C h a p t e r 5 

Laboratory Techniques
for Diagnosis of
Monogenic Disorders

In the history of medical genetics, the ‘chromosome break- PCR allows analysis of DNA from any cellular source con-
through’ in the mid-1950s was revolutionary. In the past 4 taining nuclei; in addition to blood, this can include less invasive
decades, DNA technology has had a profound effect, not only samples, such as saliva, buccal scrapings, or pathological archival
in medical genetics but also in many areas of biological science material. It is also possible to start with quantities of DNA as
(Box 5.1).The seminal developments in the field are summa- small as that from a single cell, as is the case in pre-implantation
rized in Table 5.1. One of the most revolutionary developments genetic diagnosis (p. 313). Great care has to be taken with
is the technique first developed in the mid-1980s known as the PCR, however, because DNA from a contaminating extraneous
polymerase chain reaction or PCR which can be used to source, such as desquamated skin from a laboratory worker,
produce vast quantities of a target DNA fragment provided will also be amplified. This can lead to false-positive results
that the DNA sequence of that region is known. unless the appropriate control studies are used to detect this
possible source of error.
PCR (Polymerase Chain Reaction) Another advantage of PCR is the rapid turnaround time of
samples for analysis. Use of the heat-stable Taq DNA poly-
DNA sequence information is used to design two oligonucleo­ merase isolated from the bacterium Thermophilus aquaticus,
tide primers (amplimers) of approximately 20 bp in length which grows naturally in hot springs, generates PCR products
complementary to the DNA sequences flanking the target in a matter of hours. Real-time PCR machines have reduced
DNA fragment. The first step is to denature the double- this time to less than 1 hour, and fluorescence technology is
stranded DNA by heating. The primers then bind to the used to monitor the generation of PCR products during each
complementary DNA sequences of the single-stranded DNA cycle, thus eliminating the need for gel electrophoresis.
templates. DNA polymerase extends the primer DNA in
the presence of the deoxynucleotide triphosphates (dATP,
dCTP, dGTP, and dTTP) to synthesize the complementary Application of DNA Sequence
DNA sequence. Subsequent heat denaturation of the double- Polymorphisms
stranded DNA, followed by annealing of the same primer There is an enormous amount of DNA sequence variation in
sequences to the resulting single-stranded DNA, will result in the human genome (p. 9). Two main types, SNPs and hyper-
the synthesis of further copies of the target DNA. Some 30–35 variable tandem repeat DNA length polymorphisms, are pre-
successive repeated cycles result in more than 1 million copies dominantly used in genetic analysis.
(amplicons) of the DNA target, sufficient for direct visualiza-
tion by ultraviolet fluorescence after ethidium bromide stain- Single Nucleotide Polymorphisms
ing, without the need to use indirect detection techniques Approximately 1 in 1000 bases within the human genome
(Figure 5.1). PCR is mostly used to amplify DNA fragments shows variation. SNPs are most frequently biallelic and occur
up to 1 kb, although long-range PCR allows the amplification
of larger DNA fragments of up to 20 kb to 30 kb.
Table 5.1  Development of DNA Technology
Decade Development Examples of Application
Box 5.1 Applications of DNA Technology 1980s Recombinant DNA Recombinant erythropoietin
technology, (1987), DNA
Gene structure/mapping/function Southern blot, fingerprinting (1984),
Population genetics and Sanger and DNA sequence of
Clinical genetics sequencing Epstein–Barr virus
Preimplantation genetic diagnosis genome (1984)
Prenatal diagnosis 1990s Polymerase chain Diagnosis of genetic
Presymptomatic diagnosis reaction (PCR) disorders
Carrier detection 2000s Capillary sequencing Human genome sequence
Diagnosis and pathogenesis of disease and microarray (2003)
Genetic technology
Acquired—infective, malignant 2010s Next-generation First acute myeloid leukemia
Biosynthesis (e.g., insulin, growth hormone, interferon, sequencing (AML) cancer genome
immunization) sequenced (2008)
Treatment of genetic disease Human genome sequenced
Gene therapy at a cost of approx.
Agriculture (e.g., nitrogen fixation) $1000 (2014)

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