United States Patent [191 Gilham et al.
[451 May 1, 1973 [54] POLYNUCLEOTIDE ANALYSIS [75] Inventors: Peter Thomas Gilham; Herbert Lee Weith, both of West Lafayette, lnd.
[73] Assignee: Purdue Research Lafayette, Ind.
[22] Filed: Aug. 27, 1971 [2l] App]. No.: 175,756
Foundation,
[52] US. Cl. ....195/103.5 R, 195/28 N, 260/2115 R OTHER PUBLICATIONS Method in EnZymOl gY, Volume Xll, Nucleic Acids Part B Pages 224-235 (1968).
Primary Examiner-Alvin E. Tanenholtz Attorney-Joseph C. Schwalbach et al.
[ 5 7 ABSTRACT Sequential analysis of a polynucleotide to determine the particular order of nucleoside units therein can be conveniently carried out by adsorbing a polynucleotide on a strongly basic anion-exchange material, oxidizing the terminal nucleoside of the polynucleotide with a periodate, removing any excess periodate by reaction with L-rhamnose, treating the adsorbed polynucleotide with an amine to remove the terminal nucleoside residue from the polynucleotide molecule and with a phosphatase to remove the resulting terminal phosphate group from the remaining polynucleotide molecule, separating the so-produced nucleoside residue from the adsorbed polynucleotide for subsequent identification and then repeating the above procedure for each remaining nucleoside unit of the polynucleotide.
4 Claims, No Drawings POLYNUCLEOTIDE ANALYSIS BACKGROUND OF THE INVENTION Polynucleotides or polyribonucleotides are known to be long chain polymers containing various individual nucleoside or ribonucleoside units. Each nucleoside unit consists of a ribose containing a purine or pyrimidine substituent. The ribose portions of adjacent nucleosides are linked through phosphate groups. It is often of importance in biochemical and medical research to know the specific order in which the nucleoside units are attached in the formation of the polynucleotide molecule. Various techniques have been proposed in the prior art for degradation of the polynucleotide molecule into separate nucleoside fragments which can then be individually analyzed to deter mine the purine or pyrimidine bases from which they were formed. The final desired analytical result is the particular sequence of bases in the polynucleotide chain.
One technique proposed for analysis ofa polynucleotide involved exonucleolytic enzymes which allegedly would split off the terminal nucleoside units one at a time for subsequent analysis. This enzymatic technique was not successful because the proposed enzymes had variable and non-reproducible activity and produced inaccurate results.
A stepwise chemical and enzymatic degradation procedure was then proposed. This process involved reaction with a phosphatase to remove the terminal 3' phosphate group of the polynucleotide, oxidation of the unsubstituted cis-hydroxyl groups of the terminal nucleoside unit to dialdehyde groups, followed by alkaline catalyzed elimination of the terminal nucleoside fragment. The so-produced fragment was then identified for its purine or pyrimidine substituent. This procedure was then repeated for each nucleoside unit of the polynucleotide molecule. This proposed procedure had several disadvantages. First, there was no simple and efficient means for separating the liberated nucleoside fragment since all the reaction components and products were in solution. Second, great care must be taken to avoid the simultaneous presence in the reaction mixture of the phosphatase, periodate and alkali. Otherwise, the cleavage of the nucleoside fragments might occur in an uncontrolled manner to produce erroneous results.
A process improvement was then suggested to employ ion exchange chromatography to separate the liberated nucleoside residue from the remaining polynucleotide molecule after each degradation cycle. This was successful but had the disadvantages of being quite time consuming and of sustaining significant material losses. It could therefore be used only for a relatively few degradation cycles and thus could not be used for analysis of more complex polynucleotide molecules.
Attempts to precipitate the liberated nucleoside residues in order to separate them from the remaining polynucleotide molecule have also been unsuccessful due to excessive manipulation and consequent losses of material.
It is an object of the present invention to provide an accurate and convenient process for the sequential degradation of a polynucleotide into distinct reproducible nucleoside fragments which can subsequently be identified as to their purine or pyrimidine bases.
SUMMARY or THE INVENTION In accordance with the present invention, a process is provided for the sequential analysis of a polynucleotide which comprises (1) adsorbing on a strongly-basic anion-exchange material a polynucleotide having its terminal 3' phosphate group previously removed, (2) treating the adsorbed polynucleotide with a periodate to oxidize the unsubstituted cis-hydroxyl groups of the terminal nucleoside unit of the polynucleotide to dialdehyde groups, (3) adding L-rhamnose to react with and remove any remaining periodate material, (4) treating the adsorbed polynucleotide with an amine to remove the terminal nucleoside unit from the polynucleotide molecule and at substantially the same time with a phosphatase to remove the resulting terr minal 3' phosphate group from the remaining polynucleotide molecule, (5) separating the soproduced nucleoside residue from the adsorbed polynucleotide for subsequent identification, and then repeating the above steps (2) through (5) inclusive for each remaining nucleoside unit of the polynucleotide.
