Whole genome amplification of small amounts of DNA to determine the molecular karyotype of cells

Abstract

Molecular genetic studies often face the need to perform uniform scaling of extremely small amounts of DNA from single cells. This, however, requires the development of specialized methods. Over the past 30 years, various approaches based on the use of degenerate primers have been proposed. However, the methods are not devoid of limitations. These limitations, among other things, are determined by the properties of polymerases. In the present study, we propose the use of two-step whole genome amplification to obtain reliable data on the molecular karyotype of the original sample based on the analysis of 20 picograms of DNA. In the protocol, at the first stage of DNA fragment flanking and amplification with the displacement of the second strand, a pair of partially degenerate primers was used: 5’-TGTGTTGGGTGTGTTTGGNNNNNNGG and 5’-TGTGTTGGGTGTGTTTGGNNNNNNTTT. At the second stage of the polymerase chain reaction, the use of a primer based on the constitutive part is sufficient: 5’-TGTGTTGGGTGTGTTTGG. The article provides reaction conditions and compares the use of various polymerases and their combinations. It discusses the possibility of using a blend of Bst and Pfu polymerases in the presence of 10 mM magnesium ions for the first stage. It also shows the potential for using Klentaq1 polymerase carrying the D732N substitution for the development of whole genome amplification methods. It has been demonstrated that the proposed method allows scaling the initial extremely small amount of DNA to obtain a sample suitable for analysis by massive parallel sequencing (next generation sequencing, NGS) using standard commercial data interpretation protocols.