Abstract
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 paper, we describe a novel assay allowing a single-cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL 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 were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation, and low cost. It is a promising approach in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).
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Song, Q., Yang, H., Zou, B., Kambara, H., Zhou, G. (2016). Pyrosequencing on a Single Cell. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_34
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DOI: https://doi.org/10.1007/978-1-4939-3308-2_34
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