Fig 1.
The expression and purification of TaqS (A) and NeqSSB-TaqS (B) DNA polymerases. The proteins were analyzed on a 10% polyacrylamide gel (SDS-PAGE). Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), molecular masses highlighted. Lane 1: the sonicated extract of induced cells; Lane 2: heat treatment; Lane 3: a by-product after the second washing with the use of the buffer B; Lane 4: purified protein after elution with the buffer C.
Fig 2.
Characterization of a fusion NeqSSB-TaqS DNA polymerase in comparison to a TaqS DNA polymerase.
The effect of (A) MgCl2, (B) KCl, (C) (NH4)2SO4, (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the TaqS DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq-TaqS DNA polymerase does not have the end bar.
Fig 3.
Amplification efficiency for DNA polymerases depending on the composition of salt in PCR.
Differences in the amplification efficiency for the fusion NeqSSB-TaqS DNA polymerase (A) and TaqS DNA polymerase (B) depending on the composition of salt in the PCR buffer (10 mM KCl plus 0; 10; 20; 30; or 40 mM of (NH4)2SO4. Lane M: the DNA molecular size marker HyperLadder II (Bioline, UK).
Fig 4.
Evaluation of PCR amplification rate.
Comparison of the PCR amplification rates of a fusion NeqSSB-TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a TaqS DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).
Fig 5.
Determination of processivity based on the melting temperatures of DNA products created in the presence of a heparin trap.
(A) Melting curves of the resulting products for the DNA polymerases. (B) Melting temperature of the elongated products.
Fig 6.
The primer-template binding for NeqSSB-TaqS and TaqS DNA polymerases.
The binding of fusion NeqSSB-TaqS (A) and native TaqS (B) DNA polymerases to a primer-template measured at various annealing PCR temperatures (indicated in each lane at the top of the gels). The amplified products were analyzed on a 2% agarose gel stained with ethidium bromide.
Fig 7.
DNA polymerase tolerance to PCR inhibitors.
The effect of blood (A), lactoferrin (B) and heparin (C) inhibitors on DNA amplification with the use of the genomic DNA of S.aureus as a template and primers for specific nuc gene detection. The effect of whole human blood on the DNA amplification with the use of primers for the amplification of a human CCR5 gene (D). No inhibitors were used in control reactions. Lane M: DNA standards ladder (100–1000 bp). The amplified products were analyzed on a 2% agarose gel stained with ethidium bromide.
Fig 8.
A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT)76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT)76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT)76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT)76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT)76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol NeqSSB-TaqS DNA polymerase, respectively.