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J. Am. Chem. Soc. 120, 607-608 (1998) Abstract | Full Article
An equimolar mixture of the four 13C and 15N labeled dNMPs was purchased from Martek Biosciences Corporation (Rockville, MD). Guanylate kinase from porcine brain, myokinase from chicken muscle, pyruvate kinase from rabbit muscle, phosphoenolpyruvate, levigated alumina, Celite (diatomaceous earth), Polymin P, and nonlabeled dNMPs and dATP were obtained from Sigma. Centriprep-3 centrifugal concentrators were purchased from Amicon.
The kinasing fraction of E. coli was prepared based on a protocol published by Hurlbert and Furlong [Methods Enzymol. 1967, 12, 193-202] and Zimmer and Crothers [Proc. Natl. Acad. Sci. USA 1995, 92, 3091-3095].
Six liters of E. coli JM101 cells were grown at 37 C in a minimum medium composed of one liter of 1.5 g of KH2PO4 / 13.5 g of Na2HPO4 / 0.2 g MgSO4 7H2O / 2 g NH4Cl / 10 mg CaCl2 / 0.5 mg FeSO4 7H2O / 4% glucose. The following steps were done on ice or at 4 C. Before growth ceased (optical density of 0.8 at 540 nm), cells were harvested by centrifugation for 30 min at 5000 rpm in a Sorvall GS-3 rotor. Cells were lysed by grinding with levigated alumina (2.5 g per g of cells) and the ground paste was extracted two times with 3 ml of 10 mM K2HPO4, pH 7.2 / 2 mM glutamine / 5 mM EDTA per g of cells. Alumina and cell debris were separated from the lysate by centrifugation for 35 min at 8000 rpm in a Sorvall SA 600 rotor. The cell lysate was then centrifuged for 60 min at 47,500 rpm in a Beckman ultracentrifuge using a type 70.1 Ti rotor. The ribosomes were precipitated from the supernatant with streptomycin sulfate (50 mg per g of cells) by centrifugation for 15 min at 15,000 rpm in a Sorvall SA 600 rotor. Polymin P (0.1% v/v) and Celite (1% w/v) were added to the supernatant before another centrifugation step at 15,000 rpm in a SA 600 rotor for 20 min. The supernatant was then fractionated at ammonium sulfate concentrations of 0.21, 0.3 and 0.35 g per ml. Each fraction was centrifuged for 15 min at 8000 rpm in a SA 600 rotor. The last fraction was resuspended in 50 mM glycine, pH 8.0 / 2 mM glutamine / 0.2 mM EDTA (1 ml per g of cells) and stored at -80 C. The approximate protein concentration was 5 mg/ml (Bio-Rad dye-binding assay).
13C,15N-labeled dNTPs were prepared from the corresponding labeled dNMPs by phosphorylation with a mixture of enzymes and the kinase activity from JM101 E. coli cells according to the method of Zimmer and Crothers (1995). The reactions were done in 80 mM Tris HCl, pH 7.5 / 20 mM KCl / 20 mM MgCl2 / 10 mM DTT / 50 mM phosphoenolpyruvate / 100 mM ATP / 10 mM dNMPs (2.5 mM each) / 50 units of pyruvate kinase per ml / 20 units of myokinase per ml / 0.1 unit of guanylate kinase per ml / the E. coli kinasing fraction (1 mg / ml) for 4 hr at 37 C. The reactions were monitored by HPLC with a Vydac nucleotide analysis column. The reaction mixture was then extracted with 1 vol of phenol, three times with 1 vol of chloroform, and once with 1 vol of diethyl ether. The labeled dNTPs were stored at -80 C.
The exonuclease free mutant D424A of the Klenow polymerase was expressed in E. coli and purified according to the procedure of Joyce and Derbyshire [Methods Enzymol. 1995, 262, 3-13]. The original purification procedure involves three chromatographic steps, but in our own experience the two first steps, Mono Q chromatography followed by Phenyl Superose chromatography, are sufficient to produce enzyme sufficiently pure for DNA synthesis.
The oligonucleotide templates were synthesized on a Pharmacia LKB gene assembler using solid state b-cyanoethyl phosphoramidite chemistry. The DNA was purified by perfusion anion exchange chromatography using an HQ POROS resin on a Biocad Sprint (PerSeptive Biosystems Inc. Framingham, MA) in 10 mM NaOH with a gradient of 0.4-0.7 M NaCl, then desalted on a Waters C18 Sep-Pak cartridge with elution using a mixture of 50% H2O / 50% methanol.
The polymerization reactions were carried out in 10
mM Tris HCl at pH 7.5, 5 mM MgCl2, 3 units of Klenow
polymerase D424A per nanomole of DNA template, 0.1 mM template and 0.5
mM of each labeled dNTP (i.e. 2 mM of the labeled mixture) for the first
step reaction or 0.75 mM for the second step reaction. The general rule
is to use 3 times the theoretical amount of labeled nucleotides needed
(Zimmer and Crothers, 1995). The reaction mixtures were incubated for
3 hours at 37 C and the enzymatic reaction stopped by heating 1 min at
95 C. The completion of the reactions was checked by 19% polyacrylamide
gel electrophoresis.
It should be noted that the specific activity of the purified
Klenow polymerase was estimated from the amount of protein, assuming
that 1 mg of enzyme corresponds to 10000 units.
The first enzymatic reaction was performed with 0.75 mmole of hairpin DNA. The elongation reaction was
followed by a desalting step with a Centriprep-3 centrifugal concentrator,
which both removes salts and unincorporated dNTPs. The product was cleaved
from the template-loop in 0.4 mM KOH for 3 hours at 55 C. It was then
purified by anion exchange chromatography using an HQ POROS resin on
a Biocad Sprint in 10 mM NaOH with a gradient of 0.4-0.7 M NaCl. The
purified DNA was desalted on a C18 Sep-Pak cartridge and used for the
second enzymatic reaction. The product of this reaction was purified in
a similar manner. From the initial 0.75 mmole
of hairpin DNA, 0.4 mmole of extended primer
was obtained which then yielded 0.21 mmole
of the final purified product.