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Deterministic whole-genome amplification of single cells

: Czyz, Zbigniew Tadeusz; Klein, Christoph A.


Kroneis, Thomas (Ed.):
Whole genome amplification : Methods and protocols
New York: Springer Science+Business Media, 2015 (Methods in molecular biology 1347)
ISBN: 978-1-4939-2989-4 (Print)
ISBN: 978-1-4939-2990-0 (Online)
ISBN: 1-4939-2989-5
Aufsatz in Buch
Fraunhofer ITEM ()
whole-genome amplification (WGA); single cell analysis; ligation-mediated PCR; multiple displacement amplification (MDA)

This chapter describes a single-cell whole genome amplification method (WGA) that has been originally published under the name "Single Cell Comparative Genomic Hybridization (SCOMP)" (Klein et al., Proc Natl Acad Sci U S A 96(8):4494-4499, 1999). The method has recently become available commercially under the name "Ampli1() WGA Kit." It is a PCR-based technique for whole genome amplification (WGA) allowing comprehensive and quite uniform amplification of DNA from low quantities of input DNA material, in particular single cells. The method is based on a ligation-mediated adaptor linker PCR approach. In contrast to other PCR-based WGA approaches, both the primer design and mechanism underlying the fragmentation of genome are nonrandom, enabling high priming efficiency and deterministic fragmentation of template DNA. This is particularly important for the design of (diagnostic) assays targeting specific loci. Here, we describe the WGA protocol for amplification of single-cell genomes designed to provide high-quality material in quantity sufficient for a number of locus-specific and genome-wide downstream assays [e.g., targeted Sanger sequencing, restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR), and array comparative genomic hybridization (CGH)].