For diseases involving a heterogeneous spectrum of mutations
identified, such as Cystic Fibrosis, -Thalassemia or
Haemophilia A, the development of a mutation-based PGD
strategy is not practical because it requires time and
resources for standardization of PCR protocols unique for
the specific mutations of interest. For these kinds of
monogenic diseases, the use of a diagnostic strategy capable
of detecting a wide spectrum of mutations and compound
genotypes is more feasible.
PGD lab conceived and implemented the innovation of using
Minisequencing technique for mutation detection on single
cells. This procedure is now widely used by most of the
centers performing PGD testing.
Minisequencing technique is based on the single
dideoxynucleotide extension of unlabelled oligonucleotide
primers. The primer extension reaction is performed starting
from the purified amplified target. Specific minisequencing
primers, which are exactly one base short of the mutation
sites, are used for each mutation under investigation.
Primers bind to the complementary templates in the presence
of fluorescent-labelled ddNTPs and AmpliTaq? DNA Polymerase.
The polymerase then adds a single ddNTP at the 3? end of
each primer, complementarily according to the sequence.
Since the reaction contains only template, primer, dye-labelled
ddNTP and does not include dNTPs, interruption of the
reaction occurs after only one incorporation of a dideoxy
terminator. This process is repeated in successive rounds of
extension and termination, thus the resulting products,
varying in colour, can then be analyzed by electrophoresis (Figure 1).
The colours of the final peaks are determined
by the specific genotype at this locus, making it possible
to identify the base variation. The mutation site can thus
be reliably differentiated between homozygous wild type and
mutant (one peak of a specific colour) or heterozygous. In
the latter case, two differently-coloured peaks occur in the
electropherogram, one derived from the normal base and the
other from the mutated base.
The minisequencing method involves the use of a
fluorescence-based DNA mutation analysis protocol. When
compared with conventional methods, fluorescence-based
protocols offer many distinct advantages for clinical PGD.
Firstly, fluorescence permits PGD sensitivity, as well as
accuracy and reliability, to be substantially increased
(Findlay et al., 1995 and 1998). Secondly, packaged computer
software allows easy data interpretation and management.
Furthermore, fluorescence-based systems are highly amenable
to multiplexing, which has great potential regarding
detection of multiple mutation sites. This strategy can be
accurate enough to distinguish different kinds of mutations,
such as single nucleotide substitution and small deletions
Minisequencing results of a Beta Thalassemia PGD case,
in which the mutations of interest were Cod.8 del-AA and Cod
The main benefit of minisequencing is the use of a mutation
analysis protocol based on a common procedure for each
mutation to be analyzed. This is very important for the PGD
of genetic diseases characterized by a heterogeneous
spectrum of mutations identified, such as Cystic Fibrosis,
B-Thalassemia or Haemophilia A. In fact, for this kind of
monogenic diseases, the use of a common procedure able to
detect a wide spectrum of mutations, and compound genotypes
becomes more feasible. The development of different PGD
protocols specific for each mutation to be analysed is thus
avoided. With the use of minisequencing for mutation
detection, the PGD strategy is the same for the diverse
clinical cases involving different mutations: external and
nested PCR, purification of PCR products, minisequencing
reaction and capillary electrophoresis. The only difference
between one PGD case and another that involves PCR
amplification of the same DNA region, but with a different
mutation panel, lies in the use of different minisequencing
primers that can be easily designed and whose application
does not require the need of extensive trial testing.
Another useful feature of minisequencing is that different
mutation sites can be investigated simultaneously
("multiplexing?), even if they are located in different
regions of the gene, depending on the number of primers used
in the minisequencing reaction. In multiplex reactions,
multiple primer/template combinations are used in a single
tube reaction format, and products are analyzed in a single
Establishing a diagnosis