Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis.
A comprehensive genomic analysis of single cells is instrumental for numerous applications in tumor genetics, clinical diagnostics and forensic analyses. Here, we provide a protocol for single-cell isolation and whole genome amplification, which includes the following stages: preparation of single-cell suspensions from blood or bone marrow samples and cancer cell lines; their characterization on the basis of morphology, interphase fluorescent in situ hybridization pattern and antibody staining; isolation of single cells by either laser microdissection or micromanipulation; and unbiased amplification of single-cell genomes by either linker-adaptor PCR or GenomePlex library technology. This protocol provides a suitable template to screen for chromosomal copy number changes by conventional comparative genomic hybridization (CGH) or array CGH. Expected results include the generation of several micrograms of DNA from single cells, which can be used for CGH or other analyses, such as sequencing. Using linker-adaptor PCR or GenomePlex library technology, the protocol takes 72 or 30 h, respectively.
Geigl
Jochen B
JB
Institute of Human Genetics, Center for Applied Biomedicine, Medical University of Graz, Harrachgasse 21/8, A-8010 Graz, Austria.
Speicher
Michael R
MR
D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D015723:Gene Library; D023281:Genomics / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods