory tests (e.g. biochemical and
histological). In current practice, the DNA laboratory has an
increasing role in the initial diagnosis of many diseases
by analysis of specific genes associated with mendelian
disorders.
Saturday, April 11, 2009
molecular genetics laboratories
Over 20 regional molecular genetics laboratories provide a
service to the regions of the UK with many additional
laboratories providing genetic tests in areas such as
mitochondrial disease and haemoglobinopathies. The following
chapter summarises the standard techniques of DNA analysis
employed by molecular laboratories for the provision of
services to the clinician.
service to the regions of the UK with many additional
laboratories providing genetic tests in areas such as
mitochondrial disease and haemoglobinopathies. The following
chapter summarises the standard techniques of DNA analysis
employed by molecular laboratories for the provision of
services to the clinician.
DNA extraction
Genomic DNA is usually isolated from EDTA-anticoagulated
whole blood, often using an automated method. In addition,
DNA can also be readily isolated from fresh or frozen tissue
samples, chorionic villus biopsies, cultured amniocytes and
lymphoblastoid cell lines. Smaller quantities of DNA can be
recovered from buccal mouthwash samples and fixed
embedded tissues, although the recovery is considerably less
reliable. The increased use of the polymerase chain reaction
(PCR) means that for a small proportion of analyses, blood
volumes of 1 ml are adequate. In many instances however,
larger volumes of blood are still required because numerous
tests are required when analysing large or multiple genes and
not all tests use PCR based methods of analysis.
whole blood, often using an automated method. In addition,
DNA can also be readily isolated from fresh or frozen tissue
samples, chorionic villus biopsies, cultured amniocytes and
lymphoblastoid cell lines. Smaller quantities of DNA can be
recovered from buccal mouthwash samples and fixed
embedded tissues, although the recovery is considerably less
reliable. The increased use of the polymerase chain reaction
(PCR) means that for a small proportion of analyses, blood
volumes of 1 ml are adequate. In many instances however,
larger volumes of blood are still required because numerous
tests are required when analysing large or multiple genes and
not all tests use PCR based methods of analysis.
Genomic DNA
Genomic DNA remains stable for many years when frozen.
This enables storage of samples for future analysis of genes that
are not yet isolated, and is crucial when organising the
collection of DNA samples for long term studies of inherited
conditions.
This enables storage of samples for future analysis of genes that
are not yet isolated, and is crucial when organising the
collection of DNA samples for long term studies of inherited
conditions.
The polymerase chain reaction (PCR)
The use of PCR in the analysis of an inherited condition was
first demonstrated in the detection of a common -globin
mutation in 1985. Since then, PCR has become an
indispensable technique for all laboratories involved in DNA
analysis. The technique requires the DNA sequence in the gene
or region of interest to have been elucidated. This limitation is
becoming increasingly less problematic with the pending
completion of the entire human DNA sequence.
first demonstrated in the detection of a common -globin
mutation in 1985. Since then, PCR has become an
indispensable technique for all laboratories involved in DNA
analysis. The technique requires the DNA sequence in the gene
or region of interest to have been elucidated. This limitation is
becoming increasingly less problematic with the pending
completion of the entire human DNA sequence.
advantage of the PCR
The main advantage of the PCR method is that the regions
of the gene of interest can be amplified rapidly using very small
quantities of the original DNA sample. This feature makes the
method applicable in prenatal diagnosis using chorionic villus
or amniocentesis samples and in other situations in which
blood sampling is not appropriate.
of the gene of interest can be amplified rapidly using very small
quantities of the original DNA sample. This feature makes the
method applicable in prenatal diagnosis using chorionic villus
or amniocentesis samples and in other situations in which
blood sampling is not appropriate.
genomic DNA
In practice, because of the way genomic DNA is organised
into coding sequences (exons) separated by non-coding
sequences (introns), analysis of even a small gene usually
involves multiple PCR amplifications. For example, the breast
cancer susceptibility gene, BRCA1, is organised into 24 exons,
with mutations potentially located in any one of them. Analysis
of BRCA1 therefore necessitates PCR amplification of each
exon to enable mutation analysis.
into coding sequences (exons) separated by non-coding
sequences (introns), analysis of even a small gene usually
involves multiple PCR amplifications. For example, the breast
cancer susceptibility gene, BRCA1, is organised into 24 exons,
with mutations potentially located in any one of them. Analysis
of BRCA1 therefore necessitates PCR amplification of each
exon to enable mutation analysis.
