A solid or semisolid, at least two-phase active ingredient-containing formulation in which there is multiparticulate incorporation of one of the two phases into a matrix of the other phase, and at least one of the phases contains at least one active ingredient, obtainable by introducing particles of one phase into the other phase in a plastic state, and shaping the material while still plastic.

The present invention relates to solid, at least two-phase active ingredient-containing formulations in which there is multiparticulate incorporation of one of the two phases into a matrix of the other phase, and at least one of the phases contains at least one active ingredient, obtainable by introducing particles of one phase into the other phase in a plastic state, and shaping the material while still plastic. The invention furthermore relates to a process for producing such forms.
A problem which frequently occurs in pharmaceutical technology is to introduce mutually incompatible active ingredients into a drug form.
This is solved in the prior art by producing laminated or multilayered tablets. These tablet forms permit not only incompatible active ingredients to be separated but also the initial and maintenance doses to be separated in controlled release drug forms. These drug forms are normally obtained by conventional compressing. However, this requires specially designed tableting machines and at least two filling and compression stations (cf. xe2x80x9cPharmazeutische Technologiexe2x80x9d, Georg Thieme Verlag, 4th edition, 1993, pages 300 et seq.). This process is, however, elaborate and costly.
It is furthermore known to produce mixed granule tablets by compressing a mixture of differently pretreated granules. This entails, for example, processing untreated medicinal substances (initial dose) together with medicinal substances enveloped in fats or coated with lacquer (maintenance dose) (cf. R. Voigt, Lehrbuch der pharmazeutischen Technologie, 1987, page 225). This process is also relatively elaborate.
EP-A 580 860 discloses that it is possible to meter water or solvent in during the extrusion process. However, metering in of active ingredients is not described.
It is an object of the present invention to find a simple process for producing active ingredient-containing forms which permits the introduction of mutually incompatible active ingredients or active ingredients of different release into one drug form or the production of forms with multiphase release characteristics.
We have found that this object is achieved by the formulations defined at the outset.
It is preferred according to the invention to incorporate active ingredient-containing particles into a melt which may likewise contain active ingredient or else be free of active ingredient.
Possible particles according to the invention are granules, pellets or crystal particles, and crystal particles are preferably coated.
The particles can be obtained in a conventional way, for example by wet granulation of one or more active ingredients with conventional additives. If the active ingredient automatically results as granules in the preparation process, it can also be employed without further treatment with additives. Granules can also be obtained in a conventional way by melt extrusion of an active ingredient-containing polymer melt and subsequent shaping by hot or cold cut, prilling or drop-formation processes. Pellets can also be produced by conventional processes, for example by dry granulation. Coated or uncoated active ingredient crystals can also be obtained by processes known to the skilled worker. The particle size is not critical. For ease of handling of the particles it is advisable to use particles with a size of the order of from 0.01 to 3 mm, preferably 0.5 to 2 mm.
Particles for the purpose of this invention are also microtablets. Microtablets can likewise be produced in a conventional way.
As already mentioned, the particles can consist of pure active ingredient without containing other additives. If additive-containing particles are employed, the nature of the additives depends in particular on the release rate required for the particulate phase, ie. whether the particulate phase is to be rapid or slow release.
If coated active ingredient crystals are used, the coating may have purely stabilizing or else release-slowing properties. It is also possible to employ coatings which dissolve in particular pH ranges.
It is possible in this way to obtain combination drug forms in which the active ingredient(s) can be released from the particulate portions at various points in the digestive tract. Examples of possible coatings of this type are polyacrylates or methacrylic acid copolymers (Eudragit types).
The content of the aliquot added as particles can be from 0.01 to 90%, preferably 0.1 to 70%, particularly preferably 0.5 to 50%, of the total weight of the finished active ingredient-containing form.
In the formulation according to the invention there is multiparticulate incorporation of the particles in another phase. Thus the other phase forms the matrix for the particulate phase. The matrix phase may contain active ingredient or be free of active ingredient. An active ingredient-containing matrix phase is preferred.
It is possible in principle to employ all substances which can be melted or softened to constitute the matrix phase as long as they do not decompose under the processing conditions. The required thermoplasticity can also be brought out by adding suitable auxiliaries.
Examples of suitable constituents of the matrix are melt-processable polymeric binders. Suitable as such are uncrosslinked homo- or copolymers of N-vinylpyrrolidone with Fikentscher K values of from 12 to 120, preferably 20 to 100, with suitable comonomers preferably being vinyl esters such as vinyl propionate or vinyl butyrate or, in particular, vinyl acetate, or else N-vinylimidazole or N-vinylcaprolactam.
Further suitable binders are cellulose derivatives such as cellulose ethers, for example cellulose alkyl ethers such as methyl- or ethylcellulose or hydroxyalkylcelluloses such as hydroxypropylcellulose, also cellulose esters such as cellulose acetate, cellulose phthalate, cellulose acetate propionate, cellulose acetate phthalate or the like. Also suitable as binders are acrylate- or methacrylate-containing polymers, for example Eudragit types.
Also suitable according to the invention are matrix polymers which can be absorbed or degraded in the body. These include polylactic acid and copolymers thereof, poly(ortho)esters. Polyamides, polyphosphazenes or polyurethanes are also suitable.
Likewise suitable as matrix polymers are starch or dextrins.
Suitable matrixes according to the invention are also those composed of sugar alcohols such as erythritol, sorbitol, maltitol, mannitol, isomalt, mono- or disaccharides such as fructose or glucose.
Also suitable as matrix constituents are fatty acid glycerides and/or fatty acid polyethylene glycol esters. The latter can also be packed as semisolid extrudates with the incorporated particles into capsules. It is particularly advantageous that the softening point of these substances is relatively low and thus reliable metering in is ensured without the risk of melting the added particles. Such easily softening formulations are likewise suitable for producing suppositories and chewable compositions.
Suitable matrix polymers are also polyethylene glycols with molecular weights in the range from 1000 to 20,000.
It is furthermore possible to incorporate conventional physiologically tolerated ancillary substances into the matrix, for example bulking agents, lubricants, mold release agents, plasticizers, blowing agents, stabilizers, dyes, flavorings or flow regulators.
Examples of bulking agents are inorganic bulking agents such as the oxides of magnesium, aluminum, silicon, titanium etc. in a concentration of from 0.01 to 50, preferably from 0.20 to 20, % of the total weight of the drug form.
Examples of lubricants are stearates of aluminum, calcium and magnesium, and talc and silicones in a concentration of from 0.1 to 5, preferably from 0.1 to 3, % of the total weight of the form.
Examples of disintegration promoters which can be employed are sodium carboxymethyl starch and crospovidone. It is also possible to employ wetting agents such as sodium lauryl sulfate and sodium docusate. Salts such as Na2CO3 or NaHCO3 can also be employed.
Examples of plasticizers comprise low molecular weight poly(alkylene oxides), such as poly(ethylene glycols), poly(propylene glycols), poly(ethylene/propylene glycols); organic plasticizers with a low molecular weight such as glycerol, pentaerythritol, glycerol monoacetate, diacetate or triacetate, propylene glycol, sodium diethyl sulfosuccinate and similar compounds added in concentrations of from 0.5 to 15, preferably from 0.5 to 5, % of the total weight of the drug form.
Different coloring of the various phases allows the forms to be made attractive with high recognizability. Examples of dyes are known azo dyes, organic and inorganic pigments, or coloring agents of natural origin. Inorganic pigments are preferred, in concentrations of from 0.001 to 10, preferably from 0.5 to 3, % of the total weight of the drug form.
Flavorings and aromas such as vanillin are preferably present in the carrier matrix.
It is also possible furthermore to add other additives which improve the flow properties of the mixture or act as mold release agents, eg. animal or vegetable fats, preferably in their hydrogenated form, especially those which are solid at room temperature. These fats preferably have a melting point of 50xc2x0 C. or above. Triglycerides of C12, C14, C16 and C18 fatty acids are preferred. The same function can also be carried out by waxes such as carnauba wax. These additives can be added alone without addition of bulking agents or plasticizers. These fats and waxes can advantageously be admixed alone or together with mono- and/or diglycerides or phosphatides, especially lecithin. The mono- and diglycerides are preferably derived from the fat types described above, ie. C12, C14, C16 and C18 fatty acids. The total amount of fats, waxes, mono- and diglycerides and/or lecithins is 0.1-30, preferably 0.1-5, % of the total weight of the drug form.
Examples of flow regulators which can be used are aerosils or talc.
It is also possible furthermore to add stabilizers such as antioxidants, light stabilizers, hydroperoxide destroyers, radical scavengers and stabilizers against microbial attack.
Ancillary substances for the purpose of the invention also mean substances for producing a solid solution with the pharmaceutical active ingredient. Examples of these ancillary substances are pentaerythritol and pentaerythritol tetraacetate, polymers such as polyethylene oxides and polypropylene oxides and their block copolymers (poloxamers), phosphatides such as lecithin, homo- and copolymers of vinylpyrrolidone, surfactants such as polyoxyethylene 40 stearate, and citric and succinic acids, bile acids, sterols and others as indicated, for example, in J. L. Ford, Pharm. Acta Helv. 61, 69-88 (1986).
Bases or acids added to control the solubility of an active ingredient are also regarded as pharmaceutical ancillary substances (see, for example, K. Thoma et al., Pharm. Ind. 51, 98-101 (1989)).
