USRE25785E - Process for preparing a nucleic acid - Google Patents

Description

United States Patent 25,785 PROCESS FOR PREPARING A NUCLEIC ACID Roland F. Beers, Jr., 1406 Carrollton Ave, Baltimore, Md.

No Drawing. Original No. 3,001,913, dated Sept. 26, 1961, Ser. No. 722,627, Mar. 20, 1958. Application for reissue Apr. 1, 1963, Ser. No. 270,141

7 Claims. (Cl. 195-29) Matter enclosed in heavy brackets II appears in the original patent but forms no part of this reissue specification; matter printed in italics indicates the additions made by reissue.

This invention relates to an improved process for the separation of deoxyribonucleic acid (DNA) from mixtures containing deoxyribonucleic acid and ribonucleic acid (RNA). Particularly, the invention relates to a process for the separation of deoxyribonncleic acid from mixtures containing both ribonucleic acid and deoxyribonucleic acid which comprises the selective destruction of ribonucleic acid by phosphorylysis and separating the deoxyribonucleic acid from the mixture.

It has been found that mixture of ribonucleic acid and deoxyribonucleic acid may be resolved to obtain highly purified deoxyribonucleic acid by a process which involves selective destruction of ribonucleic acid by the enzyme polynucleotide phosphorylase (polyase), in the presence of ortho-ph'osphate ions at a pH within the range of 7.5 to 9. Briefly, this new and improved process comprises the steps of contacting a mixture of ribonucleic acid and deoxyribonucleic acid with a source of orthophosphate and polyase for the desired period of time, thereafter adding a salt solution to protect the deoxyribonucleic acid from damage and to facilitate the subsequent separation of deoxyribonucleic acid from protein, heating the mixture to a temperature of about 85 to 85 for about to 30 minutes, and separating and recovering the deoxy ribonucleic acid from the ribonucleic acid and other impurities.

The key to the operability of the instant process is the use of orthophosphate ions in the presence of polyase. This combination, which is essential in the process, splits the polymer of ribonucleic acid into small fragments which may be readily separated from the deoxy'ribonucleic acid which is not attacked by the combination. In the absence of orthop-hosphate there is little destruction of ribonucleic acid except under conditions which also extensively destroy the dcoxyribonucleic acid. Except at the proper pH range polyase is inactive. Also of particular importance are (1) the necessity of carrying out the procesS with ribonucleic acid which has not been degraded or substantially altered from its native form; (2) the heating step during which the destruction of ribonucleic acid is rendered more complete and the separation of deoxyribonucleic acid from other impurities, such as proteins, is facilitated; and (3) the separation of deoxyribonucleic acid from ribonucleic acid fragments by fractional precipitation with ethanol or acetone at a proper salt concentration.

The inventive process is more specifically described as follows: (In this illustration the destruction of ribonucleic acid is carried out in the bacterial cells undergoing concurrent lysis, thereby preventing any substantial degradation of ribonucleic acid into a form which cannot be attacked by the polyase-orthophosphate system. The

Reissued June 1, 1965 cells contain the necessary amount of polyase for carrying out the process.)

Cells of Micrococcus lysodeikticus are suspended in a solution of sodium chloride and the pH of the solution is adjusted to approximately pH 8.5 Lysozyme is added to the suspension along with the desired amount of a salt of orthophosphoric acid. The mixture is incubated for from 30 minutes to 1 hour at a temperature within the range of 30 to 40 C. in order for the action of the polyase-orthophosphate system to take place on the ribonucleic acid.

After this incubation time additional salt solution is added and the volume of the mixture increased to facilitate the separation of the highly viscous dcoxyribonucleic acid from the subsequently formed insoluble matter. The mixture is then heated for from 15 to 30 minutes at a temperature within the range of to C. The heating should be as brief as possible, consistent with the volume of material and source and rate of heating. A flow system can be used in which the mixture is passed througt coils immersed in boiling Water and ice water at a ratr sufficient to render the major fraction of the proteins in soluble by the heating.