Post-PCR analysis
It should be noted that the PCR process itself is usually merely
a starting point for an investigation by providing a sufficient
quantity of DNA for further analysis. After completion of
thermal cycling, the first step in analysis is to determine the
success of amplification using agarose gel electrophoresis
(AGE). The DNA is separated within the gel depending on its
size; large DNA molecules travel slowly through the gel in
contrast to small DNA molecules that travel faster. The DNA is
detected within the gel with the use of a fluorescent dye
(ethidium bromide) as a pink fluoresent band when
illuminated by ultraviolet light. By varying the agarose
concentration in the gel, this approach can be used for the
analysis of PCR products from less than 100 to over 10 000 base
pairs in size.
a starting point for an investigation by providing a sufficient
quantity of DNA for further analysis. After completion of
thermal cycling, the first step in analysis is to determine the
success of amplification using agarose gel electrophoresis
(AGE). The DNA is separated within the gel depending on its
size; large DNA molecules travel slowly through the gel in
contrast to small DNA molecules that travel faster. The DNA is
detected within the gel with the use of a fluorescent dye
(ethidium bromide) as a pink fluoresent band when
illuminated by ultraviolet light. By varying the agarose
concentration in the gel, this approach can be used for the
analysis of PCR products from less than 100 to over 10 000 base
pairs in size.
presence or absence of a PCR product
As well as showing the presence or absence of a PCR
product, an agarose gel can also be used to determine the size
of the product. In some instances, agarose gel electrophoresis
alone is sufficient to demonstrate that a mutation is present.
For example, a 250 base pair PCR product containing a
deletion mutation of 10 bases will be readily detected by
agarose gel electrophoresis. Determining the exact position of
the deletion, however, requires additional analysis.
product, an agarose gel can also be used to determine the size
of the product. In some instances, agarose gel electrophoresis
alone is sufficient to demonstrate that a mutation is present.
For example, a 250 base pair PCR product containing a
deletion mutation of 10 bases will be readily detected by
agarose gel electrophoresis. Determining the exact position of
the deletion, however, requires additional analysis.
Agarose gel electrophoresis
Agarose gel electrophoresis is of sufficient resolution to
allow the rapid detection of the deletion of whole exons, which
is often seen in affected male DMD patients. In this approach,
a number of exons of the DMD gene are simultaneously
amplified in a “multiplex” PCR approach. Samples with exon
deletions are readily detected by the absence of specific bands
when analysed by agarose gel electrophoresis.
allow the rapid detection of the deletion of whole exons, which
is often seen in affected male DMD patients. In this approach,
a number of exons of the DMD gene are simultaneously
amplified in a “multiplex” PCR approach. Samples with exon
deletions are readily detected by the absence of specific bands
when analysed by agarose gel electrophoresis.
analysis of PCR products
For analysis of PCR products below 1000 bp, polyacrylamide
gel electrophoresis is often used, which allows separation of
DNA molecules that differ from each other in size by only a
single base. The DNA can be detected in the gel by a variety of
methods including ethidium bromide staining and silver
staining however, many laboratories now use fluorescently
tagged primers to generate labelled PCR products that can be
visualised by laser-induced fluorescence. It is this technology
that has been developed into the high-throughput DNA
sequencing instruments that have been the workhorses of the
Human Genome Sequencing Project.
gel electrophoresis is often used, which allows separation of
DNA molecules that differ from each other in size by only a
single base. The DNA can be detected in the gel by a variety of
methods including ethidium bromide staining and silver
staining however, many laboratories now use fluorescently
tagged primers to generate labelled PCR products that can be
visualised by laser-induced fluorescence. It is this technology
that has been developed into the high-throughput DNA
sequencing instruments that have been the workhorses of the
Human Genome Sequencing Project.
Sequence-specific amplification
One of the properties of the short synthetic pieces of DNA
(oligonucleotides) used as primers in PCR is their sequence
specificity. This can be exploited to design PCR primers that
only generate a product when they are perfectly matched to
their target sequence. Conversely, a mismatch in the region of
sequence where the primer binds, prevents PCR amplification
from proceeding. In this way, an assay can be designed to
detect the presence or absence of specific known mutations.
This approach (known as ‘ARMS’ or Amplification Refractory
Mutation System) is often used to detect common cystic fibrosis
mutations and certain mutations involved in familial breast
cancer.
