Rapid detection of the deltaF508 mutation in single cells using DHPLC: implications for preimplantation genetic diagnosis.
OBJECTIVE
Practice of preimplantation genetic diagnosis (PGD) requires efficient amplification and analysis techniques. We have tested Denaturing High Performance Liquid Chromatography (DHPLC) to screen the deltaF508 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/deltaF508--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 deltaF508 mutation in single cells and is therefore appropriate for clinical application of preimplantation genetic diagnosis of cystic fibrosis.
Girardet
Anne
A
Laboratoire de Génétique Moléculaire, Centre Hospitalo-Universitaire, Montpellier, France. girardet@igh.cnrs.fr
Cathala
Philippe
P
Claustres
Mireille
M
D002851:Chromatography, High Pressure Liquid; D003550:Cystic Fibrosis / Q000175:diagnosis*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D005260:Female; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008214:Lymphocytes; D009154:Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019836:Preimplantation Diagnosis
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator