Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling).
OBJECTIVE
Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis.


METHODS
Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined.


RESULTS
Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization.


CONCLUSIONS
Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.
Rechitsky
S
S
Reproductive Genetics Institute, Illinois Masonic Medical Center, Department of Obstetrics and Gynecology, Chicago 60657, USA.
Freidine
M
M
Verlinsky
Y
Y
Strom
C M
CM
D000483:Alleles; D053523:Amelogenin; D000755:Anemia, Sickle Cell / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D016477:Artifacts; D001483:Base Sequence; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D003746:Dental Enamel Proteins / Q000235:genetics; D005069:Evaluation Studies as Topic; D005240:Feasibility Studies; D005260:Female; D005347:Fibroblasts / Q000737:chemistry*; D005819:Genetic Markers; D005914:Globins / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007453:Iodide Peroxidase / Q000235:genetics; D008297:Male; D008832:Microchemistry; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D009394:Nephritis, Hereditary / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011296:Prenatal Diagnosis / Q000379:methods*; D017384:Sequence Deletion; D012743:Sex Preselection; D014842:von Willebrand Diseases / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D014841:von Willebrand Factor / Q000235:genetics
D053523:Amelogenin; C505032:CFTR protein, human; D017931:DNA Primers; D003746:Dental Enamel Proteins; D005819:Genetic Markers; D014841:von Willebrand Factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005914:Globins; D004247:DNA; D007453:Iodide Peroxidase