Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR

Abstract

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.

Figure 1

Primary screening of mutagenized library. Clones were arrayed in parallel with 9 mm spacing and the single colony assay was incubated at 37°C for 45 min (A) or 68°C for 10 min (B). Examples of cold-sensitive phenotype, temperature-sensitive phenotype and wild-type are labeled. Clone Cs3 is marked with an arrow in both panels. (C) Relative activity of cold-sensitive mutants versus wild-type Klentaq1, at low versus high temperature, was best demonstrated and confirmed by serial dilutions of purified enzymes. Cs3C and Cs3AC mutant Klentaq1 (singly and doubly mutated derivatives of Cs3) were tested in standard 50 µl DNA polymerase assays, in parallel with Klentaq1 enzyme (KT1). (D) The ratio of dCTP incorporation at 74 versus 37°C was calculated from the input data for (C).

Figure 1

Primary screening of mutagenized library. Clones were arrayed in parallel with 9 mm spacing and the single colony assay was incubated at 37°C for 45 min (A) or 68°C for 10 min (B). Examples of cold-sensitive phenotype, temperature-sensitive phenotype and wild-type are labeled. Clone Cs3 is marked with an arrow in both panels. (C) Relative activity of cold-sensitive mutants versus wild-type Klentaq1, at low versus high temperature, was best demonstrated and confirmed by serial dilutions of purified enzymes. Cs3C and Cs3AC mutant Klentaq1 (singly and doubly mutated derivatives of Cs3) were tested in standard 50 µl DNA polymerase assays, in parallel with Klentaq1 enzyme (KT1). (D) The ratio of dCTP incorporation at 74 versus 37°C was calculated from the input data for (C).

Figure 1

Primary screening of mutagenized library. Clones were arrayed in parallel with 9 mm spacing and the single colony assay was incubated at 37°C for 45 min (A) or 68°C for 10 min (B). Examples of cold-sensitive phenotype, temperature-sensitive phenotype and wild-type are labeled. Clone Cs3 is marked with an arrow in both panels. (C) Relative activity of cold-sensitive mutants versus wild-type Klentaq1, at low versus high temperature, was best demonstrated and confirmed by serial dilutions of purified enzymes. Cs3C and Cs3AC mutant Klentaq1 (singly and doubly mutated derivatives of Cs3) were tested in standard 50 µl DNA polymerase assays, in parallel with Klentaq1 enzyme (KT1). (D) The ratio of dCTP incorporation at 74 versus 37°C was calculated from the input data for (C).

Figure 2

Amplification of 513 bp at the co-receptor CCR5 locus from human genomic DNA with Klentaq1 and the Cs3 triple mutant. (A) Relative activity of wild-type Klentaq and the Cs3 triple mutant during pre-PCR incubation as assayed by TCA insolubility. Each reaction contained 1 µCi of [³²P]dCTP. Sixty minutes at 25°C was followed by 10 min at 37°C and a warming step of 15 min at 66°C. Parallel aliquots from the three reactions, catalyzed by Klentaq1, Klentaq or the Cs3 mutant enzyme, were taken at time points as follows: lane 1, 10 min; lane 2, 20 min; lane 3, 40 min; lane 4, 60 min (at 25°C); lane 5, additional 10 min at 37°C; lane 6, 15 min at 66°C; lanes 7 and 8, after three and six PCR cycles, respectively. The aliquots were dotted on filters, washed with 5% TCA and exposed to X-ray film. (B) After completion of the PCR (total 35 cycles), products from the very same radiolabeled reactions were electrophoresed in a 6% acrylamide/urea gel and autoradiographed on X-ray film. The correct amplified product (513 bp), shown by an arrow, appears only for Cs3. The larger, unintended products in all lanes are not reproducible and may have been caused somehow by the aliquot-taking for this experiment. (C) Non-radioactive amplicons catalyzed by Klentaq1 and the Cs3 triple mutant analyzed on a 1.4% agarose gel. Magnesium was added early to lanes 1 and 2 (before the 30°C preincubation for 30 min) and only at 68°C (manual hot start) to lanes 3 and 4.

Figure 2

Amplification of 513 bp at the co-receptor CCR5 locus from human genomic DNA with Klentaq1 and the Cs3 triple mutant. (A) Relative activity of wild-type Klentaq and the Cs3 triple mutant during pre-PCR incubation as assayed by TCA insolubility. Each reaction contained 1 µCi of [³²P]dCTP. Sixty minutes at 25°C was followed by 10 min at 37°C and a warming step of 15 min at 66°C. Parallel aliquots from the three reactions, catalyzed by Klentaq1, Klentaq or the Cs3 mutant enzyme, were taken at time points as follows: lane 1, 10 min; lane 2, 20 min; lane 3, 40 min; lane 4, 60 min (at 25°C); lane 5, additional 10 min at 37°C; lane 6, 15 min at 66°C; lanes 7 and 8, after three and six PCR cycles, respectively. The aliquots were dotted on filters, washed with 5% TCA and exposed to X-ray film. (B) After completion of the PCR (total 35 cycles), products from the very same radiolabeled reactions were electrophoresed in a 6% acrylamide/urea gel and autoradiographed on X-ray film. The correct amplified product (513 bp), shown by an arrow, appears only for Cs3. The larger, unintended products in all lanes are not reproducible and may have been caused somehow by the aliquot-taking for this experiment. (C) Non-radioactive amplicons catalyzed by Klentaq1 and the Cs3 triple mutant analyzed on a 1.4% agarose gel. Magnesium was added early to lanes 1 and 2 (before the 30°C preincubation for 30 min) and only at 68°C (manual hot start) to lanes 3 and 4.

