A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement

Abstract

A change of an aspartic acid to asparagine of Taq (Thermus aquaticus) DNA polymerase is a gain of function mutation that supports faster PCR: the extension times for PCR amplification can be 2-3 times shorter. Surprising results from negative controls led to the discovery of strand-displacement ability and reverse transcriptase activity of Taq D732N DNA polymerase. We demonstrate that the mutant enzyme can, by itself, catalyze RT-PCR, and RT-LAMP assays. Residue 732 is on the surface of the enzyme, not near the active site.

Keywords: RNA detection; RT-LAMP; RT-PCR; Taq DNA polymerase; diagnostics; polymerase chain reaction with reverse transcription.

FIGURE 1

PCR speed tests. PCR performance of wild-type Taq pol on the left half of each agarose gel is compared to TaqD732N on the right half. The gap size (distance between 3′-ends of the primer pairs) was 270, 502, 1000 or 1500 as labeled. Thermal cycler machine settings for annealing/extension time in seconds was tested in pairs with the shorter time on the top half of each gel. (A) 2 and 8 s (B) 5 and 20 s. Other timings 1, 10, 15, 18, 25, and 30 s are shown in Supplementary Figure 2.

FIGURE 2

Strand-displacement by mutant enzymes with D732N. (A) Half-dumbbell test. Catalyzed DNA synthesis begins at the primer (arrow) and proceeds to and through the loop and back again, only if the polymerase is capable of strand displacement, to make a product 2.4 kb in size. The two D732N mutant versions of Taq (full-length A111 and Klentaq1) are marked with asterisk (*). Bst pol was expected to be able to strand-displace, and was included as a positive control. We explain unextended 1.2 kb template as due to inefficiency of the half-dumbbell construction at unknown point(s) during its preparation. (B) Discovery of LAMP ability. A multiply mutant form of Taq pol carrying D732N, named KTflnC4RR (lanes KT*, see text) was tested compared Manta (lanes M), a brand of the large fragment of Bst DNA polymerase (Enzymatics). The banded amplification product which is typical for LAMP appeared at 3 h incubation at 60°C for both enzymes but only for KT* enzyme at 70°C. The middle lane size standards were 1, 2, 4 kb.

FIGURE 3

RT-LAMP of MS2 RNA using single-enzyme Taq-D732N or Klentaq1-D732N. Shown are real-time fluorescence traces of reactions with 1/3 μl test enzyme but no RT (reverse transcriptase) enzyme and no Mn⁺⁺, using Eva Green fluorescence as indicator of double-strand DNA amplification. Blue circles/line: complete 26 μl reaction, with 3 ng MS2 RNA. Black lines: MS2 RNA preincubated with RNase I. Red lines, no MS2 RNA added. Taq and KT1: Controls wild-type Taq (full-length) and Klentaq1 (N-terminal deletion of 278 a.a.) DNA polymerase. Buffer conditions are 50 mM Tris–HCl pH 8.55, 8 mM ammonium sulfate, 200 μM each dNTP, 0.75 M betaine, 0.025% Brij-58, 68°C. Melt curves for this experiment are shown in Supplementary Figure 2.

FIGURE 4

Sharpening of fuzzy bands produced by Taq D732N catalyzing RT-LAMP. MS2 RT-LAMP under conditions as for Figure 3, reacted for only 51 min, catalyzed by four differently mutated enzymes. Codon 732 was D732N and enzyme was full-length Taq pol for all lanes. Lane 1: D119D, i.e., wild-type at codon 119. Lanes 2 and 3: mutations D119A and D119N. Improved clarity of the banding pattern, to make it similar to that observed for standard LAMP, was the result if these mutations near a metal-binding site of the 5′-exonuclease (5′-flap endonuclease) were present in the enzyme. As shown, mutation D142A failed to have this effect.

FIGURE 5

RT-PCR catalyzable by single enzyme Taq D732N but not wild-type Taq. No reverse transcriptase was included, nor was any manganese ion. Two MS2 phage RNA targets, P and K were amplified from 5 pg phage RNA with 0.1 or 0.05 μl D732N mutant or twice as much volume of wild-type Taq (NEB) in 35 μl reactions for 35 PCR cycles. The amplified products were run in a 2.5% agarose gel stained with ethidium bromide, along with a 100 bp DNA ladder (white/black reversed for clarity as printed). As negative control, no RNA was included for the reactions analyzed on the bottom half of the gel. The size of the clearly and successfully amplified bands are 405 and 544 bp (primer-primer gap sizes 359 and 500).