Jump to main content
Jump to site search

Issue 17, 2013
Previous Article Next Article

Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing

Author affiliations

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 the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).

Graphical abstract: Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing

Back to tab navigation

Supplementary files

Publication details

The article was received on 23 Apr 2013, accepted on 04 Jun 2013 and first published on 04 Jun 2013


Article type: Paper
DOI: 10.1039/C3AN00821E
Citation: Analyst, 2013,138, 4991-4997
  •   Request permissions

    Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing

    Q. Song, H. Yang, B. Zou, T. Kajiyama, H. Kambara and G. Zhou, Analyst, 2013, 138, 4991
    DOI: 10.1039/C3AN00821E

Search articles by author

Spotlight

Advertisements