Abstract
The development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses. This has led to a continual requirement of improved DNA amplification protocols and the elaboration of new tailored methods. As key elements in WGA, identification and engineering of novel, faithful and processive DNA polymerases is a driving force in the field. We have engineered the B-family DNA polymerase of virus Bam35 with a C-terminal fusion of DNA-binding motifs. The new protein, named B35-HhH, shows faithful DNA replication in the presence of magnesium or an optimised combination of magnesium and manganese divalent cofactors, which enhances the replication of damaged DNA substrates. Overall, the newly generated variant displays improved amplification performance, sensitivity, translesion synthesis and resistance to salt, which are of great interest for several applications of isothermal DNA amplification. Further, rolling-circle amplification of abasic site-containing minicircles provides a proof-of-concept for using B35-HhH for processive amplification of damaged DNA samples.
Highlights
The development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses
Prominent among these is bacteriophage Φ29 DNA polymerase (Φ29DNAP)[8], a B-family DNA polymerases (DNAPs) characterised by a high fidelity and an outstanding processive DNA synthesis ability, coupled with an intrinsic strand displacement capacity
As is the case for Φ29DNAP, this processivity is likely defined by a high stability of the DNAP/DNA complex by virtue of a ring-shaped, positively charged, internal structure formed by thumb, palm, TPR1, and TPR2 s ubdomains[30,31]
Summary
The development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses. During the last 60 years, the extensive biochemical characterisation of numerous A- and B-family DNA replicases has facilitated their use as tools for many applications in molecular biology and b iotechnology[6,7] Prominent among these is bacteriophage Φ29 DNA polymerase (Φ29DNAP)[8], a B-family DNAP characterised by a high fidelity and an outstanding processive DNA synthesis ability, coupled with an intrinsic strand displacement capacity. These features enabled the development of the so-called MDA or Multiple Displacement Amplification method[9], which uses random hexamer primers and allows the amplification of DNA substrates without prior sequence information. Amplification artifacts can arise, leading to chimeric formation of false structural variants as well as allelic dropout where one allele is differentially amplified over the other in a heterozygous sample, which is a major obstacle for the clinical application of single-cell WGA1 3
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