DESCRIPTION OF THE ENVENTION The polynucleotides useful as raw materials in the sequential analysis process of the present invention are well known polyribonucleotide compounds which occur naturally in biological materials or can be produced synthetically. In order to be initially useful in this process the polynucleotide must have its terminal 3' phosphate group removed. This is conveniently accomplished through the known use of an alkaline phosphatase.
The strongly basic anion-exchange materials useful in the present invention are well-known and are commercially available. They are prepared, for example, by suspension polymerization of styrene and divinylbenzene. The resulting polymer beads are reacted with chloromethyl ether, in the presence of aluminum chloride or zinc chloride catalyst, to introduce CHgCl groups on the benzene rings of the polymer.
This product is then aminated with trimethylamine, for example, to form a highly ionized quaternary ammonium group on the benzene rings.
Strongly basic anion-exchange resins having quaternary ammonium reactive groups are sold under the following illustrative tradenames by the indicated suppliers.
Tradename Supplier Dowex 1 Dow Chemical Co. Dowex 2 Dow Chemical Co. Dowex 21 K Dow Chemical Co. Amberlite IRA-400 Rohm and Haas Co. Amberlite C6400 Rohm and Haas Co. Amberlite [RA-401 Rohm and Haas Co. Nalcite SBR National Aluminate Co. Nalcite SBR? National Aluminate Co.
Duolite A-lOl D Duolitc A-l02 D Pen'nutit S-l00 Permutit 8-200 Diamond Alkali Co. Diamond Alkali Co. The Permutit Co. The Permutit Co.
The periodate compounds useful to oxidize the cishydroxyl groups of the dephosphorylated terminal nucleoside unit of the polynucleotide are well-known, and the general reaction conditions are known.
The use of hydroxyl-containing materials, such as ethylene glycol and butane-2, 3-diol,to react with excess periodate is also known. It is preferred in the process of the present invention to employ L-rhamnose since this material has been found to be most efficient and is the fastest reacting substance for this purpose. This tends to reduce the overall process time, which is an advantage over the prior art.
The use of alkaline materials, such as amines, to degrade the polynucleotide by removal of the terminal nucleoside fragment is known in the art. It is preferred in the process of the present invention to employ a mixture of cyclohexylamine and N,N,N,N'-tetramethylglycinamide-HCl since this mixture provides improved pH control at the desired level of pH 8.5 during this step of the overall process.
While the temperature conditions under which this process is carried out are not narrowly critical, it is preferred that the reaction of the polynucleotide with the periodate, the treatment with the L-rhamnose and the separation of the degraded nucleoside fragment from the adsorbed polynucleotide be carried out at about 1 C. and the amine reaction with the polynucleotide to degrade and remove the terminal nucleoside fragment be carried out at about 45 C.
The principal point of technical advancement of the present invention resides in the adsorption of the polynucleotide on an insoluble support, reacting various materials with this insolubilized form of polynucleotide and easily separating the soluble degraded nucleoside fragments from the insolubilized remaining portion of the polynucleotide. It is important, therefore, at the time that reaction products are to be separated from the polynucleotide that all of the remaining polynucleotide be adsorbed by the anionexchange material. This is accomplished by dilution of the liquid in contact with the anion-exchange material to the point that the concentrations of anions, other than'those of the polynucleotide, are reduced to a level such that they do not displace the polynucleotide being adsorbed by the anion-exchange material. The specific conditions under which a polynucleotide is released from the anion-exchange material and readsorbed by it are dependent on the size of the polynucleotide molecule. For example, a polynucleotide having ten nucleoside units is released from the anion-exchange material when the competitive anion concentration exceeds about 1 molar. Such a polynucleotide is completely readsorbed when the displacing anion concentration is reduced by dilution to about 0.1 molar. A polynucleotide containing only two nucleoside units is released when the competitive anion concentration exceeds about 0.4 molar and is completely readsorbed when the competitive anion concentration is below about 0.05 molar.
When the nucleoside fragment is separated from the polynucleotide, it can be analyzed for its purine or pyrimidine base by well-known methods. For example, the effluent from the degradation cycle containing the terminal nucleoside unit, amine and phosphatase is evaporated to dryness. Formic acid is added, and the resulting reaction mixture is heated in an autoclave.
This acid treatment converts the terminal nucleoside residue into free purine or pyrimidine base which is then identified by anion exchange chromatography.
The process of the present invention is described in additional detail in the following illustrative example.
