PREIMPLANTATION GENETIC DIAGNOSIS (PGD): APPLICATION OF THE
"MINISEQUENCING METHOD"
Francesco Fiorentino1, Cristina Magli2, Daniele Podini1, Andrea Nuccitelli1, Anna P. Ferraretti2, Moncef
BenKhalifa3 Marina Baldi3,Luca Gianaroli2.
1
"GENOMA" Molecular Genetics Laboratory, Rome - Italy; 2 S.I.S.ME.R. - Reproductive Medicine Unit
- Bologna - Italy; 3Consultorio di Genetica, Rome - Italy
OBJECTIVE
Preimplantation Genetic Diagnosis (PGD) is presently a valid alternative for couples at high risk of
pregnancies with genetic anomalies. The intrinsic difficulties of this diagnostic approach require the use
of extremely sensitive and automatized techniques that provide unambiguous and reliable results.
Techniques involving gel analysis are used for mutation screening in the majority of PGD cases to detect
the presence or absence of restriction sites, electrophoretic mobility shift, as in Denaturing Gradient Gel
Electrophoresis, or Heteroduplex analysis. Such techniques may be sensitive, but are unwielding , labour
intensive and often need to be validated properly.
Mutation analysis by means of sequencing of the genetic regions of interest is, at present, a better method
of genetic analysis, since it permits the exact determination and direct visualization of a specific mutation.
While this technique guarantees the maximum interpretative trustworthiness its application in PGD
analysis is time expensive and moreover, requires good quality amplification products.
In order to overcome some of these limitation, especially when dealing with larger blastomere numbers,
we have applied a new method of genetic analysis, called "ddNTP Primer Extension" or
"Minisequencing", to PGD of monogenic disorders. This technique permits the exact determination and
direct visualization of a specific mutation without direct sequencing, while still maintaining the same
qualitative characteristics of sequence analysis.
In this work, we present the results obtained by the application of the “minisequencing” method in the
PGD of different genetic pathologies, such as Cystic Fibrosis,  Thalassemia, Hemophilia A, and
Retinoblastoma. In order to evaluate the reliability of this method, 245 blastomeres were analyzed
simultaneously with both traditional automated sequence analysis and with the “minisequencing” method.
MATERIALS AND METHODS
Each blastomere first underwent cell lysis (65°C for 10’) by the addition of an alkaline lysis buffer to
each test tube. The PCR strategy consisted in an initial multiplex external amplification followed by
nested PCR, specific for each region concerned by mutations. After EXOI/SAP purification, PCR
products were analyzed both by direct sequencing and “minisequencing” methods, followed by capillary
electrophoresis on automatic DNA sequencer ABI PRISM 310. In minisequencing, a primer extension
reaction is performed, starting from a specific primer that is designed to anneal directly adjacent to the
mutation site, by the incorporation of a single fluorescent ddNTP which is complementary to the variant
base in the template. This process is repeated in successive rounds of extension and termination to
generate the fluorescently labeled fragment for analysis, that is resolved and detected by capillary
electrophoresis. Thus, the mutation site can reliably be differentiated between homozygotic wild type,
homozygotic mutant or heterozygote, by the dye labeled ddNTP incorporated.
RESULTS AND CONCLUSIONS
Analysis of 245 blastomeres, carried out simultaneously with both traditional automated sequence
analysis and “minisequencing”, evidenced perfectly overlapping data. “Minisequencing” has proved to be
a very useful method in PGD analysis, due to its elevated sensitivity, automation, and easy data
interpretation. The method is extremely efficient, providing interpretable results in 97.9% of the cases
(240/245) of the blastomeres investigated, with respect to 89.8% (220/245) obtained with sequence
analysis (P<0.001). Moreover, the possibility of simultaneous investigation of more than one mutation
site ("multiplexing") significantly reduces time for analysis (9h for minisequencing with respect to 15h
for sequence analysis).
Our results indicate that “minisequencing” method, in combination with automated analysis, is a reliable,
sensitive, efficient and easy-to-approach analysis, applicable to PGD on a single cell.