Suitable active ingredients for the purpose of this invention are in principle all active ingredients which do not decompose under the processing conditions.
The process according to the invention is suitable, for example, for processing the following active ingredients:
acebutolol, acetylcysteine, acetylsalicylic acid, acyclovir, alprazolam, alfacalcidol, allantoin, allopurinol, ambroxol, amikacin, amiloride, aminoacetic acid, amiodarone, amitriptyline, amlodipine, amoxicillin, ampicillin, ascorbic acid, aspartame, astemizole, atenolol, beclomethasone, benserazide, benzalkoniumhydrochloride, benzocaine, benzoic acid, betamethasone, bezafibrate, biotin, biperiden, bisoprolol, bromazepam, bromhexine, bromocriptine, budesonide, bufexamac, buflomedil, buspirone, caffeine, camphor, captopril, carbamazepine, carbidopa, carboplatin, cefachlor, cefalexin, w cefadroxil, cefazolin, cefixime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, selegiline, chloramphenicol, chlorhexidine, chlorpheniramine, chlortalidone, choline, cyclosporin, cilastatin, cimetidine, ciprofloxacin, cisapride, cisplatin, clarithromycin, clavulanic acid, clomipramine, clonazepam, clonidine, clotrimazole, codeine, cholestyramine, cromoglycic acid, cyanocobalamin, cyproterone, desogestrel, dexamethasone, dexpanthenol, dextromethorphan, dextropropoxiphene, diazepam, diclofenac, digoxin, dihydrocodeine, dihydroergotamine, dihydroergotoxin, diltiazem, diphenhydramine, dipyridamole, dipyrone, disopyramide, domperidone, dopamine, doxycycline, enalapril, ephedrine, epinephrin, ergocalciferol, ergotamine, erythromycin, estradiol, ethinylestradiol, etoposide, Eucalyptus globulus, famotidine, felodipine, fenofibrate, fenoterol, fentanyl, flavin mononucleotide, fluconazole, flunarizine, fluorouracil, fluoxetine, flurbiprofen, furosemide, gallopamil, gemfibrozil, gentamicin, Gingko biloba, glibenclamide, glipizide, clozapine, Glycyrrhiza glabra, griseofulvin, guaifenesin, haloperidol, heparin, hyaluronic acid, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, ipratropium hydroxide, ibuprofen, imipenem, indomethacin, iohexol, iopamidol, isosorbide dinitrate, isosorbide mononitrate, isotretinoin, ketotifen, ketoconazole, ketoprofen, ketorolac, labetalol, lactulose, lecithin, levocarnitine, levodopa, levoglutamide, levonorgestrel, levothyroxine, lidocaine, lipase, imipramine, lisinopril, loperamide, lorazepam, lovastatin, medroxypregesterone, menthol, methotrexate, methyldopa, methylprednisolone, metoclopramide, metoprolol, miconazole, midazolam, minocycline, minoxidil, misoprostol, morphine, multivitamin mixtures or combinations and mineral salts, n-methylephedrine, naftidrofuryl, naproxen, neomycin, nicardipine, nicergoline, nicotinamide, nicotine, nicotinic acid, nifedipine, nimodipine, nitrazepam, nitrendipine, nizatidine, norethisterone, norfloxacin, norgestrel, nortriptyline, nystatin, ofloxacin, omeprazole, ondansetron, pancreatin, panthenol, pantothenic acid, paracetamol, penicillin g, penicillin v, phenobarbital, pentoxifylline, phenoxymethylpenicillin, phenylephrine, phenylpropanolamine, phenytoin, piroxicam, polymyxin b, povidone-iodine, pravastatin, prazepam, prazosin, prednisolone, prednisone, propafenone, propranolol, proxyphylline, pseudoephedrine, pyridoxine, quinidine, ramipril, ranitidine, reserpine, retinol, riboflavin, rifampicin, rutoside, saccharin, salbutamol, salcatonin, salicylic acid, simvastatin, somatropin, sotalol, spironolactone, sucralfate, sulbactam, sulfamethoxazole, sulfasalazine, sulpiride, tamoxifen, tegafur, teprenone, terazosin, terbutaline, terfenadine, tetracycline, theophylline, thiamine, ticlopidine, timolol, tranexamic acid, tretinoin, triamcinolone acetonide, triamterene, trimethoprim, troxerutin, uracil, valproic acid, vancomycin, verapamil, vitamin e, folinic acid, zidovudine, zotepine.
Active ingredients for the purpose of the invention are also vitamins and minerals, and crop treatment agents and insecticides. The vitamins include vitamins of A group, of the B group, by which are meant, besides B1, B2, B6 and B12 and nicotinic acid and nicotinamide, also compounds with vitamin B properties such as adenine, choline, pantothenic acid, biotin, adenylic acid, folic acid, orotic acid, pangamic acid, carnitine, p-aminobenzoic acid, myo-inositol and lipoic acid, and vitamin C, vitamins of the D group, E group, F group, H group, I and J groups, K group and P group. Active ingredients for the purpose of the invention also include therapeutic peptides.
In a few cases there may be formation of solid solutions. The term xe2x80x9csolid solutionxe2x80x9d is familiar to the skilled worker, for example from Chiou and Riegelman, J. Pharm. Sci. 60, 1281-1302 (1971). In solid solutions, the active ingredient is in the form of a molecular dispersion in the matrix.
The active ingredient content per dose unit and the concentration may be varied within wide limits depending on the activity and release rate. The only condition is that they are sufficient to achieve the required effect. Thus, the active ingredient concentration can be in the range form 0.1 to 90, preferably from 0.5 to 60, .% by weight. These data likewise apply to the food supplement sector, such as vitamin products.
The formulations according to the invention can be produced as described below:
Firstly, the matrix components are converted into a plastic state. This may entail either a premix of all the components being plasticized, or first the polymeric binder being softened and then the other components, ie. active ingredients and/or other ancillary substances, being added thereto. The plasticization is effected by input of energy. Depending on the composition of the matrix, the components soften in the range from 40 to 190xc2x0 C., preferably 50 to 150xc2x0 C. The suitable temperature range in each case depends on the glass transition temperature of the polymeric binders, the properties of the active ingredients which are added where appropriate, and any plasticizers which are added. This is preferably done in the absence of solvents such as water or organic solvents. However, it may also be advisable to add small amounts of water as plasticizer. The optimal temperature range can be established by a few simple tests. The mixture of matrix components should soften so that the corresponding plastic material has a specific viscosity of from 0.0007 to 10,000 Pa.s, preferably 0.001 to 3000 Pa.s (at 150xc2x0 C.).
The softening process can take place in an extruder, a kneader or a mixing reactor, with the plastic material being homogenized by longitudinal and transverse mixing.
The plasticization preferably takes place in an extruder having one or more screws which may rotate in the same direction or opposite directions, especially in a twin screw extruder. The latter can be operated with or without kneading elements, but use of kneading elements is preferred because mixing is better.
The particulate phase is added during the plasticization process. On use of an extruder, this can take place in the hot zone (hot feed) or in the cold zone. The particles are preferably fed in continuously via weigh feeders.
The temperature at which addition of the particulate phase takes place depends on the nature of the active ingredients and the type of release profiles required. If, for example, pellets are added, it may be desirable for these to melt on the surface because this leads to an increase in the surface area, which facilitates onset of the diffusion process after administration. On the other hand, however, the temperature should be low enough for separate phases to be maintained. The required temperature can be established by a few simple tests.
After incorporation of the particulate phase into the matrix phase, the still plastic material is shaped to the required dosage forms.
If, for example, it is wished to incorporate the particulate phase into a crystalline sugar matrix, it is advisable for the plastic material to be cooled, before the shaping, with stirring to such an extent that it can still be shaped but extrusion does not result in a quenched, amorphous, glassy melt.
The still plastic material can be extruded through a die or breaker plate and then shaped in a conventional way to tablets, chewable tablets, pastilles, buccal tablets, sublingual tablets, chewing compositions such as chewing gum or suppositories, or be packed in capsules. Shaping preferably takes place by calendering or by injection molding. Multilayer tablets can also be produced by the conventional coextrusion process. It is also possible to produce multilayer tablets by injection molding.
It is possible with the aid of the process according to the invention to produce in a simple manner dosage forms with very different release profiles.
The compositions have at least two phases, but may also contain several phases if different particulate phases are used.
It is thus possible, for example, to incorporate mutually incompatible active ingredients each as separate particulate phases. In this case, the surrounding matrix performs the function of an envelope and may be free of active ingredient.
In the case of active ingredients with very different physical properties, optimal particulate formulations of each can likewise be incorporated into one dosage form in a simple manner.
It is possible and particularly advantageous to produce dosage forms with stepped release profiles. Thus, for example, slow-release granules or pellets of an active ingredient can be incorporated into an instant release matrix, particularly beneficially into a solid solution, which likewise contains this active ingredient. It is possible in this way to achieve a rapid rise in level and a prolonged plateau-like release profile with one dosage form. Any desired sequence profiles can be adjusted by using granules of one and the same active ingredient but with different dissolving characteristics. It is also possible to adjust profiles with a delayed onset of release of active ingredient and repeat profiles.
Compared with conventional forms obtained by compression, the forms obtained from plasticized materials display better and, on production of solid forms, lower porosity and better mechanical stability.

