Short Communication: Rapid Detection of the ΔF508 Mutation in Single Cells Using DHPLC: Implications for Preimplantation Genetic Diagnosis

摘要

>Purpose: Practice of preimplantation genetic diagnosis (PGD) requires efficient amplification and analysis techniques. We have tested Denaturing High Performance Liquid Chromatography (DHPLC) to screen the Δ F508 mutation in heterozygous single cells in order to assess its usefulness for PGD of cystic fibrosis.>Methods: One hundred and two single lymphocytes—including N/N and N/ΔF508—were studied. F508 locus was amplified by nested PCR followed by the analysis of PCR products by DHPLC in non-denaturing conditions.>Results: On the basis of PCR-amplified product analysis, total efficiency of amplification was 98.78% (101/102), and allele dropout (ADO) rate was 3.7% (3/81). For each sample, results were obtained in less than 4 min with high resolution.>Conclusions: DHPLC is a rapid and efficient technique to detect the ΔF508 mutation in single cells and is therefore appropriate for clinical application of preimplantation genetic diagnosis of cystic fibrosis.