Engineering Polymerases for New Functions

Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA

“…During the last 60 years, many fields related to genetics and molecular biology have greatly benefited from the characterisation of innumerable DNAPs. Further, for many applications, those DNAPs featuring optimal properties have been engineered from natural enzymes by means of several directed evolution approaches 6,17 . Among them, fusions of single-or double-stranded DNA-binding domains have often improved DNA amplification proficiency of DNAPs in PCR and MDA protocols 18,20,41,42 .…”

“…The presence of modified bases in DNA samples blocks DNA synthesis by most DNAPs and thus strongly impairs DNA amplification of damaged DNA from environmental or ancient DNA 14 , 15 . Those shortcomings, along with the recent exponential growth of sequencing capacities, have motivated the development of new DNA amplification methods that have been nourished by novel and tailored DNAPs with enhanced proficiency and a variety of improved features, including thermostability, resistance to inhibitors or even amplification of “reluctant” samples 6 , 16 , 17 . Among the new-engineered DNAPs, protein fusions with single- or double-stranded DNA-binding proteins have been successfully created for improved MDA and PCR applications 18 – 21 .…”

Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

“…DNA polymerase is an omnipresent enzyme that synthesizes complementary DNA strands from an existing template in living cells. DNA polymerases are widely used for a variety of molecular techniques that rely on DNA manipulation, including polymerase chain reaction (PCR), molecular cloning, sequencing, DNA labeling, and mutagenesis among others (Gibbs et al, 2009; Killelea et al, 2009; Coulther et al, 2019). Several thermostable DNA polymerases have been isolated and studied in prokaryotes (Cho et al, 2012; Kim et al, 2007; Lee et al, 2009; Terpe, 2013; Zhang et al, 2015).…”

“…Several thermostable DNA polymerases have been isolated and studied in prokaryotes (Cho et al, 2012; Kim et al, 2007; Lee et al, 2009; Terpe, 2013; Zhang et al, 2015). These enzymes are grouped into eight families: A, B, C, D, X, Y, RT, and AEP based on their amino acid sequences (Killelea et al, 2009; Coulther et al, 2019). Most thermostable DNA polymerases primarily used in PCR procedures belong to the A‐ and B‐family polymerases from thermophilic bacteria and archaea, respectively.…”

Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

“…[6][7][8] The related verification and condition optimization are still necessary, including component quantities (such as DNA polymerase, primers, templates and dNTPs) and thermal conditions (such as annealing and extension temperatures). In addition, many other efforts have been used to increase PCR efficiency, specificity and robustness, including hot-start strategy with heat-activated polymerase or primer, [9][10][11] directed-evolved DNA polymerase, 12 chemical additives (including DMSO, betaine, metal nanoparticles, quantum dots and nano-polymers), 13,14 supplementary recombinase and helicase, 15,16 and modified or immobilized primers. 17 These efforts are time-consuming and labour-intensive.…”