使用Phi29 DNA聚合酶通过等温多链置换进行全基因组扩增。

Whole Genome Amplification by Isothermal Multiple Strand Displacement Using Phi29 DNA Polymerase.

作者信息

Kroneis Thomas, El-Heliebi Amin

机构信息

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden.

出版信息

Methods Mol Biol. 2015;1347:111-7. doi: 10.1007/978-1-4939-2990-0_8.

DOI:10.1007/978-1-4939-2990-0_8

PMID:26374313

Abstract

The here described method of isothermal whole genome amplification (iWGA) uses a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit) that amplifies minute quantities of DNA by multiple strand displacement upon random hexamer primer binding. Starting from genomic DNA or single cells this amplification yields up to 5 μg of iWGA product with fragment lengths of 10 kb and longer. As this amplification lacks the need of fragmenting DNA, its products are well suited for many downstream applications (e.g. sequencing and DNA profiling). On the contrary, degraded DNA samples are not supported by the nature of the amplification and are not well suited.

摘要

这里所描述的等温全基因组扩增（iWGA）方法使用了一种基于Phi29 DNA聚合酶的试剂盒（Illustra GenomiPhi V2 DNA扩增试剂盒），该试剂盒通过在随机六聚体引物结合后进行多链置换来扩增微量DNA。从基因组DNA或单细胞开始，这种扩增可产生高达5μg的iWGA产物，片段长度为10kb及以上。由于这种扩增无需对DNA进行片段化，其产物非常适合许多下游应用（如测序和DNA分析）。相反，降解的DNA样本不适合这种扩增性质，不太适用。