Improvement Of Late-Pcr To Prepare Pyrosequencing Template

Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this chapter, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponentialPCR (imLATE-PCR) on a modifi ed glass slide with highly sensitive pyrosequencing. Due to the signifi cantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1-mu L chip PCR which directly used a single cell as starting material. As a proof-of-concept, the 1555A> G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731 C>T of the BRCA1 gene on genome DNA from a single cell have been successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation, and low cost. The approach has promise for effi cient use in the fi elds of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).