Preclinical models for human pre-embryo biopsy and genetic diagnosis. II. Polymerase chain reaction amplification of deoxyribonucleic acid from single lymphoblasts and blastomeres with mutation detection.
OBJECTIVE: To demonstrate the use of the polymerase chain reaction in the amplification of deoxyribonucleic acid (DNA) from single human lymphoblasts and mouse blastomeres. Amplified target genes for diagnosis of sickle cell anemia and Tay-Sachs are shown. Similarly, the sparce fur mouse model for ornithine transcarbamylase deficiency was used as an X-linked system for demonstration of mutation detection after biopsy of a single blastomere. A new diagnostic method for the detection of the ornithine transcarbamylase mutation using the restriction enzyme Mse I is presented. Accuracy and reproducibility were assured. DESIGN: Polymerase chain reaction proficiency test for amplification from single cells was studied. Also, accuracy of mutation detection systems was demonstrated. SETTING: Laboratories of The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. PATIENTS, PARTICIPANTS: We used the sparse fur mouse model and human blood cells. INTERVENTIONS: Pre-embryo biopsy, polymerase chain reaction amplification, and mutation detection were performed. MAIN OUTCOME MEASURES: Accuracy and reproducibility of DNA amplification without contamination, as well as efficient diagnostic analysis, from both single somatic and embryonic cells were shown. RESULTS: DNA amplification from single cells was uniformly rapid (6 to 10 hours) reproducible (n = 220) and accurate (n = 52). CONCLUSIONS: Our findings support the feasibility of clinical application for pre-embryo biopsy and genetic diagnosis of specific heritable diseases.