Multiphase active ingredient-containing formulations


Polyethylene having a broad molecular weight distribution is extruded in pellet form using a pelletizing extruder having one or more zones essentially filled with the polyethylene and two or more zones partially filled with the polyethylene. The polyethylene is fed into the extruder at a temperature sufficient to melt the polyethylene, and a mixture of an inert gas and oxygen is fed into at least one partially filled zone of the extruder with the exception of the feed zone. The mixture contains about 1 to about 21% by volume oxygen based on the volume of the gas mixture.

This invention relates to a process for the extrusion of polyethylene into pellets useful in the production of blown film.
Ziegler-type catalysts have undergone development over the years to improve the economy and quality of various polyethylene products. This development has tended towards narrowing the molecular weight distribution of the resin; however, narrow molecular weight distributions are not desirable for blown film resins, i.e., the narrow molecular weight distribution does not improve the processability of the resins into blown film in terms of bubble stability. Rather, bubble stability is a characteristic of broad molecular weight distribution resins.
A particularly good technique for producing broad molecular weight distribution polyethylenes is through the use of a two stage polymerization process similar to those mentioned in U.S. Pat. Nos. 5,047,468 and 5,149,738. Briefly, this process is one for the in situ blending of polymers wherein an ethylene copolymer is prepared in a high melt index reactor and an ethylene copolymer is prepared in a low melt index reactor, the two reactors being connected in series. The process typically comprises continuously contacting, under polymerization conditions, a mixture of ethylene and one or more alpha-olefins with a catalyst system in two gas phase, fluidized bed reactors connected in series, said catalyst system comprising: (i) a supported magnesium/titanium based catalyst precursor; (ii) an aluminum containing activator compound; and (iii) a hydrocarbyl aluminum cocatalyst, the polymerization conditions being such that an ethylene copolymer having a melt index in the range of about 0.1 to about 3000 grams per 10 minutes is formed in the high melt index reactor and an ethylene copolymer having a melt index in the range of about 0.001 to about 1 gram per 10 minutes is formed in the low melt index reactor, each copolymer having a density of about 0.860 to about 0.965 gram per cubic centimeter and a melt flow ratio in the range of about 14 to about 70, with the provisos that:
(a) the mixture of ethylene copolymer matrix and active catalyst formed in the first reactor in the series is transferred to the second reactor in the series;
(b) other than the active catalyst referred to in proviso (a) and the cocatalyst referred to in proviso (e), no additional catalyst is introduced into the second reactor;
(c) in the high melt index reactor:
(1) the alpha-olefin is present in a ratio of about 0.02 to about 3.5 moles of alpha-olefin per mole of ethylene; and
(2) hydrogen is present in a ratio of about 0.05 to about 3 moles of hydrogen per mole of combined ethylene and alpha-olefin;
(d) in the low melt index reactor:
(1) the alpha-olefin is present in a ratio of about 0.02 to about 3.5 moles of alpha-olefin per mole of ethylene; and
(2) hydrogen is, optionally, present in a ratio of about 0.0001 to about 0.5 mole of hydrogen per mole of combined ethylene and alpha-olefin; and
(e) additional hydrocarbyl aluminum cocatalyst is introduced into the second reactor in an amount sufficient to restore the level of activity of the catalyst transferred from the first reactor to about the initial level of activity in the first reactor.
While the resins produced by these two stage processes can be turned into products having superior mechanical strength and other advantageous physical and chemical characteristics, the resins generally do not achieve a high level of bubble stability.
It is well known that one way to achieve this characteristic is to tailor the broad molecular weight distribution resin after it is produced. Tailoring is simply controlled light crosslinking, which can be used in the fabrication of fibers, films, molded products, and the like to provide products having excellent physical and chemical properties for particular applications. It is often achieved by the use of additives, which are homogenized, alloyed and/or combined with the resin through various extrusion/mixing/pelletizing systems. In many cases, the additive is a critical factor in the commercial success of the final product.
Useful tailoring additives are free radical generators such as organic peroxides and oxygen. Unfortunately, excessive amounts of free radical generators can cause chain scission, which is characterized by a rupture of chemical bonds in the backbone and side chains of the polymer. The result is a decrease in the solid state strength of the resin product. Thus, the amount of free radical generator must be carefully controlled. In addition, organic peroxides increase operating costs because of the cost of the peroxides; additional equipment needed to safely handle the peroxides; and the presence of undesirable by-products, particularly when the tailored polymer will be used in FDA applications. In the case of oxygen tailoring, when melt temperatures become to high, the solid state strength of the blown film becomes unacceptably low. Also, high amounts of oxygen, e.g., 21 percent based on the volume of the gases used in extrusion systems, are often required due to the design of the equipment. But too much oxygen in the presence of unmelted polymer in the form of granules or powders can lead to dust explosions.
An object of this invention, therefore, is to provide a process for the tailoring of broad molecular weight distribution polyethylene whereby processability is enhanced in terms of bubble stability and high solid state strength is achieved in blown film while avoiding the drawbacks of the organic peroxides, and the high melt temperature and dust explosion problems of oxygen. Other objects and advantages will become apparent hereinafter.
According to the present invention such a process has been discovered. The process is one for the extrusion of polyethylene having a broad molecular weight distribution wherein the polyethylene is prepared in pellet form, said extrusion taking place in a pelletizing extruder having one or more zones essentially filled with polyethylene and two or more zones partially filled with polyethylene including a feed zone comprising (i) introducing the polyethylene into the extruder at a temperature sufficient to melt the polyethylene; (ii) introducing a mixture of an inert gas and oxygen into at least one of the partially filled zones other than the feed zone, said mixture containing about 1 to about 21 percent by volume oxygen based on the volume of the gaseous mixture; (iii) passing the molten polyethylene through each zone at melt temperature; and (iv) extruding the polyethylene into pellets and cooling same.
As noted, the polyethylene is preferably produced in two staged reactors connected in series wherein a mixture of resin and catalyst precursor is transferred from the first reactor to the second reactor in which another copolymer is prepared and blends in situ with the copolymer from the first reactor. The polyethylene can also be produced in one reactor as described, for example, in U.S. Pat. No. 4,302,565, or in three or more reactors provided that a broad molecular weight distribution polyethylene is made by the process.
The resin can be extruded into pellets in a conventional extruder adapted for that purpose. Extruders and processes for extrusion are described in U.S. Pat. Nos. 4,814,135; 4,857,600; 5,076,988; and 5,153,382. Examples of various extruders, which can be used in forming the pellets are single screw and multiscrew types. A typical pelletizing extruder can be illustrated by a two stage twin screw melter/mixer with a feed section and a vent section, a gear pump, a pelletizing device, and various other sections. Another typical pelletizing extruder can be illustrated by a two stage single screw extruder. Thus, the term xe2x80x9cextruderxe2x80x9d, as used in this specification, is considered to include conventional extruders and mixers, both of which are adapted to form pellets. A typical single screw type extruder can be described as one having a hopper at its upstream end and a die at its downstream end. The hopper feeds into a barrel, which contains a screw. At the downstream end, between the end of the screw and the pellet forming die, is a screen pack and a breaker plate. The screw portion of the two stage extruder is considered to be divided up into many sections, a feed section, a compression section, a metering section, which is followed by another feed section or a vent section, another compression section, a final metering section, and multiple heating sections from the rear heating section to the front heating section, the multiple heating-sections running from upstream to downstream. There can be one or more vent sections. If the extruder has more than one barrel, the barrels are connected in series. The length to diameter ratio of typical single screw extruders is in the range of about 16:1 to about 36:1, and the length to diameter ratio of typical twin screw mixers is in the range of about 5:1 to about 30:1.
In the process of this invention, the extrusion is carried out at a temperature sufficient to melt the resin. The extrusion generally takes place at temperatures in the range of about 140 to about 320 degrees C., and is preferably carried out at temperatures in the range of about 160 to about 280 degrees C. The temperature in the extruder is also sufficient to maintain the resin in the molten state until the pellets are formed and cooled.
In twin screw mixer/gear pump extrusion systems, which are typically starved fed, i.e., the feed section is partially filled, polymer temperature can be controlled by controlling the speed of the mixer, by adjusting feed rate, by adjusting mixing/temperature/residence time control devices, i.e., commercially available control devices uniquely designed by each twin screw manufacturer which are commonly referred to as xe2x80x9cgate positionxe2x80x9d in Kobe(trademark) LCM twin screw mixers, xe2x80x9cslot positionxe2x80x9d in JSW(trademark) CMP twin screw mixers, etc., and by adjusting gear pump suction pressure, U.S. Pat. No. 4,452,750. Typically, increasing speed, decreasing feed rate, closing xe2x80x9cmixingxe2x80x9d control devices, and increasing gear pump suction pressure increases polymer temperature for a given type and grade of polymer. Further, experience has shown that physical properties such as density, melt index, and flow index affect polymer temperatures. In most cases, as density increases, melt index decreases, flow index decreases, melt flow ratio decreases and molecular weight distribution decreases, polymer temperatures increase. Inlet feed temperature of resin being processed also has an effect on polymer temperature, In general, as inlet feed temperature increases, polymer temperature increases. It should be noted that in high capacity twin screw mixers, barrel metal temperature has only a minor effect on polymer temperature. This phenomenon is due to the low overall heat transfer coefficient of polymers, i.e., polymers are typically good insulators.
