Establishment And Application Of Haplotype Analysis And Single Cell PCR In Spinal Muscular Atrophy

Objectives: 1.To optimize the prenatal diagnosis system of spinal muscular atrophy (SMA). 2.To establish the technique of single cell PCR. 3.To apply single cell PCR in the gene diagnosis of SMA. Methods: 1.PCR-PAGE was applied to analyze the polymorphisms of STR loci that tightlylinkage to survival motor neuron (SMN) gene. 2.PCR-restriction enzyme digestion, DHPLC, STR linkage analysis andGeneScan were applied to prenatal diagnosis of SMA. 3.Nest-PCR, PEP and fluorescent PCR were applied to amplify singlelymphocyte. 4.PCR-restriction enzyme digestion technique was applied to detect the deletionof SMN 1 gene in single cell. Results: 1.Three STR loci picked out from eleven STR loci tightly linkage to SMN genehad abundant polymorphism and high rate of heterozygosis. 2.Three STR loci (D5S435, D5F149, D5S351) were applied in linkage analysis,which made the results of prenatal diagnosis more accurate and reliable. 3.Amplification rate of nest-PCR and PEP was 90% and 85%, respectively. 4.Exon 7 and 8 of SMN1 gene in single cell were amplified by nest-PCR and PEP, then the PCR products were digested by restriction enzyme, the results of single cell were consistent with those of whole blood.Conclusions: 1.Prenatal diagnosis of SMA was more accurate and reliable by the combination of PCR-restriction enzyme digestion, DHPLC, STR linkage analysis and GeneScan.2.PCR-restriction enzyme digestion of single cell can be applied in gene diagnosis of SMA, which is the basis of implantation genetic diagnosis of SMA and also provides the technical support for implantation genetic diagnosis of other genetic disease.