Single cell polymerase chain reaction for preimplantation genetic diagnosis: methods, strategies, and limitations.

Abstract

We provide an overview of the methodology involved in single cell polymerase chain reaction (PCR), especially for single lymphocytes or cultured lymphoblasts and blastomeres. The first step toward single cell PCR is isolation of single cells; the protocols given can be carried out using basic instruments such as a stereomicroscope. We also describe the alkaline lysis method for cell lysis as well as the design and execution of single cell PCR, either in simplex or in multiplex. Finally, we briefly discuss the different methods for analyzing PCR products and obtaining an accurate diagnosis. Special attention is given to measures that avoid contamination with extraneous DNA and reduce allele dropout.