In two stage single screw extruders, which are typically flood fed, i.e., the feed section is essentially filled with particulate (unmolten) polymer, temperature control is typically adjusted by screw speed, extruder discharge pressure and barrel temperatures. In contrast to high capacity twin screw mixers, the overall capacity of single screw extruders is much lower on a rate to barrel diameter basis and therefore barrel temperature control has an increased effect on polymer temperature.
In flood fed extruders, although the feed section is filled with particulate polymer, typically in granular or pellet form, the gaseous mixture can still be successfully introduced due to the porosity of the particulates. In fact, depending on the design of the feed section, the flow rate of the gaseous mixture may need to be controlled to insure that the particulates in the feed section are not inadvertently fluidized, possibly reducing the overall capacity of the extruder.
For the purposes of this specification, the zones of the pelletizing extruder have been divided into essentially filled zones and partially filled zones. The xe2x80x9cessentiallyxe2x80x9d filled zones are those sections of the extruder that are almost completely filled with polyethylene, usually in the molten state. The xe2x80x9cpartiallyxe2x80x9d filled zones are those sections of the extruder that are partially filled with polyethylene. In the feed section, the polyethylene is in particulate form. In other sections, the polyethylene is generally in a molten state. The term xe2x80x9cpartiallyxe2x80x9d filled means that about 5 to about 95 percent of the zone (on a volume basis) is filled with polyethylene. Preferably about 5 to about 40 percent of the zone is filled, and most preferably about 10 percent of the zone is filled with polyethylene. In starved fed extruders, the partially filled zones are generally the feed and vent sections or zones. The zones, other than the partially filled zones, are operated as essentially filled zones.
The atmosphere in at least one of the partially filled zones with the exception of the feed zone is a mixture of an inert gas (such as nitrogen) and oxygen wherein the oxygen can be present in the range of about 1 to about 21 percent by volume based on the volume of the gaseous mixture, and is preferably in the range of about 1 to about 4 percent by volume. The preferred method for controlling the oxygen concentration is by continuously mixing air and nitrogen via flow control valves to achieve the desired oxygen concentration and continuously replenishing the partially filled sections with the newly made gas mixture.
In order to reduce the cost and keep the tailoring operation as simple as possible, which, in turn, usually improves the overall operation, like gaseous mixtures can be simultaneously introduced into each partially filled zone. Alternatively, depending on the degree of tailoring required, gaseous mixtures with higher or lower oxygen concentrations can be separately introduced into each partially filled zone.
It will be understood that the design and operation of each extrusion system will depend on the resin to be processed, the desired through-put rates, and the desired properties of the ultimate product. In general, however, the design and operation will be conventional provided that the requirements of subject invention are met.
The polyethylene used in the process of the invention generally has a broad molecular weight distribution, which can be reflected in an Mw/Mn ratio of about 2 to about 44, preferably about 10 to about 30. Mw is the weight average molecular weight; Mn is the number average molecular weight; and the Mw/Mn ratio can be referred to as the polydispersity index, which is a measure of the breadth of the molecular weight distribution.
As noted above, the process of the invention enhances the bubble stability of the polyethylene while maintaining acceptable solid state strength of the blown film in terms of dart drop strength.
High speed extrusion bubble stability is an important factor in blown film extrusion processes. A blown film extrusion process is one in which molten polymer is forced through an annular die to form a continuous cylindrical tube. Outside the die, the tube is blown out to a larger diameter than the die diameter, cooled by an air ring, and then flattened before wind-up. The ratio of the final diameter of the cylindrical tube to the diameter of the annulus is called the blow-up ratio (BUR). The combination of the extrusion rate, BUR, and the speed with which the tube is drawn out determines the final film thickness. The ability to produce thinner films at higher rates of extrusion is governed by many factors including the molecular and rheological characteristics of the molten polymer, dependence of properties on temperature, and the design of the air ring.
The entire cylindrical tube between the die and the collapsing frame (where the tube is flattened) is called the xe2x80x9cbubblexe2x80x9d. This is a cylindrical bubble in contrast to traditionally familiar spherical bubbles in which air, or some other gas, is enveloped by a thin liquid film. Here, air is used to blow a thin molten polymer film. Although the cross-section (circular) of the bubble does not change, the bubble looks conical from the outside because of a change in the diameter until the solidification at the frost-line height (FLH). Sometimes, e.g., with high density polyethylene, the bubble looks like a cylindrical semi-molten tube with a diameter approximately equal to the ring diameter before being blown out into a larger diameter. Past the FLH, however, the shape does not change from a circular cylindrical tube of constant diameter. Note that between the die and the FLH the bubble is always semi-molten.
One would like to produce a final film with close tolerance in film thickness under stable conditions of operation, i.e., the shape of the bubble remains the same without any change over time. The highest extrusion rate and the highest wind-up speed that can be used without changing the shape of the bubble provides the operating constraints for a given resin in a given system (extruder, air ring, cooling air temperature, etc.). For example, at a given extrusion rate, if one can increase the wind-up speed to make thinner film without losing the shape of the bubble, then the resin is said to have good high speed extrusion bubble stability.
Often, the bubble stability within a given extrusion system is directly related to the molecular characteristics which influence the behavior of the bubble. For example, it is easier to stretch chewing gum into a very thin thread without breakage than a rubber band. A rubber band stiffens as it is stretched and, beyond a certain point, it will break. However, the stiffening of the rubber band also gives it better stability since it will be less sensitive to external disturbances. This is not the case with the chewing gum since it deforms very easily and is very sensitive to disturbances. In the blown film concept, one would like to have rubber band behavior as the bubble is being blown and stretched rather than a chewing gum behavior. In this invention, the addition of oxygen in the two or more partially filled zone has been found to provide better high speed extrusion bubble stability, i.e., the film can be drawn at higher wind-up speeds to produce thinner films. Further, when the resin pellets are subjected to conventional blown film extrusion, the dart drop strength of the blown film is also improved. Dart drop strength is measured in accordance with ASTM D-1709. The dart drop is reported in grams for a given film thickness. The combination of enhanced bubble stability and dart drop strength is unexpected.
The preferred two stage process can be described as follows:
The copolymers produced in each of the reactors are copolymers of ethylene and at least one alpha-olefin comonomer having 3 to 8 carbon atoms, preferably one or two alpha-olefin comonomers. The alpha-olefins can be, for example, propylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene.
It will be understood that the in situ blend can generally be characterized as a bimodal resin. In some cases, however, it is difficult to distinguish two peaks. The properties of bimodal resins are strongly dependent on the proportion of the high molecular weight component, i.e., the low melt index component. For a staged reactor system, the proportion of the high molecular weight component is controlled via the relative production rate in each reactor. The relative production rate in each reactor can, in turn, be controlled by a computer application program, which monitors the production rate in the reactors (measured by heat balance) and then manipulates the ethylene partial pressure in each reactor and catalyst feed rate in order to meet the production rate, the production rate split, and catalyst productivity requirements.
Various transition metal catalyst systems can be used to provide the polyethylenes used in subject process. One is a magnesium/titanium based catalyst system, which can be exemplified by the catalyst system described in U.S. Pat. No. 4,302,565. The precursor can be supported or unsupported. Another catalyst system is one where the precursor is formed by spray drying and used in slurry form. Such a catalyst precursor, for example, contains titanium, magnesium, and aluminum halides, and an electron donor, and is attached to the surface of silica. The precursor is then introduced into a hydrocarbon medium such as mineral oil to provide the slurry form. This is described in U.S. Pat. No. 5,290,745. Metallocene catalyst systems such as those described in U.S. Pat. Nos. 5,317,036 and 5,272,236 can also be used. In the case of these metallocene catalyst systems, the melt flow ratio can be about 14 to about 70.
The electron donor, if used in the catalyst precursor, is an organic Lewis base, liquid at temperatures in the range of about 0xc2x0 C. to about 200xc2x0 C., in which the magnesium and titanium compounds are soluble. The electron donor can be an alkyl ester of an aliphatic or aromatic carboxylic acid, an aliphatic ketone, an aliphatic amine, an aliphatic alcohol, an alkyl or cycloalkyl ether, or mixtures thereof, each electron donor having 2 to 20 carbon atoms. Among these electron donors, the preferred are alkyl and cycloalkyl ethers having 2 to 20 carbon atoms; dialkyl, diaryl, and alkylaryl ketones having 3 to 20 carbon atoms; and alkyl, alkoxy, and alkylalkoxy esters of alkyl and aryl carboxylic acids having 2 to 20 carbon atoms. The most preferred electron donor is tetrahydrofuran. Other examples of suitable electron donors are methyl formate, ethyl acetate, butyl acetate, ethyl ether, dioxane, di-n-propyl ether, dibutyl ether, ethyl formate, methyl acetate, ethyl anisate, ethylene carbonate, tetrahydropyran, and ethyl propionate.
While an excess of electron donor is used initially to provide the reaction product of titanium compound and electron donor, the reaction product finally contains about 1 to about 20 moles of electron donor per mole of titanium compound and preferably about 1 to about 10 moles of electron donor per mole of titanium compound.
An activator compound is optional, but is often used with any of the titanium based catalyst precursors. The activator can have the formula AlRaXbHc wherein each X is independently chlorine, bromine, iodine, or ORxe2x80x2; each R and Rxe2x80x2 is independently a saturated aliphatic hydrocarbon radical having 1 to 14 carbon atoms; b is 0 to 1.5; c is 0 or 1; and a+b+c=3. Preferred activators include alkylaluminum mono- and dichlorides wherein each alkyl radical has 1 to 6 carbon atoms and the trialkylaluminums. A particularly preferred activator is a mixture of diethylaluminum chloride and tri-n-hexylaluminum. If it is desired to use an activator, about 0.10 to about 10 moles, and preferably about 0.15 to about 2.5 moles, of activator can be used per mole of electron donor. The molar ratio of activator to titanium can be in the range of about 1:1 to about 10:1 and is preferably in the range of about 2:1 to about 5:1.
