Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.
The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.
Galbiati
Silvia
S
Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Restagno
Gabriella
G
Foglieni
Barbara
B
Bonalumi
Sara
S
Travi
Maurizio
M
Piga
Antonio
A
Sbaiz
Luca
L
Chiari
Marcella
M
Damin
Francesco
F
Smid
Maddalena
M
Valsecchi
Luca
L
Pasi
Federica
F
Ferrari
Augusto
A
Ferrari
Maurizio
M
Cremonesi
Laura
L
D004247:DNA / Q000097:blood; D004252:DNA Mutational Analysis; D030342:Genetic Diseases, Inborn / Q000175:diagnosis*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011296:Prenatal Diagnosis / Q000379:methods*; D017422:Sequence Analysis, DNA
D020135:Peptide Nucleic Acids; D004247:DNA