Afiter the incubation and heating steps, during whicl time a major fraction of the proteins are rendered insol uble and the ribonucleic acid is degraded into small frag ments, the mixture is cooled to room temperature, am the solids separated by centrifugati'on. The supernatan is mixed with an equal volume of an organic solvent t precipitate the deoxyribonucleic acid as a fibrous mass which is removed from the liquid, dissolved in a simila salt solution and shaken several times with successive poi tions of chloroform accompanied by denaturation of th protein and removal of this impurity as a gel suspensior The ribonucleic acid fragments are removed with the prc tein. The protein-free deoxyribonucleic acid solution i mixed with an equal volume of organic solvent to re precipitate the deoxyribonucleic acid, which is redissolve in a 2% salt solution, reprecipitated with the organi solvent, etc., the cycle being repeated three times or out all the inorganic orthophosphate has been removed l: this procedure. The same procedure also removes ti last residual ribonucleic acid which has not been remove by the deproteination step. After the final precipitatic the white fibrous deoxyribonucleic acid is washed wit ethanol, ace-tone and/ or ether and dried in vacuo.

In its preferred form the invention contemplates t] phosphorylysis of the ribonucleic acid within the Micr coccus lysodeikticus cells by the endogenous polyase a1 orthophosphate. This phosphorylysis may be carri: out at a pH range of 7.5 to 9. However, it is preferrt to use a pH within the range of 8.3 and 8.7, and a pH 8.5 is especially preferred. This corresponds to the ma: mum activity of the enzyme.

The lysozyme used in the lysing step is not critic Crystalline lysozyme or dried egg white may be used.

The phosphorylysis step is carried out in the presence a salt of orthophosphoric acid. Any of the tollowi salts or acid may be used: M PO M HPO MH P( where M is an alkali or alkaline earth metal. The proci is applicable to any bacteriological material which Ct tains suificient endogenous polyase, sutficient deoxyril nucleic acid to make the process practical, and which c be fragmented by any of several well known metht which do not cause extensive damage to the endogenl eaves 'solution 'of'Na l-IPO and ml. :of 0.5 molar trishydroxymethylaminomethane (buffer), final pH 8.5. Two hundred mg. of crystalline lysozyme were added and the mixture incubated for minutes at 37 C. Following the incubation period 230 ml. of a 20% sodium chloride solution was added, and the solutions mixed thoroughly and heated rapidly to 95 C. for minutes.

The mixture was cooled to room temperature and centrifuged at 5 C. for 30 minutes at 28,000 g. The supernatant from the centrifugation was mixed with 1 volume of 95% ethanol. The fibrous precipitate which formed was removed and dissolved in 100 ml. of 10% NaCl.

The solution was then shaken with four volumes of chloroform for 30 minutes on a mechanical shaker, the chloroform layer removed and the emulsion of denatured protein and deoxyribonucleic acid centrifuged at 10,000 g. for ten minutes. The supernatant containing the deoxyribonucleic acid was removed and shaken again with 4 volumes of chloroform, centrifuged as above. This process was repeated for a minimum of three times or until no further gel interface of protein was formed. The

supernatant was mixed with approximately an equal volume of 95% ethanol and the precipitated fibrous deoxyribonucleic acid removed, dissolved in 50 ml. of 2% NaCl, reprecipitated with 50 ml. ethanol, redissolved in 2% NaCl as before, this cycle repeated 3 times. The final precipitate containing the purified deoxyribonucleic acid was washed with 95% ethanol, acetone, ether, and dried in vacuo. a

In Table I below are set out data for two preparations (Examples 4 and 5) carried out in accordance with the process of the instant invention. For comparison the effects of varying lengths of lysis time in the absence of orthophosphate are given (Examples 1, 2 and 3). The results of a low temperature extraction after incubation at 37 are also given (Example 6).

