Abstract
Single cell genome analysis methods are powerful tools to define features of single cells and to identify differences between them. Since the DNA amount of a single cell is very limited, cellular DNA usually needs to be amplified by whole-genome amplification before being subjected to further analysis. A single nucleus only contains two haploid genomes. Thus, any DNA damage that prevents amplification results in loss of damaged DNA sites and induces an amplification bias. Therefore, the assessment of single cell DNA quality is urgently required. As of today, there is no simple method to determine the quality of a single cell DNA in a manner that will still retain the entire cellular DNA for amplification and downstream analysis. Here, we describe a method for whole-genome amplification with simultaneous quality control of single cell DNA by using a competitive spike-in DNA template.
Highlights
Single cell genome analysis has become increasingly important and has rapidly evolved over the past decade
multiple displacement amplification (MDA) results in whole genome amplification from tiny samples like a single cell with high genome coverage and low error rates due to the proofreading and strand-displacement activities of Phi[29] polymerase[16,17]
If cellular DNA stems from a non-damaged cell, the double-stranded DNA contains only a very few blocking sites for DNA synthesis
Summary
Single cell genome analysis has become increasingly important and has rapidly evolved over the past decade. Real-time PCR assays are limited to a small number of genomic loci which may behave differently compared to the whole genome. Most important, applying these methods results in the consumption of the single. Cell genome that would not be available for WGA and deep genome analysis None of these methods can be used for quality control of a single cell genome. We have developed a new method that combines a quality assay of the single cell target DNA and whole-genome-amplification (WGA) for further downstream analysis. Fragment lengths or distances between polymerase stop sites of Control-DNA and single cell DNA are compared during the WGA reaction.
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