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Open Access Highly Accessed Methodology article

Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues

Dan Frumkin1, Adam Wasserstrom1, Shalev Itzkovitz2, Alon Harmelin3, Gideon Rechavi4 and Ehud Shapiro12*

Author Affiliations

1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

2 Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel

3 Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel

4 Sheba Cancer Research Center and the Institute of Hematology, The Chaim Sheba Medical Center, Tel Hashomer and the Sackler School of Medicine, Tel Aviv University, Israel

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BMC Biotechnology 2008, 8:17  doi:10.1186/1472-6750-8-17

Published: 20 February 2008

Abstract

Background

Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues.

Results

Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to ~700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53.

Conclusion

Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.