The cocatalyst, generally a hydrocarbyl aluminum cocatalyst, can be represented by the formula R3Al or R2AlX wherein each R is independently alkyl, cycloalkyl, aryl, or hydrogen; at least one R is hydrocarbyl; and two or three R radicals can be joined to form a heterocyclic structure. Each R, which is a hydrocarbyl radical, can have 1 to 20 carbon atoms, and preferably has 1 to 10 carbon atoms. X is a halogen, preferably chlorine, bromine, or iodine. Examples of hydrocarbyl aluminum compounds are as follows: triisobutylaluminum, tri-n-hexylaluminum, di-isobutyl-aluminum hydride, dihexylaluminum dihydride, di-isobutyl-hexylaluminum, isobutyl dihexylaluminum, trimethyl-aluminum, triethylaluminum, tripropylaluminum, triisopropylaluminum, tri-n-butylaluminum, trioctylaluminum, tridecylaluminum, tridodecylaluminum, tribenzylaluminum, triphenylaluminum, trinaphthylaluminum, tritolylaluminum, dibutylaluminum chloride, diethylaluminum chloride, and ethylaluminum sesquichloride. The cocatalyst compounds can also serve as activators and modifiers.
As noted above, it is preferred not to use a support. However, in those cases where it is desired to support the precursor, silica is the preferred support. Other suitable supports are inorganic oxides such as aluminum phosphate, alumina, silica/alumina mixtures, silica modified with an organoaluminum compound such as triethylaluminum, and silica modified with diethyl zinc. A typical support is a solid, particulate, porous material essentially inert to the polymerization. It is used as a dry powder having an average particle size of about 10 to about 250 microns and preferably about 30 to about 100 microns; a surface area of at least 200 square meters per gram and preferably at least about 250 square meters per gram; and a pore size of at least about 100 angstroms and preferably at least about 200 angstroms. Generally, the amount of support used is that which will provide about 0.1 to about 1.0 millimole of titanium per gram of support and preferably about 0.4 to about 0.9 millimole of titanium per gram of support. Impregnation of the above mentioned catalyst precursor into a silica support can be accomplished by mixing the precursor and silica gel in the electron donor solvent or other solvent followed by solvent removal under reduced pressure. When a support is not desired, the catalyst precursor can be used in liquid form.
Activators can be added to the precursor either before and/or during polymerization. In one procedure, the precursor is fully activated before polymerization. In another procedure, the precursor is partially activated before polymerization, and activation is completed in the reactor. Where a modifier is used instead of an activator, the modifiers are usually dissolved in an organic solvent such as isopentane and, where a support is used, impregnated into the support following impregnation of the titanium compound or complex, after which the supported catalyst precursor is dried. Otherwise, the modifier solution is added by itself directly to the reactor. Modifiers are similar in chemical structure and function to the activators. For variations, see, for example, U.S. Pat. No. 5,106,926. The cocatalyst is preferably added separately neat or as a solution in an inert solvent, such as isopentane, to the polymerization reactor at the same time as the flow of ethylene is initiated.
U.S. Pat. No. 5,106,926 provides another example of a magnesium/titanium based catalyst system comprising:
(a) a catalyst precursor having the formula MgdTi(OR)eXf(ED)g wherein R is an aliphatic or aromatic hydrocarbon radical having 1 to 14 carbon atoms or CORxe2x80x2 wherein Rxe2x80x2 is a aliphatic or aromatic hydrocarbon radical having 1 to 14 carbon atoms; each OR group is the same or different; X is independently chlorine, bromine or iodine; ED is an electron donor; d is 0.5 to 56; e is 0, 1, or 2; f is 2 to 116; and g is 1.5d+2;
(b) at least one modifier having the formula BX3 or AlR(3xe2x88x92e)Xe wherein each R is alkyl or aryl and is the same or different, and X and e are as defined above for component (a)
wherein components (a) and (b) are impregnated into an inorganic support; and
(c) a hydrocarbyl aluminum cocatalyst.
The precursor is prepared from a titanium compound, a magnesium compound, and an electron donor. Titanium compounds, which are useful in preparing these precursors, have the formula Ti(OR)eXh wherein R, X, and e are as defined above for component (a); h is an integer from 1 to 4; and e+h is 3 or 4. Examples of titanium compounds are TiCl3, TiCl4, Ti(OC2H5)2Br2, Ti(OC6H5) Cl3, Ti(OCOCH3) Cl3, and Ti(OCOC6H5) Cl3. The magnesium compounds include magnesium halides such as MgCl2, MgBr2, and MgI2. Anhydrous MgCl2 is a preferred compound. About 0.5 to 56, and preferably about 1 to 10, moles of the magnesium compounds are used per mole of titanium compounds.
The electron donor, the support, and the cocatalyst are the same as those described above. As noted, the modifier can be similar in chemical structure to the aluminum containing activators. The modifier has the formula BX3 or AlR(3xe2x88x92e)Xe wherein each R is independently alkyl having 1 to 14 carbon atoms; each X is independently chlorine, bromine, or iodine; and e is 1 or 2. One or more modifiers can be used. Preferred modifiers include alkylaluminum mono- and dichlorides wherein each alkyl radical has 1 to 6 carbon atoms; boron trichloride; and the trialkylaluminums. About 0.1 to about 10 moles, and preferably about 0.2 to about 2.5 moles, of modifier can be used per mole of electron donor. The molar ratio of modifier to titanium can be in the range of about 1:1 to about 10:1 and is preferably in the range of about 2:1 to about 5:1.
The entire catalyst system, which includes the precursor or activated precursor and the cocatalyst, is added to the first reactor. The catalyst is admixed with the copolymer produced in the first reactor, and the mixture is transferred to the second reactor. Insofar as the catalyst is concerned, only cocatalyst is added to the second reactor from an outside source.
The polymerization in each reactor is, preferably, conducted in the gas phase using a continuous fluidized process. A typical fluidized bed reactor is described in U.S. Pat. No. 4,482,687.
A relatively low melt index (or high molecular weight) copolymer can be prepared in the first reactor. The first reactor is generally smaller in size than the second reactor because only a portion of the final product is made in the first reactor. The mixture of polymer and an active catalyst is usually transferred from the first reactor to the second reactor via an interconnecting device using nitrogen or second reactor recycle gas as a transfer medium. Alternatively, the low molecular weight copolymer can be prepared in the first reactor and the high molecular weight copolymer can be prepared in the second reactor.
In the high molecular weight reactor:
Because of the low values, instead of melt index, flow index is determined and those values are used in this specification. The flow index can be in the range of about 0.01 to about 30 grams per 10 minutes, and is preferably in the range of about 0.1 to about 20 grams per 10 minutes. The molecular weight of this polymer is, generally, in the range of about 135,000 to about 550,000. The density of the copolymer can be at least 0.860 gram per cubic centimeter, and is preferably in the range of 0.900 to 0.940 gram per cubic centimeter. The melt flow ratio of the polymer can be in the range of about 20 to about 70, and is preferably about 22 to about 45.
Melt index is determined under ASTM D-1238, Condition E. It is measured at 190xc2x0 C. and 2.16 kilograms and reported as grams per 10 minutes. Flow index is determined under ASTM D-1238, Condition F. It is measured at 190xc2x0 C. and 10 times the weight used in determining the melt index, and reported as grams per 10 minutes. Melt flow ratio is the ratio of flow index to melt index.
In the low molecular weight reactor:
A relatively high melt index (or low molecular weight) copolymer is prepared in this reactor. The high melt index can be in the range of about 1 to about 3000 grams per 10 minutes, and is preferably in the range of about 50 to about 1000 grams per 10 minutes. The molecular weight of the high melt index copolymer is, generally, in the range of about 15,800 to about 35,000. The density of the copolymer prepared in this reactor can be at least 0.900 gram per cubic centimeter, and is preferably in the range of 0.910 to 0.975 gram per cubic centimeter. The melt flow ratio of this copolymer can be in the range of about 20 to about 70, and is preferably about 20 to about 45.
The blend or final product, as removed from the second reactor, can have a melt index in the range of about 0.02 to about 3.5 grams per 10 minutes, and preferably has a melt index in the range of about 0.04 to about 2.0 grams per 10 minutes. The melt flow ratio can be in the range of about 30 to about 160, and is preferably in the range of about 30 to about 125. The molecular weight of the final product is, generally, in the range of about 90,000 to about 550,000. The density of the blend is at least 0.915 gram per cubic centimeter, and is preferably in the range of 0.916 to 0.960 gram per cubic centimeter.
The weight ratio of copolymer prepared in the high molecular weight reactor to copolymer prepared in the low molecular weight reactor can be in the range of about 0.67:1 to about 2:1, and is preferably in the range of about 0.75:1 to about 1.6:1. This is also known as the split.
The magnesium/titanium based catalyst system, ethylene, alpha-olefin, and hydrogen are continuously fed into the first reactor; the polymer/catalyst mixture is continuously transferred from the first reactor to the second reactor; ethylene, alpha-olefin, and hydrogen, as well as cocatalyst are continuously fed to the second reactor. The final product is continuously removed from the second reactor.
