OPTIMISATION DE L'ANALYSE GENETIQUE DE LA CELLULE UNIQUE

2, 3 Abstract : The molecular genetic analysis of microdissected cells by laser, a method for selecting a starting material of pure DNA or RNA uncontaminated. Our study focuses on technical pre-PCR (polymerase chain reaction) for the amplification of DNA from a single cell (leukocyte) isolated from human blood after laser microdissection and aims to optimize the yield of DNA extracted of this cell to be amplified without errors and provide reliable genetic analyzes. This study has allowed us to reduce the duration of cell lysis in order to perform the step of expanding genomic PEP (primer extension preamplification) directly after lysis the same day and the quality of genomic amplification and eliminate purification step of the product PEP, step with a risk of contamination and risk of loss of genetic material related to manipulation. This approach has shown that the combination of at least 3 STR (short tandem repeat) markers for genetic analysis of single cell improves the efficiency and accuracy of PCR and minimizes the loss of allele (allele drop out; ADO). This protocol can be applied to large scale and an effective means suitable for genetic testing for molecular diagnostic from isolated single cell (cancerous - fetal).