TABLE I Example 1 2 3 4 5 6 Lysis time (in hr.) 1. 0 0. 5 5.0 0.5 0.5 0.5 Orthophosphate Temperature 01 extn.,

deg ees 95 95 5 Yield (gm.) (percent) 0.78 0. 82 0. 49

P 3. 66 3. 95 3. 84 e (260 my) 7, 420 7, 400 7, 430 529E 3 2. 05 1. s1 3. 2s 6;,(230 Inn) u) 4. 63 2. as 3. as 3.60 a. so 4. 53 5;,(290 mp) 1 234 175 180 RNA (percent). 0.57 3. 0 12.0 DNA (percent) 31 20 91 98. 7 97. 0 87. 0 Orthophosphate (percent) 0. (i5 0 1. 0

The following points should be noted in the table:

(1) Examples 1 and 2 illustrate the essential requirement of orthophosphate for removal of ribonucleic acid and ,deoxyribonucleic acid.

(2) Examples 2 and 3 illustrate the destructive effect of prolonged lysis on the yield of deoxyribonucleic acid.

(3) Example 3 illustrates the possible separation of ribonucleic acid from deoxyribonucleic acid without added orthophosphate following prolonged incubation of the mixture, but the potential advantages of this are destroyed by (a) the poor yields of deoxyribonucleic acid and (b) the very poor quality of deoxyribonucleic acid as evidensed by its failure to precipitate as a fibrous precipitate in ethanol and the absence of any significant viscosity of concentrated solutions of this material.

(4) Example 6 illustrates the desirability of including a heating step in the extraction procedure, although fractionation of the material with acetone alone will remove a substantial portion of the residual ribonucleic acid not removed with ethanol.

(5) Examples 4, 5, and 6 illustrate the highly polymerized state of the deoxyribonucleic acid preparations as evidenced by their intrinsic viscosities, 7 The probable molecular weights of these preparations are in the vicinity of ten million.

(6) The degree of purity of deoxyribonucleic acid according to the various methods of isolation and purification are indicated by the absorption spectra data, the nitrogen/phosphorus ratios and the intrinsic viscosities.

As was stated above the pH of the phosphorylysis step has a bearing on the rate and extent to which ribonucleic acid is phosphorylysed and rendered susceptible to removal by fractionation procedures. This is illustrated in Table II. Cells were lysed as above in the absence of orthophosphate and when lysis was ind ed to be complete (approximately 15 minutes), the mixture was centrifuged for 30 minutes at 28,000 g. The supernatant contained substantial quantities of ribonucleic acid and polyase. The rate of phosphorylysis of the ribonucleic acid was determined at varying pH in the presence of 0.1 M. Na HPO 0.4 M. KCl, 0.1 M. trishydroxymethylaminomethane buffer, 0.0061 M. MgCl at 37.

TABLE II Relative rates pH: of phosphorylysis 7.0 6.75 7.5 11.0 8.0 12.7 8.5 14.5 9.0 9.75 9.5 6.75

It will be seen that the preferred pH for ribonucleic acid destruction is at 8.5 although an operable range is w between 7.5 and 9.0, a pH of 8.3 to 8.7 is preferred.

KCl and MgCl were added in this experiment because of their activating effect on the enzyme. They are not nefiessary in the phosphorylysis process using Whole lysed ce s.

The optimum concentration of orthophosphate was determined in a similar experiment. The reaction was carried out in 0.2 M. KCl, 0.002 M. MgCl 0.1 M. trishydroxymethylaminomethane, pH 8.5, with a cell extract as prepared in Table II plus 0.2% of a crude nucleic acid preparation obtained from the cells.

. TABLE III Concn. of Na HPO Relative rate of added: phosphorylysis 0.000 1.30 0.0017 M. 1.55 0.0033 1.97 0.0083 t 2.80 0 .017 3.76 0.033 2.90 0.083 2.50

It will be seen that the preferred concentration of orthophosphate in this experiment is approximately 0.02 M. However, in view of the large quantity of ribonucleic acid present in the Whole cells, it has been judged desirable to increase the concentration to 0.1 M. Also to be noted is the fact that phosphorylysis will take place in the absence of orthophosphate. This reflects the fact that the cells contain a substantial amount of orthophosphate which can act to degrade the ribonucleic acid given sufficient time, as illustrated by Example 3 of Table I. Thus the degree of purity and quality of deoxyribonucleic acid depends upon the concentration of orthophosphate and the time of phosphorylysis. Table IV contains data of this nature. The procedures of lysis, extraction and purification are the same as in Table I but with smaller quantities of material.