In the low melt index, as reflected in flow index ,reactor, i.e., the first reactor:
The mole ratio of alpha-olefin to ethylene can be in the range of about 0.01:1 to about 0.4:1, and is preferably in the range of about 0.02:1 to about 0.3:1. The mole ratio of hydrogen (if used) to ethylene can be in the range of about 0.001:1 to about 0.3:1, and is preferably in the range of about 0.017:1 to about 0.18:1. The operating temperature is generally in the range of about 60xc2x0 C. to about 100xc2x0 C. Preferred operating temperatures vary depending on the density desired, i.e., lower temperatures for lower densities and higher temperatures for higher densities.
In the high melt index reactor, i.e., the second reactor:
The mole ratio of alpha-olefin (if used) to ethylene can be in the range of about 0.005:1 to about 0.6:1, and is preferably in the range of about 0.01:1 to about 0.42:1. The mole ratio of hydrogen to ethylene can be in the range of about 0.2:1 to about 3:1, and is preferably in the range of about 1:1 to about 2.2:1. The operating temperature is generally in the range of about 70xc2x0 C. to about 110xc2x0 C. As mentioned above, the temperature is preferably varied with the desired density.
The pressure, i.e., the total pressure in the reactor, can be in the range of about 200 to about 450 psi and is preferably in the range of about 280 to about 400 psig. The ethylene partial pressure in the first reactor is at least about 25 psi and is preferably at least about 30 psi. The upper limit of ethylene partial pressure in the first reactor and the ethylene partial pressure in the second reactor are set according to the amount of copolymer it is desired to produce in each of these reactors, i.e., to achieve the split mentioned above. It is noted that increasing the ethylene partial pressure in the first reactor leads to an increase in ethylene partial pressure in the second reactor. Generally, the ethylene partial pressure roughly doubles in the second reactor over the first reactor. The balance of the total pressure is provided by alpha-olefin other than ethylene and an inert gas such as nitrogen.
A typical fluidized bed reactor can be described as follows:
The bed is usually made up of the same granular resin that is to be produced in the reactor. Thus, during the course of the polymerization, the bed comprises formed polymer particles, growing polymer particles, and catalyst particles fluidized by polymerization and modifying gaseous components introduced at a flow rate or velocity sufficient to cause the particles to separate and act as a fluid. The fluidizing gas is made up of the initial feed, make-up feed, and cycle (recycle) gas, i.e., comonomers and, if desired, modifiers and/or an inert carrier gas.
The essential parts of the reaction system are the vessel, the bed, the gas distribution plate, inlet and outlet piping, a compressor, cycle gas cooler, and a product discharge system. In the vessel, above the bed, there is a velocity reduction zone, and, in the bed, a reaction zone. Both are above the gas distribution plate.
A typical fluidized bed reactor is described in U.S. Pat. No. 4,482,687.
The gaseous feed streams of ethylene, other gaseous alpha-olefins, and hydrogen, when used, are preferably fed to the reactor recycle line as well as liquid alpha-olefins and the cocatalyst solution. Optionally, the liquid cocatalyst can be fed directly to the fluidized bed. The partially activated or completely activated catalyst precursor is preferably injected into the fluidized bed as a solid or a mineral oil slurry. In the case of partial activation, activator is added to the reactor. The product composition can be varied by changing the molar ratios of the comonomers introduced into the fluidized bed. The product is continuously discharged in granular or particulate form from the reactor as the bed level builds up with polymerization. The production rate is controlled by adjusting the catalyst feed rate.
The hydrogen:ethylene molar ratio can be adjusted to control average molecular weights. The alpha-olefins (other than ethylene) can be present in a total amount of up to 15 percent by weight of the copolymer and, if used, are preferably included in the copolymer in a total amount of about 1 to about 10 percent by weight based on the weight of the copolymer.
Several steps can be taken, in addition to temperature control, to prevent agglomeration. The product discharge line between the reactor and the product pot is often plugged up with chunks between intervals of product drops. A continuous purge flow of nitrogen or reactor gas in the line prevents the plugging problem. Also, coating the reactor surface with a low surface energy material is shown to be beneficial to slow down the rate of fouling build up. In addition, control of the electrostatic level in the bed prevents static induced particle agglomeration. Static can be adjusted to a satisfactory level by controlled use of reaction rate, avoidance of quick change of gas composition, selective use of static-neutralizing chemicals and surface passivation with aluminum alkyls.
It is preferred to control the static in the reactor system during start-up. If the static is not controlled, static induced layers of catalyst rich fines can form on the reactor surfaces. These fines may, in turn, induce localized hot spots and the formation of chunks. Reactor surface passivation with an aluminum alkyl minimizes the formation of fines layers. This passivation is accomplished by first building up the aluminum alkyl concentration in the start-up bed to about 300 to 1000 ppm based on the weight of the bed, and then fluidizing the bed with purified nitrogen or ethylene for several hours. At the end of this passivation period, the reactor is purged while maintaining the circulation, the reaction conditions are established, and the reaction is kicked off by feeding catalyst into the system. If static still persists, additional purges or selective use of static neutralizing chemicals may become necessary to reduce the level of static.
The residence time of the mixture of reactants including gaseous and liquid reactants, catalyst, and resin in each fluidized bed can be in the range of about 1 to about 12 hours and is preferably in the range of about 1.5 to about 5 hours.
The advantages of the invention are found in the film prepared from the broad molecular weight distribution resin in the ease of processability brought about by the enhanced high speed extrusion bubble stability and the solid state strength of the blown film. Thus, the resin can be extruded to thinner gauges at higher extrusion speeds, and the resulting film has an enhanced dart drop strength.
Conventional additives, which can be introduced into the blend, are exemplified by antioxidants, ultraviolet absorbers, antistatic agents, pigments, dyes, nucleating agents, fillers, slip agents, fire retardants, plasticizers, processing aids, lubricants, stabilizers, smoke inhibitors, viscosity control agents, and crosslinking agents, catalysts, and boosters, tackifiers, and anti-blocking agents. Aside from the fillers, the additives can be present in the blend in amounts of about 0.1 to about 10 parts by weight of additive for each 100 parts by weight of polymer blend. Fillers can be added in amounts up to 200 parts by weight and more for each 100 parts by weight of the blend.
Patents and patent application Ser. No. 08/671,166 mentioned in this specification are incorporated by reference herein.
The invention is illustrated by the following examples.

Pelletizing of broad molecular weight polyethylene

Union Carbide Chemicals and Plastics Technology Corporation


The present invention relates to a method of identifying a base at a target position in a sample nucleic acid sequence wherein a primer, which hybridizes to the sample nucleic acid immediately adjacent to the target position, is provided and the sample nucleic acid and primer are subjected to a polymerase reaction in the presence of a nucleotide whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and said incorporation is detected, characterized in that, a single-stranded nucleic acid binding protein is included in the polymerase reaction step.

The present invention relates to methods of nucleic acid sequencing and in particular to sequencing-by-synthesis methods, ie. those methods based on the detection of nucleotide incorporation during polymerase extension, rather than on analysis of the nucleotide sequence itself, and to the improvements derivable in such methods by the use of a single-stranded DNA binding protein.
DNA sequencing is an essential tool in molecular genetic analysis. The ability to determine DNA nucleotide sequences has become increasingly important as efforts have commenced to determine the sequences of the large genomes of humans and other higher organisms. The two most commonly used methods for DNA sequencing are the enzymatic chain-termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert. Both methods rely on gel electrophoresis to resolve, according to their size, DNA fragments produced from a larger DNA segment. Since the electrophoresis step as well as the subsequent detection of the separated DNA-fragments are cumbersome procedures, a great effort has been made to automate these steps. However, despite the fact that automated electrophoresis units are commercially available, electrophoresis is not well suited for large-scale genome projects or clinical sequencing where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great and several alternative strategies have been described, such as scanning tunnel electron microscopy (Driscoll et al., 1990, Nature, 346, 294-296), sequencing by hybridization (Bains et al., 1988, J. Theo. Biol. 135, 308-307) and single molecule detection (Jeff et al., 1989, Biomol. Struct. Dynamics, 7, 301-306), to overcome the disadvantages of electrophoresis.
Techniques enabling the rapid detection of a single DNA base change are also important tools for genetic analysis. In many cases detection of a single base or a few bases would be a great help in genetic analysis since several genetic diseases and certain cancers are related to minor mutations.
Sequencing-by-synthesis methods are useful ways of determining the sequence of a DNA molecule of up to a hundred or more bases or the identity of a single nucleotide within a sample DNA molecule. During typical sequencing-by-synthesis methods the four different nucleotides (adenine, thymine, guanine and cytosine) are conveniently added cyclically in a specific order; when the base which forms a pair (according to the normal rules of base pairing, A-T and C-G) with the next base in the single-strand target sequence is added, it will be incorporated into the growing complementary strand by a polymerase and this incorporation will trigger a detectable signal. The event of incorporation can be detected directly or indirectly. In direct detection, nucleotides are usually fluorescently labelled allowing analysis by a fluorometer. (U.S. Pat. No. 48,638,449, U.S. Pat. No. 5,302,509, Metzker et al. Nucl. Acids Res. (1994) 22: 4259-4267, Rosenthal International Patent Application No. WO 93/213401, WO 91/06678, Canard et al. Gene (1994) 148: 1-6). One such strategy of sequencing-by-synthesis called base addition sequencing scheme (BASS) is based on nucleotide analogues that terminate DNA synthesis. BASS involves repetitive cycles of incorporation of each successive nucleotide, in situ monitoring to identify the incorporated base, and deprotection to allow the next cycle of DNA synthesis.
