ENZYMATIC AMPLIFICATION OF SPECIFIC DEOXYRIBONUCLEIC-ACID SEQUENCES FROM SINGLE CELLS - EVALUATION OF A SIMPLIFIED AND RAPID METHOD FOR USEIN PREIMPLANTATION GENETIC DIAGNOSIS

Objective: To develop a simplified polymerase chain reaction (PCR) pro tocol on single cells for the purpose of preimplantation genetic diagn osis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene , La Jolla, CA), for reducing the time to complete PCR amplification. Design: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. Results: Amplification of the 149 bp se gment using fetal cells was 100% correct. Results on single lymphocyte s were concordant in all but one of the 15 male cases. However, 2 of t he 25 female cases were identified as male suggesting the occurrence o f DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negative s. No false positives were detected for female cells. Amplification us ing the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. Conclusion: These data show that this PCR assay performed directly, without DNA extraction or purification and withou t re-amplification is a practical and effective approach for amplifica tion of specific DNA sequences in single cells. Furthermore, the simpl ified PCR protocol significantly reduced the time to complete DNA ampl ification. The reduced time is expected to facilitate the management o f a routine program for preimplantation genetic diagnosis.