Rapid Preparat,On of DNA Polymerase

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Rapid Preparat,On of DNA Polymerase Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tips PCR depends on the use of a thermo- HCI (pH 8.0), 0.25% (vol/vol) sodium Rapid stable DNA polymerase. (1'2) Several ther- lactate, 4.2 mM succinic acid, 5.9 mM glu- Preparat,on of mostable DNA polymerases are commer- tamic acid, 3.8 mM KzHPO4, 0.48 mM cially available from a number of MgSO 4, 0.38 mM CaC12, and 8.5 mM Thermus flavus thermophilic bacterial cells, including NaC1. (8) The fermenter vessel and its Thermus aquaticus, Thermus flavus, and heating lines were wrapped in alumi- DNA Polymerase members of the Pyrococcus family. Other num foil to keep the temperature near polymerases have been purified from 70~ Thermus thermophilus (3'4) and Thermus Growth was monitored by A65o and caldophilus. (s) The polymerase that we allowed to continue until stationary Robert A. Harrell II and found to be most suitable for our phase (-24 hr postinoculation). Once Ronald P. Hart needs (6,7) was isolated from the thermo- stationary phase was reached, the cul- philic bacteria T. flavus (e.g., HotTub, ture was pumped into 0.5-liter centrifuge Department of Biological Sciences, Amersham, or Pyrostase, Molecular Ge- bottles and the cells collected by centrif- Rutgers University, Newark, New Jersey netics Resources). A T. flavus DNA poly- ugation at 4000 rpm using a JS-42 rotor 07102 merase has been isolated and character- in a J6-B centrifuge (Beckman). The pel- ized previously. (8'9) Unfortunately, the lets were resuspended in buffer A (0.14 M published procedure for DNA poly- NaC1 and 0.01 M Tris-HC1 pH at 8.0) and merase isolation was laborious and inef- consolidated. Approximately 30 grams ficient. Therefore, we developed a rapid of cells were obtained from this 5-liter preparation method suited to our needs. culture. The cell pellet was stored frozen A flow diagram outlining our procedure at - 80~ is given in Figure 1. Enzyme Assay MATERIALS AND METHODS During isolation of DNA polymerase, ac- Bacterial Culture tivity was assessed by a primer extension T. flavus was obtained from American assay. Activi W was determined by incor- Type Culture Collection (ATCC 33923; poration of [~-32p]dATP into high-mo- also known as T. aquaticus Wpe AT62) lecular-weight DNA on a primed single- and grown from a 50-ml seed culture in stranded M13 phage DNA template. a 5-liter Bioflo II Fermenter (New Brun- Enzyme fractions were assayed by addi- swick Scientific, Inc.) at 65-70~ on syn- tion of 680 ng of single-stranded thetic medium containing 2% (wt/vol) M13mpl9 DNA and 5x10 4 ng of 17- peptone, 0.2% (wt/vol) yeast extract, mer M13 universal primer (U.S. Bio- 0.1% (wt/vol) beef extract, 50 mM Tris- chemical) in a 5-~1 reaction containing 1 Culture of Thermus flavus t Collect ceils by centrifugation Sonicate cells to release cytosol PEI test of crude extract to determine appropriate concentration for precipitation of DNA polymerase t PEI precipitation of crude extract Recovery of DNA polymerase from PEI precipitate Ion exchange chromatography Ammonium sulfate precipitation FIGURE 1 Flow chart of T. flavus DNA polymerase preparation. 372 PCR Methods and Applications 3:372-3759 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/94 $5.00 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press lllllll Technical Tips saturating for enzyme; 1 unit is the amount of enzyme that will incorporate 10 nmoles of dNTP into acid insoluble product at 70~ in 30 rain) of enzyme or no added enzyme. Units of T. flavus DNA polymerase were determined by compar- ing enzyme reactivity in a primer exten- sion assay with dilutions of Pyrostase (data not shown). Units of Pyrostase and Taq polymerase were determined by their manufacturer. RESULTS Isolation of DNA Polymerase T. flavus DNA polymerase was partially purified by a combination of polycation precipitation and column chromatogra- phy. Frozen cell pellets were thawed by FIGURE 2 Ten percent SDS-PAGE of S-Sepharose column fractions. Fraction numbers are shown at the top of each lane. Conductivity testing indicated the KCI concentrations shown. Fractions 30-36 exhibited maximal levels of DNA polymerase activity (see Figs. 3 and 4). (Right) The 94-kD band that best correlates with enzyme activity levels and further protein purification (Fig. 5). (M) Marker: 20 kD rabbit muscle myosin, 116 kD E. coli f3-galactosidase, 97 kD rabbit muscle phos- phorylase, 66 kD bovine albumin, and 45 kD ovalbumin. (CL) Column load. ~l of enzyme fraction, 50 mM HEPES, 1.5 and dCTP, 1 p~M of each primer, and 50 mM MgCl, 50 mM KCI, 100 ~M each of pg of pBSVIP template DNA (C. Liu and dGTP, dTTP, and dCTP, and 10 p.M dATP R.P. Hart, unpubl.). The primers were including 1 ~Ci of [~-32P]dATP (DuPont VIP472 (5'-AGGAAGTCTGGAGAATCTC- NEN). The reactions were incubated for CCTCA-3') and VIP228 (5'-GGAGTTT- 1 min at 37~ then for 3 min at 65~ TCACCAGCGACTACAGT-3'), designed The reactions were stopped by addition from rat VIP cDNA sequence. (11) The re- of 10 p.1 of deionized formamide con- actions were amplified for 30 cycles of taining 0.1% (wt/vol) xylene cyanol. 