(oligonucleotides) used as primers in PCR is their sequence
specificity. This can be exploited to design PCR primers that
only generate a product when they are perfectly matched to
their target sequence. Conversely, a mismatch in the region of
sequence where the primer binds, prevents PCR amplification
from proceeding. In this way, an assay can be designed to
detect the presence or absence of specific known mutations.
This approach (known as ‘ARMS’ or Amplification Refractory
Mutation System) is often used to detect common cystic fibrosis
mutations and certain mutations involved in familial breast
cancer.
Oligonucleotide ligation assay (OLA)
In the OLA reaction, two oligonucleotide probes are hybridised
to a DNA sample so that the 3 terminus of the upstream oligo
is adjacent to the 5 terminus of the downstream oligo. If the 3
terminus of the first primer is perfectly matched to its target
sequence, then the probes can be joined together with a DNA
ligase. In contrast no ligation can occur if there is a mismatch
at the 3 terminus of the first oligo. This approach has been
successfully applied to the detection of 31 common mutations
in cystic fibrosis with a commercial kit, and for the detection of
19 common mutations in the LDL receptor gene in
hypercholesterolaemia.
to a DNA sample so that the 3 terminus of the upstream oligo
is adjacent to the 5 terminus of the downstream oligo. If the 3
terminus of the first primer is perfectly matched to its target
sequence, then the probes can be joined together with a DNA
ligase. In contrast no ligation can occur if there is a mismatch
at the 3 terminus of the first oligo. This approach has been
successfully applied to the detection of 31 common mutations
in cystic fibrosis with a commercial kit, and for the detection of
19 common mutations in the LDL receptor gene in
hypercholesterolaemia.
Restriction enzyme analysis of PCR products
Restriction endonuclease enzymes are produced naturally by
bacterial species as a mechanism of protection against “foreign”
DNA. Each enzyme recognises a specific DNA sequence and
cleaves double-stranded DNA at this site. Hundreds of these
restriction enzymes are now commercially available and provide
a rapid and reliable method of detecting the presence of a
specific DNA sequence within PCR products. This property
becomes especially relevant when a mutation either creates or
destroys the enzyme’s recognition site. By studying the size of
the products that are generated following restriction enzyme
digestion of PCR-amplified DNA (by agarose gel
electrophoresis), it is possible to accurately determine the
presence or absence of a particular mutation.
bacterial species as a mechanism of protection against “foreign”
DNA. Each enzyme recognises a specific DNA sequence and
cleaves double-stranded DNA at this site. Hundreds of these
restriction enzymes are now commercially available and provide
a rapid and reliable method of detecting the presence of a
specific DNA sequence within PCR products. This property
becomes especially relevant when a mutation either creates or
destroys the enzyme’s recognition site. By studying the size of
the products that are generated following restriction enzyme
digestion of PCR-amplified DNA (by agarose gel
electrophoresis), it is possible to accurately determine the
presence or absence of a particular mutation.
Single-stranded conformation polymorphism analysis (SSCP)
The principle of SSCP analysis is based on the fact that the
secondary structure of single-stranded DNA is dependent on its
base composition. Any change to the base composition
introduced by a mutation or polymorphism will cause a
modification to the secondary structure of the DNA strand.
This altered conformation affects its migration through a
non-denaturing polyacrylamide gel, resulting in a band shift
when compared to a sample without a mutation. The bands
of single-stranded DNA are usually visualised by silver-staining.
It should be noted that the presence of a band shift itself does
not provide any information about the nature of the mutation.
Consequently, samples that show altered banding patterns
require further investigation by DNA sequencing.
secondary structure of single-stranded DNA is dependent on its
base composition. Any change to the base composition
introduced by a mutation or polymorphism will cause a
modification to the secondary structure of the DNA strand.
This altered conformation affects its migration through a
non-denaturing polyacrylamide gel, resulting in a band shift
when compared to a sample without a mutation. The bands
of single-stranded DNA are usually visualised by silver-staining.
It should be noted that the presence of a band shift itself does
not provide any information about the nature of the mutation.
Consequently, samples that show altered banding patterns
require further investigation by DNA sequencing.
Heteroduplex analysis
Heteroduplexes are double-stranded DNA molecules that are
formed from two complementary strands that are imperfectly
matched. If a mutation is present in one copy of a gene being
amplified using PCR, heteroduplexes will be formed from
the hybridisation of the normal and the mutant PCR product.
