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Different Approaches for Noninvasive Prenatal Diagnosis of Genetic Diseases Based on PNA‐Mediated Enriched PCR
Author(s) -
GALBIATI SILVIA,
RESTAGNO GABRIELLA,
FOGLIENI BARBARA,
BONALUMI SARA,
TRAVI MAURIZIO,
PIGA ANTONIO,
SBAIZ LUCA,
CHIARI MARCELLA,
DAMIN FRANCESCO,
SMID MADDALENA,
VALSECCHI LUCA,
PASI FEDERICA,
FERRARI AUGUSTO,
FERRARI MAURIZIO,
CREMONESI LAURA
Publication year - 2006
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1368.018
Subject(s) - primer (cosmetics) , mutant , dna , microbiology and biotechnology , mutation , pyrosequencing , biology , nucleic acid , polymerase chain reaction , gene , allele , digital polymerase chain reaction , genetics , chemistry , organic chemistry
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.