EXAMPLE A 0.1 ml portion of Dowex 1 X 2 anion-exchange resin in the chloride form and having a particle size of minus 400 mesh was placed in a glass tube and positioned by plugs of glass wool. The resulting resin bed was washed with a buffer mixture of 0.5 molar sodium chloride and 0.01 molar tris (hydroxymethyl) aminomethane having a pH of 7.5 and then with cold distilled water to remove excess buffer solution. The temperature of the resin bed was maintained at about 1 C. by means ofa water bath surrounding the resin bed.
The polynucleotide to be analyzed was then treated with alkaline phosphatase to remove the terminal 3 phosphate group. An aqueous solution containing about nanomoles of the thus dephosphorylated polynucleotide was passed through the above resin bed and recirculated through the bed several times by means of a recirculating pump and associated tubing. Most of the polynucleotide was adsorbed by the resin. Any unadsorbed polynucleotide was then removed from the resin by further washing with distilled water. A 0.5 ml. portion of 0.2 molar sodium metaperiodate solution was then passed through the bed and recirculated through the bed at 1 C. for about 15 min. This periodate solution oxidized the cis-hydroxyl groups on the terminal nucleoside unit to dialdehyde groups and, because of its ionic effect, also displaced the polynucleotide from the resin. The solution being recirculated through the resin thus contained polynucleotide. A 0.5 ml. portion of 1 molar L-rhamnose solution was then added to the circulating solution, and the recirculation through the bed was continued for 5 min. during which time the L-rhamnose destroyed any previously unreacted periodate. A 4.6 ml. portion of cold distilled water was then added to the reaction vessel so as to dilute the resulting iodate ion concentration to about 0.017 molar. Recirculation of the total liquid mixture was continued for 10 minutes to allow the polynucleotide to become readsorbed by the resin bed. The liquids were then drained from the resin bed, and the resin bed was washed with 1 ml. of cold distilled water. A 0.1 ml. portion of bacterial alkaline phosphatase was then added to the resin bed followed by 0.1 ml. of an amine solution containing 1 molar cyclohexylamine and 2 molar N,N,N',N' -tetramethylglycinamide-HCI. An additional 0.1 ml. of amine solution was added and the liquids were circulated through the resin bed at 45 C. for 2 hours. This aminephosphatase mixture removed the terminal nucleoside unit from the remainder of the polynucleotide molecule and also removed the so-generated terminal 3 phosphate group. This solution, because of its ionic effect, also displaced the polynucleotide from the resin bed. A 5.0 ml. portion of distilled water was then added a to the reaction mixture so as to dilute theamine concentration to about 0.04 molar. The temperature in the resin bed was reduced to about 1 C. and the above liquid mixture was recirculated through the resin bed at 1 C. for 15 min. to allow the polynucleotide (minus its original terminal nucleoside unit) to become readsorbed by the resin bed. The diluted amine phosphatase-terminal nucleoside fragment mixture was then drained from the resin bed into a screw cap test tube. The reaction vessel and the resin bed were then washed with 1 ml. of cold distilled water which was also drained into the above test tube. The total time for the above periodate oxidation, terminal nucleoside elimination and dephosphorylation was about 200 min. The resin bed containing adsorbed polynucleotide was then treated again by the above reaction steps to eliminate a further terminal nucleoside unit. This procedure was repeated until all the neucleoside units of the polynucleotide were separately removed.
Each of the combined effluents from a single degradation cycle having an average volume of about 8 ml. was individually heated at 100 C. in a sealed tube for two hours. The resulting free purine or pyrimidine base in each test tube was individually analyzed by anion exchange chromatography.
This above procedure was employed to confirm the l. A process for the sequential analysis of a polynucleotide which comprises l) adsorbing on a strongly-basic anion-exchange material a polynucleotide having its terminal 3' phosphate group previously removed, (2) treating the adsorbed polynucleotide with a periodate to oxidize the unsubstituted cishydroxyl groups of the terminal nucleoside unit of the polynucleotide to dialdehyde groups, (3) adding L- rhamnose to react with and remove any remaining periodate material, (4) treating the adsorbed polynucleotide with an amine to remove the terminal nucleoside unit from the polynucleotide molecule and at substantially the same time with a phosphatase to removethe resulting terminal 3' phosphate group from the remaining polynucleotide molecule, (5 separating the so-produced nucleoside residue from the adsorbed polynucleotide for subsequent identification, and then repeating the above steps (2) through (5) inclusive for each remaining nucleoside unit of the polynucleotide.
2. A process according to claim 1 wherein steps (2), (3) and (5) take place at about 1 C. and step (4) takes place at about 45 C.
3. A process according to claim 1 wherein prior to steps (2), (4) and (5) the concentrations of anions, other than those of the polynucleotide, in the liquid in contact with the anion-exchange material are reduced to a level such that they do not displace the polynucleotide from being adsorbed by the anion-exchange material.
4. A process according to claim 1 wherein the quaternary ammonium reactive groups.