94~ for 30 sec, 60~ for 30 sec, and 72~ Samples were heat denatured, run on a for 3 min, followed by an 8-min exten- 6% polyacrylamide sequencing gel, and sion period at 72~ The products were visualized by autoradiography. The ac- precipitated and electrophoresed on a tivity of sample was judged by compar- 1% agarose gel. DNA polymerase activity ing the amount and size of product with was judged by the presence and relative standard reactions performed with com- intensity of specific PCR product (pre- mercial enzymes. (1~ dicted to be 245 bp). In general, the PCR assay was more useful for identifying fractions containing activity, but the PCR Conditions primer extension assay was better at T. flavus DNA polymerase was also tested judging relative enzyme activity. for activity in PCR. The target DNA was a cloned PCR product of rat vasoactive in- Temperature Stability FIGURE 3 Primer extension assay of testinal peptide cDNA prepared previ- S-Sepharose column fractions. DNA poly- The temperature stability of three DNA ously in our laboratory by cloning re- merase activity was detected by extending verse transcriptase (RT)-PCR products. polymerase preparations was compared primed M13 DNA with radiolabeled nucle- Reactions were performed in a volume of by pretreating complete PCR reactions at otide triphosphate. Products were revealed by 50 ~l containing 50 mM Tris (pH 9.0), 20 94~ then proceeding with a standard autoradiography of dried sequencing gels. mM (NH4)2SO 4, 1.5 mM MgC12, 0.005% PCR reaction. Each reaction mixture Bands at arrowhead indicate polymerase ac- BSA, 0.2 mM each of dATP, dGTP, dTTP, contained -0.2 unit (chosen to be sub- tivity. (P) Pyrostase; (-) no added enzyme. PCR Methods and Applications 373 Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Technical round= 10 sec of sonication at 20 kHz with 50 sec of rest, W-385 Sonicator (Heat Systems-Ultrasonics INC.)I, and light scattering at 650 nm was measured. Sonication was continued with an A6s o measurement after every fifth round un- til the A6s o stabilized. The solution was then centrifuged for 30 rain at 22,100g. The supernatant was clarified by centrif- ugation for 1 hr at 80,000g. Then, polyethyleneimine (PEI) was used to precipitate the DNA polymerase following a method used for isolation of overexpressed Taq polymerase. ~1~ En- gelke et al. found that the concentration of PEI used to precipitate Taq poly- merase varied between preparations. (~~ Therefore, a PEI precipitation test was done for each preparation. Concentra- tions between 0.05% and 0.8% (wt/vol) PEI were tested to determine the appro- FIGURE 4 PCR assay of S-Sepharose column fractions. DNA polymerase activity was detected by priate concentration to precipitate en- 30 cycles of PCR using 50 pg of pBSVIP plasmid DNA and two VIP-specific primers. Products were zyme activity. Once the proper concen- separated on 1% agarose gels, stained with ethidium bromide, and photographed under UV tration for precipitation was determined, illumination. (Arrowhead) Position of predicted VIP cDNA product band. (M) One-kilobase lad- the remainder of the high-speed super- der (GIBCO BRL); (P) Pyrostase; (-) no added enzyme. natant was brought to this concentra- tion by dropwise addition of 10% (wt/ vol) PEI. This mixture was incubated for addition of 2 volumes of cold buffer B 10 min at room temperature and centri- [10 mM Tris-HC1 (pH 7.9), 50 mM KC1, 1 fuged for 20 rain at 8000g. mM EDTA, 0.5 mM phenylmethylsulfo- The pellet was washed by resuspend- nyl fluoride (PMSF), 0.5% (vol/vol) ing in a glass Dounce with 0.025 M KCI in Tween 20, 0.5% (vol/vol) Nonidet-P40 buffer C [20 mM HEPES at pH 7.9, 1 mM (NP-40)] and then broken apart with a EDTA, 0.5 mM PMSF, 0.5% (vol/vol) spatula. The resuspended cells were sub- Tween 20, and 0.5% (vol/vol) NP40]. The jected to 20 rounds of sonication [1 resuspended pellet was centrifuged for FIGURE 5 Ten percent SDS-PAGE of 2 M am- monium sulfate precipitation. Pooled, peak activity S-Sepharose column fractions (SP) were precipitated by adding ammonium sul- fate to a final concentration of 2 M.
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