Amplification efficiency for DNA polymerases depending on the composition of salt in PCR.

<p>Differences in the amplification efficiency for the fusion <i>Neq</i>SSB<i>-TaqS</i> DNA polymerase (A) and <i>TaqS</i> 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).</p

Fusion of <i>Taq</i> DNA polymerase with single-stranded DNA binding-like protein of <i>Nanoarchaeum equitans</i>—Expression and characterization - Fig 1

<p>The expression and purification of <i>TaqS</i> (A) and <i>Neq</i>SSB<i>-TaqS</i> (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.</p

Determination of processivity based on the melting temperatures of DNA products created in the presence of a heparin trap.

<p>(A) Melting curves of the resulting products for the DNA polymerases. (B) Melting temperature of the elongated products.</p

DNA polymerase tolerance to PCR inhibitors.

<p>The effect of blood (A), lactoferrin (B) and heparin (C) inhibitors on DNA amplification with the use of the genomic DNA of <i>S</i>.<i>aureus</i> as a template and primers for specific <i>nuc</i> 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.</p

Evaluation of PCR amplification rate.

<p>Comparison of the PCR amplification rates of a fusion <i>Neq</i>SSB<i>-TaqS</i> DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a <i>Taq</i>S 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).</p

Sedimentation analysis of <i>Neq</i>SSB-like (A) and standard proteins (B).

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1–19: fraction number. 50 μl of 150 μM <i>Neq</i>SSB-like and the corresponding amounts of standard proteins were centrifuged in linear 15 to 30% (w/v) glycerol gradients, as described in the “Methods”. The fractions with proteins were analyzed by SDS-PAGE. The fractions at which the maximal amount of protein appears are shown by arrows in each panel. The standard proteins used are: L, lysozyme (14 kDa); CA, carbonic anhydrase (29 kDa); BSA, bovine serum albumin (66 kDa) and AD, alcohol dehydrogenase (150 kDa). The oligomerization state estimation of <i>Neq</i>SSB-like was made with these proteins.</p

The determination of ssDNA-binding activity half-life, using gel mobility shift assays, for <i>Neq</i>SSB-like and <i>Taq</i>SSB.

<p><i>Neq</i>SSB-like represented by the open circles and <i>Taq</i>SSB, shown as open triangles.</p

Interaction analysis of <i>Neq</i>SSB-like (A) with ssDNA and dsDNA (B).

<p>Different concentrations of the protein were injected with a flow rate of 30 μl/min on a streptavidin chip coated with ssDNA 60-mer and dsDNA 60-mer on separate flow channels. A flow cell with streptavidin was used as a reference. After each injection the chip was regenerated with 0.01% SDS. The different colors of the sensograms represent the concentrations of the <i>Neq</i>SSB-like injected. Solid lines state for fitted curves. The data were fitted in accordance with the Langmuir model and using BiaEval 3.0 software.</p

Binding of <i>Neq</i>SSB-like to M13 ssDNA.

<p>Lanes 1–8 contain (0.07 pmol) of M13 ssDNA and 0, 3.5, 7, 14, 28, 56, 112 and 224 pmoles of <i>Neq</i>SSB-like, respectively.</p

The expression and purification of <i>Neq</i>SSB-like from <i>E</i>. <i>coli</i> TOP10F’+pBAD/NeqSSBHT.

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1: soluble protein cell extracts after arabinose induction of protein expression (10 μl). Lane 2: <i>Neq</i>SSB-like after the Ni²⁺-affinity chromatography step (10 μl). Lane 3: <i>Neq</i>SSB-like after His-tag cleavage with TEV protease (10 μl). Lane 4: <i>Neq</i>SSB-like after chromatography on an ssDNA-cellulose column (10 μl).</p