Indirect detection usually takes advantage of enzymatic detection, e.g. measuring the release of PPi (inorganic pyrophosphate) during a polymerization reaction (WO 93/23564 and WO 89/09283). As each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can be detected enzymatically e.g. by the generation of light in the luciferase-luciferin reaction. Such methods enable a base to be identified in a target position and DNA to be sequenced simply and rapidly whilst avoiding the need for electrophoresis and the use of harmful radiolabels. These methods based on release of PPi are referred to herein as Pyrosequencing. The basic PPi-based sequencing methods have been improved by using a dATP analogue in place of dATP (WO 98/13523) and including a nucleotide-degrading enzyme such as apyrase during the polymerase reaction step, so that unincorporated nucleotides are degraded, as described in WO 98/28440.
However, these sequencing-by-synthesis methods mentioned above are not without drawbacks. A particular problem arises when the DNA to be sequenced has a number of identical adjacent bases, especially 3 or more the same. FIG. 1 shows the trace obtained when a single-stranded PCR product is sequenced according to known sequencing-by-synthesis methods (in this case involving detection of PPi). FIG. 1 shows that known methods do not provide clear results when two or more adjacent bases in the sample molecule are the same. For example, the peak height when the first set of three adenine residues are incorporated is almost the same as when four thymine residues are incorporated later; the set of three adenine residues incorporated around the middle of the sequence have the same peak height as previous doublets and the last pair of adenine residues to be incorporated have a peak height corresponding to single bases from the earlier part of the sequence.
Other problems of sequencing-by-synthesis methods include false signals which are the result of mispriming, i.e. hybridisation of the primer not to its targeted complement within the target DNA sequence but to another region which will result in generation of xe2x80x9cincorporation signalsxe2x80x9d which do not reflect the identity of the target sequence. There is an associated problem which can result in a false indication of incorporation termed xe2x80x9cminus frame incorporationxe2x80x9d, where a proportion of the growing primer originating strands are not fully extended and false positive signals appear in subsequent cycles.
Thus, there is a need further to improve sequencing-by-synthesis methods by addressing the above problems and more generally to improve the accuracy of the methods while providing methods which are simple and quick to perform, lending themselves readily to automation.
It has surprisingly been found that including a single-stranded nucleic acid binding protein in the reaction mixture improves the ratio of signals generated by one, two, three or more adjacent bases and reduces the number of false signals and generally improves the efficacy and reduces the cost of sequencing-by-synthesis methods.
In one aspect, the present invention thus provides a method of identifying a base at a target position in a sample nucleic acid sequence wherein a primer, which hybridises to the sample nucleic acid immediately adjacent to the target position, is provided and the sample nucleic acid and primer are subjected to a polymerase reaction in the presence of a nucleotide whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and said incorporation is detected, characterized in that, a single-stranded nucleic acid binding protein is included in the polymerase reaction step.
The nucleic acid to be sequenced may be any nucleotide sequence it is desirable to obtain sequence information about. Thus, it may be any polynucleotide, or indeed oligonucleotide sequence. The nucleic acid may be DNA or RNA, and may be natural, isolated or synthetic. Thus, the target DNA may be genomic DNA, or cDNA, or a PCR product or other amplicon etc. Alternatively, the target DNA may be synthetic, and genomic DNA, cDNA or a PCR product etc. may be used as primer. The target (sample) nucleic acid may be used in any convenient form, according to techniques known in the art e.g. isolated, cloned, amplified etc., and may be prepared for the sequencing reaction, as desired, according to techniques known in the art.
The DNA may also be single or double-stranded whilst a single-stranded DNA template has traditionally been used in sequencing reactions, or indeed in any primer-extension reaction, it is possible to use a double-stranded template; strand displacement, or a localised opening-up of the two DNA strands may take place to allow primer hybridisation and polymerase action to occur.
The sample nucleic acid acts as a template for possible polymerase based extension of the primer and thus may conveniently be referred to as xe2x80x9ctemplatexe2x80x9d or xe2x80x9cnucleic acid templatexe2x80x9d.
In the polymerase reaction, any convenient polymerase enzyme may be used according to choice, as will be described in more detail below. In the case of a RNA template, such a polymerase enzyme may be a reverse transcriptase enzyme. The nucleotide may be any nucleotide sutiable for a polymerase chain extension reaction e.g. a deoxynucleotide or a dideoxynucleotide. The nucleotide may optionally be labelled in order to aid or facilitate detection of nucleotide incorporation. One or more nucleotides may be used.
Nucleotide incorporation by the action of the polymerase enzyme may be detected directly or indirectly, and methods for this are well known in the art. Representative methods are described for example in U.S. Pat. No. 4,863,879 of Melamede. As mentioned above, detection of incorporation may be by means of labelled nucleotides, for example fluorescently labelled nucleotides, as is well known in sequencing procedures known in the art. Alternatively, the event of incorporation may be detected by other means e.g. indirectly. Detection of incorporation also includes the detection of absence of incorporation e.g. lack of a signal. Thus, it may be detected whether or not nucleotide incorporation takes place.
The method of the invention thus has utility in a number of different sequencing methods and formats, including mini-sequencing procedures e.g. detection of single base changes (for example, in detecting point mutations, or polymorphisms, or allelic variations etc). The method of the invention may thus be used in a xe2x80x9cfullxe2x80x9d sequencing procedure, ie. the identification of the sequential order of the bases in a stretch of nucleotides, as well in single base detection procedures.
For example, to determine sequence information in a target nucleotide sequence, different deoxynucleotides or dideoxynucleotides may be added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated.
In order to sequence the target DNA, the procedure may be repeated one or more times i.e. cyclically, as is known in the art. In this way the identity of many bases in the sample nucleic acid may be identified, essentially in the same reaction.
Hence, a sequencing protocol may involve annealing a primer as described above, performing a polymerase-catalysed primer extension step, detecting the presence or absence of incorporation, and repeating the nucleotide addition and primer extension steps etc. one or more times. As discussed above, nucleotides may be added singly or individually, or in a mixture, successively to the same primer-template mixture, or to separate aliquots of primer-template mixture, or to separate aliquots of primer-template mixture etc. according to choice, and the sequence information it is desired to obtain.
The term xe2x80x9csingle-stranded nucleic acid binding proteinxe2x80x9d as used herein is intended to refer to the class of proteins collectively referred to by the term SSB (Ann. Rev. Biochem. [1986] 55 103-136 Chase et al.). SSB has the general property of preferential binding to single-stranded (ss) over double-stranded (ds) nucleic acid molecules. The class includes E. coli single-stranded binding protein (Eco SSB), T4 gene 32 protein (T4 gp32), T7 SSB, coliophage N4 SSB, T4 gene 44/62 protein, adenovirus DNA binding protein (AdDBP or AdSSB), calf thymus unwinding protein (UP1) and the like (Coleman et al. CRC Critical Reviews in Biochemistry, (1980) 7(3), 247-289 and p5 SSB from fi-29 DNA (Lindberg et al. J. Biol. Chem. (1989) 264 12700-08) (Nakashima et al. FEBS Lett. (1974) A43 125). Any functionally equivalent or analogous proteins for example derivatives or modifications of the above-mentioned proteins, may also be used. Eco SSB or derivatives thereof are particularly preferred for use in the methods of the present invention.
Thus, modified single-stranded nucleic acid binding proteins derived by isolation of mutants or by manipulation of cloned single-stranded nucleic acid binding proteins which maintain the advantageous properties described herein, are also contemplated for use in the methods of the invention.
The term xe2x80x9cdideoxynucleotidexe2x80x9d as used herein includes all 2xe2x80x2-deoxynucleotides in which the 3xe2x80x2-hydroxyl group is absent or modified and thus, while able to be added to the primer in the presence of the polymerase, is unable to enter into a subsequent polymerisation reaction.
As described above, the method of the invention may be performed in a number of ways, and has utility in a variety of sequencing protocols. Viewed more generally, the present invention can thus be seen to provide the use of a single-stranded nucleic acid binding protein in a nucleic acid sequencing-by-synthesis method. In particular, the single-stranded nucleic acid binding protein is used to bind to the nucleic acid template.
What is meant by a DNA sequencing-by-synthesis method is defined above, namely that sequence information is derived by detecting incorporation of a nucleotide in a primer extension reaction. As explained above, such sequencing-by-synthesis protocols, include not only xe2x80x9cfullxe2x80x9d sequencing methods, but also mini-sequencing methods etc., yielding more limited sequence information.
Any sequencing-by-synthesis method, as described above, is suitable for use in the methods of the present invention but methods which rely on monitoring the release of inorganic pyrophosphate (PPi) are particularly preferred. In this case, incorporation of the nucleotide will be measured indirectly by enzymatic detection of released PPi.
PPi can be determined by many different methods and a number of enzymatic methods have been described in the literature (Reeves et al., (1969), Anal. Biochem., 28, 282-287; Guillory et al., (1971), Anal. Biochem., 39, 170-180; Johnson et al., (1968), Anal. Biochem., 15, 273; Cook et al., (1978), Anal. Biochem. 91, 557-565; and Drake et al., (1979), Anal. Biochem. 94, 117-120).