To be noted in Table IV is the effect of time and phosphate concentrations on the percent contamination of deoxyribonucleic acid by ribonucleic acid. Of less importance are the actual yields which because of the small material used were usually small as a result of mechanical losses.

The practical application of ethanol in the fraction of deoxyribonucleic acid has been demonstrated in Table 1. Two requirements must be met by the precipitating solvent to bring about a satisfactory separation of deoxyribonucleic acid from degraded ribonucleic acid. The deoxyribonucleic acid must precipitate out as a fibrous mass; the ribonucleic acid fragments must either remain soluble or precipitate out as a flocculent mass which can easily be separated from the fibrous and loosely Woven deoxyribonucleic acid. In addition to alcohol, acetone meets this requirement. Methanol, dioxane, and monomethyl ether do not.

In the following Table V acetone separation of ribonucleic acid from deoxyribonucleic acid in a preparation of mixed ribonucleic acid and deoxyribonucleic acid obtained without the addition of orthophosphate during the incubation step at 37 is illustrated. The mixture was treated in the same manner as in the examples given in Table I. Following the last ethanol precipitation aliquots of the preparation of which 36% was ribonucleic acid were dissolved in ml. of Water, 1, 2, 4 and 8% NaCl. A predetermined amount of acetone was added to each sample (that amount which will produce the fibrous precipitate of deoxyribonucleic acid). The fibrous precipitate was redissolved in 10 ml. of fresh solution and reprecipitated as before with acetone. This process was repeated for a total of three times. The results of the fractionation are as follows:

As shown by these data the separation of ribonucleic acid from deoxyribonucleic acid with acetone was excellent at low salt concentrations. The low yields at low and high salt concentrations result from the poor recoveries from the solutions; the deoxyribonucleic acid does not form a firm fibrous mat. The optimum concentrations of salt for the final purification stages is recommended at 2%. Lower salt concentrations result in progressive and extensive damage to the structure of deoxyribonucleic acid.

Although acetone has apparently successfully fractionated deoxyribonucleic acid fror ribonucleic acid Without extensive phosphorylysis of ribonucleic acid the procedure is not recommended without the phosphorylysis step because the general quality and quantity of deoxyribonucleic acid obtained is very poor and Similar to the material illustrated in Example 3 of Table I. However, in conjunction with the phosphorylysis step acetone fractionation is the method of choice.

The concentration of sodium chloride used during lysis and phosphorylysis is set at 0.5% because it has been found that this concentration of salt results in maximum rates of lysis by lysozyme. The use of trishydroxymethyl aminomethane is not necessary. The proper pH may be obtained with the addition of suitable amounts of 1.0 1* NaOH or KGH. The concentration of sodium chloride (potassium chloride may be used) used in the heating extraction step is that require to prevent the destructior of the deoxyribonucleic acid by heat. Extraction Witl water results in complete degradation of the deoxyribo nucleic acid such that no fibrous material can be re covered. The established practice in this field has been tc use a 10% solution but the limits may be as low as 2% am as high as 15%.

To reiterate briefly, the instant invention relates to a1 improved process for the separation of deoxyribonuclei acid from mixtures of deoxyribonucleic acid and ribo nucleic acid and particularly to the preparation of deoxy ribonucleic acid from the mixture of deoxyribonuclei acid and ribonucleic acid obtained from lysing Micrococ cus lysodeikticus cells. The process of the invention com prises the steps of selective destruction of ribonuclei acid in the presence of orthophosphate ions and separz tion of the deoxyribonucleic acid from the mixture.