As in SSCP analysis described above, these structures will have
altered mobility when analysed through non-denaturing
polyacrylamide gels, and are seen as band shifts when
compared to perfectly matched PCR products (or
homoduplexes).
formed from two complementary strands that are imperfectly
matched. If a mutation is present in one copy of a gene being
amplified using PCR, heteroduplexes will be formed from
the hybridisation of the normal and the mutant PCR product.
As in SSCP analysis described above, these structures will have
altered mobility when analysed through non-denaturing
polyacrylamide gels, and are seen as band shifts when
compared to perfectly matched PCR products (or
homoduplexes).
Denaturing gradient gel electrophoresis (DGGE)
The DGGE method relies on the fact that double-stranded
DNA molecules have specific denaturation characteristics, i.e.
conditions at which the double-stranded DNA disassociates into
its two single-stranded units. The denaturation of the DNA
strands can be achieved by increasing temperature or by the
addition of a chemical denaturant such as urea or formamide.
If a PCR product contains a mutation, this will subtly modify
the conditions at which denaturation occurs, which in turn
affects its electrophoretic mobility. In DGGE, a gradient of the
denaturing agent is set up so that the PCR products migrate
through the denaturant and are separated based on their
sequence specific mobility.
DNA molecules have specific denaturation characteristics, i.e.
conditions at which the double-stranded DNA disassociates into
its two single-stranded units. The denaturation of the DNA
strands can be achieved by increasing temperature or by the
addition of a chemical denaturant such as urea or formamide.
If a PCR product contains a mutation, this will subtly modify
the conditions at which denaturation occurs, which in turn
affects its electrophoretic mobility. In DGGE, a gradient of the
denaturing agent is set up so that the PCR products migrate
through the denaturant and are separated based on their
sequence specific mobility.
Denaturing HPLC (DHPLC)
While conventional SSCP and heteroduplex analysis use
polyacrylamide gel electrophoresis to separate PCR products,
DHPLC uses a high pressure system to force the products
through a column under partially denaturing conditions.
Conditions for optimum separation of normal and mutant
sequences are created by the use of buffer gradients and
specific temperatures. The DNA molecules that are
progressively eluted from the column are monitored by an
ultraviolet detector with data being collected by computer.
polyacrylamide gel electrophoresis to separate PCR products,
DHPLC uses a high pressure system to force the products
through a column under partially denaturing conditions.
Conditions for optimum separation of normal and mutant
sequences are created by the use of buffer gradients and
specific temperatures. The DNA molecules that are
progressively eluted from the column are monitored by an
ultraviolet detector with data being collected by computer.
Protein truncation test (PTT)
The key features of PTT are (i) that the analysis is based on the
protein product generated from the DNA sequence, and
(ii) the method specifically detects premature protein
truncation caused by non-sense mutations. The PCR product is
transcribed and translated in vitro by a reticulocyte lysate,
during which the nascent protein product is radiolabelled with
35S-labelled amino acids. The translation products are then
separated by polyacrylamide gel electrophoresis. Samples with
non-sense mutations are detected by their tendency to
generate smaller protein products than their normal
counterparts.
protein product generated from the DNA sequence, and
(ii) the method specifically detects premature protein
truncation caused by non-sense mutations. The PCR product is
transcribed and translated in vitro by a reticulocyte lysate,
during which the nascent protein product is radiolabelled with
35S-labelled amino acids. The translation products are then
separated by polyacrylamide gel electrophoresis. Samples with
non-sense mutations are detected by their tendency to
generate smaller protein products than their normal
counterparts.
Chemical and enzymatic cleavage of mismatch (CCM)
As outlined in previous sections, PCR products that contain
point mutations form hybrid molecules with their normal
counterparts known as heteroduplexes. The two DNA strands
in these heteroduplexes are perfectly matched except at the
site of the mutation, where base pairing cannot occur. These
mismatched sites can be recognised both by specific enzymes
and by chemicals such as osmium tetroxide and piperidine,
which cleave the DNA at the site of mismatch. This property
can therefore be used to detect mutations within a PCR
product by polyacrylamide gel electrophoresis to visualise the
cleavage products.
point mutations form hybrid molecules with their normal
counterparts known as heteroduplexes. The two DNA strands
in these heteroduplexes are perfectly matched except at the
site of the mutation, where base pairing cannot occur. These
mismatched sites can be recognised both by specific enzymes
and by chemicals such as osmium tetroxide and piperidine,
which cleave the DNA at the site of mismatch. This property
can therefore be used to detect mutations within a PCR
product by polyacrylamide gel electrophoresis to visualise the
cleavage products.
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