ClinicalBiochemistry,Vol. 29, No. 3, pp.
201-208, 1996
ELSEVIER 0009-9120(95)02022-5
Molecular Diagnosis of Genetic Diseases
M. FFRRARI, 1'2 L. CREMONESI, 2 P. CARRERA, 1 and P.A. BONINP
I. R. C. C. S. H S. Raffaele, 1Department of Laboratory Medicine and 2DIBIT - Clinical Molecular
Biology Laboratory, Milan, Italy
Introduction
D u r i n g the last 20 recombinant DNA tech-
nology has developc.dY~iarS'veryefficient and sensitive
techniques, useful in the study of molecular defects
in human diseases. DNA technology has allowed an
enormous increase of basic knowledge and has had a
great effect on diagnostics. The development of
DNA-based diagnostic l:ests is still in progress and it
will take time for experimental validation and stan-
dardization of protocols. However, their transfer-
ability to the clinical field will depend on other fac-
tors, such as simplicity of procedures, turn-around
time, and cost-effective testing.
The advent of molecular biology techniques has
expanded our ability to diagnose inherited diseases.
In fact, DNA analysis has 3 advantages: 1. The
analysis may be done, for example, on lymphocytes
and not only on the disease target cell; 2. it allows
carrier detection and, 3. it allows characterization of
the molecular defect.
Here, we report the basic methodologies that are
commonly applied to clinical molecular diagnostics.
Mutation detection methods
The spectrum of mutations found in the human
genome ranges from rearrangements involving large
parts or whole genes to single base substitutions.
Therefore, different approaches to mutation detec-
tion are required, because methods detecting large-
scale alterations will be insensitive to point muta-
tions. By combining different technologies, the most
appropriate diagnostic methods will be selected for
each disease.
Two main strategies are possible for genetic stud-
ies, depending on whether the sequence of the dis-
Correspondence: Maurizio Ferrari, M.D., Laboratorio
Centrale, via Olgettina, 60, 20132-Milano, Italy.
P r e s e n t e d at the Advanced Analytical Techniques Sym-
posium held in J e r u s a l e m , J u n e 1994.
M a n u s c r i p t received March 21, 1995; revised a n d ac-
cepted September 11, 1995.
CLINICAL BIOCHEMISTRY, VOLUME 29, JUNE 1996
Copyright© 1996The CanadianSocietyof ClinicalChemists
Printedin the USA.All rightsreserved
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eased gene is not yet available or the sequence is
already known: Southern blot analysis and in vitro
DNA amplification by polymerase chain reaction.
SOUTHERN/NORTHERN-BLOT HYBRIDIZATION
The method of Southern (1) has been the mile-
stone in molecular biology for the detection of either
large gene alterations or point mutations and poly-
morphisms altering a restriction site. This tech-
nique can be employed at the early stage of gene
characterization, even before complete sequencing is
achieved and only a restriction map is available.
In this procedure, DNA fragments originated by
restriction endonuclease digestion of whole DNA are
electrophoretically separated, according to size, on
agarose gel. The size-separated DNA molecules are
blotted onto a nylon filter by capillary action by a
high salt buffer that is passed through the gel from
a buffer-saturated paper sheet. The blotted nucleic
acids are then immobilized on the membrane by a
fixation step and hybridized to a radiolabelled
single-stranded DNA molecule, named probe, which
is complementary in sequence to the blot-trans-
ferred DNA band, or bands, to be detected. The sig-
nal corresponding to the probe hybridized to the re-
striction fragment containing the sequence of inter-
est is visualized by autoradiography. Southern blot
hybridization can be used to diagnose genetic dis-
eases resulting from mutations, either creating or
abolishing a restriction enzyme recognition site. Al-
ternatively, in case a gene has not yet been precisely
identified within a candidate region, inheritance of
the disease can be predicted by the inheritance of
polymorphic markers, provided that the diseased
gene and the markers are sufficiently close ("in link-
age") to each other on the same chromosome to be
inherited as groups. Restriction fragment length
polymorphisms (RFLPs) are useful markers for link-
age analysis. They consist of innocuous single
nucleotide changes altering a restriction enzyme
site so that an enzyme no longer cuts DNA at that
site; thus, producing a longer DNA fragment.
A modification of the same technique using mRNA
as starting material, named Northern blotting (2),
201