|
Christian Korfhage QIAGEN GmbH, Germany |
Abstract
DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell comprises only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell cycle phase. The DNA amount of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome requires a few hundred nanograms up to micrograms of genomic DNA. Consequently, accurate whole genome amplification (WGA) of single cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited, as in single cell WGA. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells).
To perform single cell WGA, we used the QIAGEN® REPLI-g® Single Cell Kit, which uses a method based on isothermal multiple displacement amplification (MDA). This technique is capable of accurate in vitro DNA replication of a single whole genome directly from single cells due to innovative lysis and the use of an optimized form of the Phi 29 Polymerase with:
(1) Proofreading activity (up to 1000-fold lower error rates compared to Taq polymerase), (2) Strong processivity (resulting in minimal enzyme dissociation at difficult structures such as GC rich regions), (3) Strong DNA displacement activity (resulting in solving hairpin structures).
Here, we describe the reliability of this single cell WGA method and its application to next-generation sequencing (NGS) and real-time PCR.
| Back to Single-cell Diagnostics |
|---|