Figure 2

Amplification of 513 bp at the co-receptor CCR5 locus from human genomic DNA with Klentaq1 and the Cs3 triple mutant. (A) Relative activity of wild-type Klentaq and the Cs3 triple mutant during pre-PCR incubation as assayed by TCA insolubility. Each reaction contained 1 µCi of [³²P]dCTP. Sixty minutes at 25°C was followed by 10 min at 37°C and a warming step of 15 min at 66°C. Parallel aliquots from the three reactions, catalyzed by Klentaq1, Klentaq or the Cs3 mutant enzyme, were taken at time points as follows: lane 1, 10 min; lane 2, 20 min; lane 3, 40 min; lane 4, 60 min (at 25°C); lane 5, additional 10 min at 37°C; lane 6, 15 min at 66°C; lanes 7 and 8, after three and six PCR cycles, respectively. The aliquots were dotted on filters, washed with 5% TCA and exposed to X-ray film. (B) After completion of the PCR (total 35 cycles), products from the very same radiolabeled reactions were electrophoresed in a 6% acrylamide/urea gel and autoradiographed on X-ray film. The correct amplified product (513 bp), shown by an arrow, appears only for Cs3. The larger, unintended products in all lanes are not reproducible and may have been caused somehow by the aliquot-taking for this experiment. (C) Non-radioactive amplicons catalyzed by Klentaq1 and the Cs3 triple mutant analyzed on a 1.4% agarose gel. Magnesium was added early to lanes 1 and 2 (before the 30°C preincubation for 30 min) and only at 68°C (manual hot start) to lanes 3 and 4.

Figure 3

Hot start PCR performance of cold-sensitive mutants of Klentaq with various targets. Four targets were amplified in a test PCR assay (where amplification typically benefits from hot start): exon 21 and Alu7.2 repeats of the human dystrophin gene (DMD), the Cryptosporidium heat shock analog protein (Crypto HSP) and the HIV-1 gag gene (HIV gag). The expected amplified products of these targets were 323, 320, 360 and 114 bp, respectively. The following enzymes and reaction set-up conditions were used: lanes 1, Klentaq, with bench start (room temperature set-up); lanes 2, Klentaq, with manual hot start; lanes 3, Cs3C mutant, with bench start; lanes 4, Cs3AC mutant with bench start. Amplified products were resolved in ethidium stained 2% agarose gel, along with a molecular weight standard DNA ladder (M).

Figure 4

Hot start performance of LA versions of the Cs mutants in long PCR. Two targets of the human tPA gene, 4.3 and 9.8 kb, that typically require ‘hot start’, were amplified with Klentaq LA (without or with manual hot start, lanes 1 and 2, respectively) or with Cs3C LA or Cs3AC LA, without hot start (lanes 3 and 4, respectively). M, λ/HindIII DNA ladder. Amplified products were resolved in ethidium stained 1% agarose gels.

Figure 5

Hot start PCR performance of Cs3AC LA mutant compared to FastStart Taq and Platinum Taq. Four targets (1–4) of mouse rRNA genes were amplified from 100 ng genomic DNA with FastStart Taq (Roche) or Platinum Taq (Invitrogen) with or without manual hot start (H.S.) or with Cs3AC LA enzyme (CS) without H.S. (details of targets and conditions are given in Materials and Methods). The expected amplified products of targets 1–4 were 998, 557, 1155 and 818 bp, respectively. PCR products were electrophoresed in ethidium stained 1.4% agarose gel, along with a λ/HindIII DNA ladder (M).

Figure 6

Fidelity of PCR products catalyzed by wild-type and cold- sensitive variants of Taq DNA polymerase when they catalyzed the amplification of a lacZ gene (bracketed on each side by a selectable marker) for 16 cycles of PCR. The percentage of lacZ^– clones was converted to errors per nucleotide incorporated as described (19), wherein the target size is assumed to be 1000 bp. The bars labeled pWB305 were determined as described (19) and the rest used plasmid pWB407. Except as noted for the top four bars, the PCR amplifications contained 1.3 M betaine. Taq, full-length, wild-type Thermus aquaticus DNA polymerase; KT1, N-terminal deletion Klentaq1; Cs3C, Klentaq1 I707L; Cs3AC, Klentaq1 I707L, E626K.

Figure 7

Looking directly through the axis of the P α-helix (residues 704–717) on the left, four amino acids mutable to cold-sensitive are rendered as orange ball and stick wild-type residues on the backbone structure (blue) of Klentaq1 and are labeled with their residue numbers. Depicted with the aid of Rasmol 2.7.2 is the closed conformation of protein database file 3KTQ (29). Also shown as yellow and green ribbons are the determined positions of a short portion of primer and template DNA. Direction of synthesis would be away from the viewer. The curved arrow indicates the proposed (29) range of motion of the fingertips. A.S., active site. Not shown is a molecule of ddCTP in the active site.

Figure 8

Close-up representation, from approximately the same point of view as Figure 7, of the region surrounding residues 707 (isoleucine, blue space-filled) and 749 (phenylalanine, orange space-filled). (A) Wild-type open configuration. (B) Wild-type closed configuration. (C) Model of mutant leucine at position 707 shows that it may now crowd Phe749 with one of its methyl groups (white arrow).