What is claimed is:

1. A process for preparing deoxyribonucleic acid whic comprises the steps of lysing cells of Micrococcus lyst deikticus in the presence of lysozyme and orthophosphat ions at a pH within the range of 8.3 to 8.7, incubating tl mixture at a temperature of from 30 to 40 for from 3 to 60 minutes, adding to the heated mixture sodium chlc ride and water, raising the temperature to one within tl range of from to 95 C. and maintaining that ten perature for from 20 to 30 minutes, separating the residi from the mixture at room temperature, precipitating tl deoxyribonucleic acid from the solution obtained with material selected from the group of ethanol and aceton and purifying the deoxyribonucleic acid thus obtained.

2. A process for preparing deoxyribonucleic acid fro the mixture of deoxyribonucleic acid and ribonucleic ac present in Micrococcus lysodeikticus cells which compris the steps of suspending Micrococcus lysodeikticus cells a solution of sodium chloride, adding to said suspensii crystalline lysozyme and a salt of orthophosphoric aci adjusting the pH of the mixture to one within the ran of from 8.3 to 8.7, incubating said mixture at about 3 C. for about 30 minutes, adding to said incubating mi ture a solution of sodium chloride and water suflicient increase the volume thereof and reduce the viscosity the mixture, heating the resulting mixture to a temperatr of about C. for from 30 to 60 minutes, cooling t heated mixture to room temperature, separating the soli from the cooled mixture, adding to the separated liqi acetone to precipitate the deoxyribonucleic acid the sodium chloride solution, redissolving the deoxyrit nucleic acid in a salt solution, treating said solution w chloroform to remove undesirable constituents, preci tating the purified deoxyribonucleic acid from said chlo: form treated solution with acetone, redissolving the p cipitated deoxyribonucleic acid in 2% sodium chlori' reprecipitating the deoxyribonucleic acid with acetone, peating cycle until deoxyribonucleic acid is freed of C( taminating orthophosphate ions and ribonucleic acid fragments, precipitating the final product with acetone and drying in [ethanol], acetone and ether and in vacuo.

' 3; A process for the separation of deoxyribonucleic acid'from a bacteriological material containing endogenous 'deoxyribonucleic acid, ribonucleic acid and polynucleotide phosphorylase which comprises fragmenting said bacteriological material without causing extensive damage to the endogenous deoxyribonucleic acid, ribonucleic acid and polynucleotide phosphorylase, selectively degrading the ribonucleic acid by contacting'the fragmented bacteriological material with orthophosphate ions at a pH within the range of from 7.5 to 9, and separating deoxyribonucleic acid from the resulting mixture. 4. A process according to claim 3 wherein the bacteriolog cal material is fragmented by lysis.

5. A process according to claim 3 wherein the bacteriological material is Micrococcus lysodeikticuc cells.

6. A process for the separation of deoxyribonucleic acid from a bacteriological material containing endogenous deoxyribonucleic acid, ribonucleic acid and polynucleotide phosphorylase which comprises fragmenting said bacteriological material without causing extensive damage to the endogenous deoxyribonucleic acid, ribonucleic acid and polynucleotide phosphorylase,

selectively degrading the ribonucleic acid by contacting the fragmented bacteriological material with orthophosphate ions at a pH within the range of from 7.5 to 9,

adding a salt solution,

heating to a temperature of about 85 to 95 C. for

about to minutes,

and separating deoxyribonucleic acid from the resulting mixture.

7. A process for the preparation of deoxyribonucleic acid which comprises the steps of lysing cells of Micrococcus lysodeikticus by lysozyme in the presence of orthophosphate ions at a pH within the range of 8.3 to 8.7,

heating the lysed mixture to a temperature of within the dange of to C. for from 20 to 30 minutes in the presence of salt,

and separating deoxyribonucleic acid from the heated mixture.

References Cited by the Examiner The following references, cited by the Examiner, are of record in the patented file of this patent or the original patent.

Palamade et aL: Cpmpt. rend., 242 June 11, 1956, pp. 2870-2872.

Beers: Nature 178, 595596,.Sept. 15, 1956.

A. LOUIS MONACELL, Primary Examiner.

ABRAHAM H. WINKELSTEIN, Examiner.