It is preferred to use luciferase and luciferin in combination to identify the release of pyrophosphate since the amount of light generated is substantially proportional to the amount of pyrophosphate released which, in turn, is directly proportional to the amount of base incorporated. The amount of light can readily be estimated by a suitable light sensitive device such as a luminometer.
Luciferin-luciferase reactions to detect the release of PPi are well known in the art. In particular, a method for continuous monitoring of PPi release based on the enzymes ATP sulphurylase and luciferase has been developed by Nyren and Lundin (Anal. Biochem., 151, 504-509, 1985) and termed ELIDA (Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay). The use of the ELIDA method to detect PPi is preferred according to the present invention. The method may however be modified, for example by the use of a more thermostable luciferase (Kaliyama et al., 1994, Biosci. Biotech. Biochem., 58, 1170-1171). This method is based on the following reactions:
The preferred detection enzymes involved in the PPi detection reaction are thus ATP sulphurylase and luciferase. Methods of detecting the light emitted are well known in the art.
In order to repeat the method cyclically and thereby sequence the sample nucleic acid and, also to aid separation of a single-stranded sample DNA from its complementary strand, it may be desirable that sample nucleic acid (DNA) is immobilised or provided with means for immobilisation attachment to a solid support.
Moreover, the amount of sample nucleic acid available may be small and it may therefore be desirable to amplify the sample nucleic acid before carrying out the method according to the invention.
The sample DNA may be amplified, for example in vitro by PCR, Self Sustained Sequence Replication (3SR), Rolling Circle Amplification or Replication (RCA or RCR), or indeed any other in vitro amplification technique, or in vivo using a vector and, if desired, in vitro and in vivo amplification may be used in combination. Whichever method of amplification is used, it may be convenient to adapt the method such that the amplified nucleic acid becomes immobilised or is provided with means for attachment to a solid support. For example, a PCR primer may be immobilised or be provided with means for attachment to a solid support. Also, a vector may comprise means for attachment to a solid support adjacent the site of insertion of the sample DNA such that the amplified sample DNA and the means for attachment may be excised together.
Immobilisation of the amplified DNA may take place as part of the amplification itself, e.g. in PCR where one or more primers are attached to a support, or alternatively one or more of the primers may carry means for immobilisation e.g. a functional group permitting subsequent immobilisation, e.g. a biotin or thiol group. Immobilisation by the 5xe2x80x2 end of a primer allows the strand of DNA emanating from that primer to be attached to a solid support and have its 3xe2x80x2 end remote from the support and available for subsequent hybridisation with the extension primer and chain extension by polymerase.
The solid support may conveniently take the form of microtitre wells, or dipsticks which may be made of polystyrene activated to bind the primer DNA (K Almer, Doctoral Theses, Royal Institute of Technology, Stockholm, Sweden, 1988). However, any solid support may conveniently be used, including any of the vast number described in the art, e.g. for separation/immobilisation reactions or solid phase assays. Thus, the support may also comprise particles, fibres or capillaries made, for example, of any polymer, e.g. agarose, cellulose, alginate, Teflon or polystyrene. Glass solid supports can also be used, e.g. glass plates or capillaries. Magnetic particles e.g. the superparamagnetic beads produced by Dynal AS (Oslo, Norway) are a preferred support since they can be readily isolated from a reaction mixture yet have superior reaction kinetics over many other forms of support.
The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups, or other moieties such as avidin or streptavidin, for the attachment of primers or the target nucleic acid. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an aminoalkylated polymer to provide amino groups. Sulphur and epoxy-based functional groups may also be used. U.S. Pat. No. 4,654,267 describes the introduction of many such surface coatings.
The assay technique is very simple and rapid, thus making it easy to automate by using a robot apparatus where a large number of samples may be rapidly analysed. Since the preferred detection and quantification is based on a luminometric reaction this can be easily followed spectrophotometrically. The use of luminometers is well known in the art and described in the literature.
As mentioned above, the sample nucleic acid, ie. the target nucleic acid to be sequenced, may be any nucleotide sequence, however obtained according to techniques known in the art, e.g. cloning, DNA isolation etc. It may for example be cDNA synthesised from RNA in the sample and the method of the invention is thus applicable todiagnosis on the basis of characteristic RNA. Such preliminary synthesis can be carried out by a preliminary treatment with a reverse transcriptase, conveniently in the same system of buffers and bases of subsequent amplification e.g. PCR steps, if used. Since the PCR procedure requires heating to effect strand separation, in the case of PCR the reverse transcriptase will be inactivated in the first PCR cycle. When mRNA is the sample nucleic acid, it may be advantageous to submit the initial sample, e.g. a serum sample, to treatment with an immobilised polydT oligonucleotide in order to retrieve all mRNA via the terminal polyA sequences thereof. Alternatively, a specific oligonucleotide sequence may be used to retrieve the RNA via a specific RNA sequence. The oligonucleotide can then serve as a primer for cDNA synthesis, as described in WO 89/0982.
Advantageously, the primer for the polymerase chain extension step (ie., the extension primer) is sufficiently large to provide appropriate hybridisation with the sequence immediately 5xe2x80x2 of the target position, yet still reasonably short in order to avoid unnecessary chemical synthesis. It will be clear to persons skilled in the art that the size of the extension primer and the stability of hybridisation will be dependent to some degree on the ratio of A-T to C-G base pairings, since more hydrogen bonding is available in a C-G pairing. Also, the skilled person will consider the degree of homology between the extension primer to other parts of the amplified sequence and choose the degree of stringency accordingly. Guidance for such routine experimentation can be found in the literature, for example, Molecular Cloning: a laboratory manual by Sambrook, J., Fritsch E. F. and Maniatis, T. (1989).
The primer is conveniently added before the sample is divided into (four) aliquots although it may be added separately to each aliquot. It should be noted that the extension primer may be identical with the PCR primer but advantageously it may be different, to introduce a further element of specificity into the system.
Where appropriate, the polymerase reaction is carried out using a polymerase which will incorporate deoxynucleotides and dideoxynucleotides, e.g. T7 polymerase, Klenow, xcfx8629 DNA polymerase or Sequenase Ver. 2.0 (USB U.S.A.). Any suitable polymerase may be used and many are known in the art and reported in the literature. However, it is known that many polymerases have a proof-reading or error checking ability and that 3xe2x80x2 ends available for chain extension are sometimes digested by one or more nucleotides. If such digestion occurs in the method according to the invention the level of background noise increases. In order to avoid this potential problem, a nonproof-reading polymerase, e.g. T7 polymerase or Sequenase may be used. Otherwise it is desirable to add to each aliquot fluoride ions or nucleotide monophosphates which suppress 3xe2x80x2 digestion by polymerase.
A fuller description of preferred embodiments of PPi based sequencing-by-synthesis methods are provided in WO 98/13523 and WO 98/28440 which are incorporated herein by reference. The use of a dATP analogue such as dATPxcex1S in place of dATP is advantageous as it does not interfere with the detection reaction as it is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for the PPi-detection enzyme luciferase. It is therefore possible to perform the chain extension and detection, or signal-generation, reactions substantially simultaneously by including the xe2x80x9cdetection enzymesxe2x80x9d in the chain extension reaction mixture and therefore the sequencing reactions can be continuously monitored in real-time, with a signal being generated and detected, as each nucleotide is incorporated.
Inclusion of a nucleotide degrading enzyme in the reaction mix is also advantageous as it means that it is not necessary to wash the template thoroughly between each nucleotide addition to remove all non-incorporated deoxynucleotides, which has the associated benefit that a template can be sequenced which is not bound to a solid support.
In a particularly preferred method of the invention, the nucleotide-degrading enzyme apyrase is included during the polymerase reaction step and it has been found that the single-stranded binding proteins used in the methods of the present invention are able to stimulate the activity of apyrase. Whilst not wishing to be bound by theory, it is believed that the xe2x80x9cSSBxe2x80x9d may play a role in reducing the inhibition of apyrase which may be observed in the presence of DNA.
Thus in a further aspect, the present invention provides a method of enhancing the activity of a nucleotide-degrading enzyme when used in a nucleic acid sequencing-by-synthesis method, which comprises the use of a single-stranded nucleic acid binding protein. More particularly, the methods comprise including or adding a single-stranded nucleic acid binding protein to the sequencing reaction mixture (i.e. the template, primer, polymerase and/or nucleotide (e.g. dNTP/ddNTP) mix).
A similar enhancing effect has been observed on the luciferase enzyme which may be used in signal detection and therefore in a further aspect the present invention provides a method of enhancing the activity of luciferase when used as a detection enzyme in a nucleic acid sequencing-by-synthesis method which comprises the use of a single-stranded nucleic acid binding protein. Again, such methods involve including or adding a single-stranded nucleic acid binding protein to the sequencing reaction mixture.
In a preferred embodiment of the present invention the single-stranded nucleic acid binding protein is added after hybridisation of the primer to the template nucleic acid molecule.
It is also preferred, not to remove the single-stranded nucleic acid binding protein after it has been added.
It is a particular advantage of the present sequencing methods that there need be no separation of the different reagents and enzymes involved in the extension and detection reactions but the labelled or unlabelled nucleotides or nucleotide analogues, sample, polymerase and where appropriate enzymes and enzyme substrates, as well as the single-stranded nucleic acid binding protein can be included in the reaction mixture and there is no need to remove the single-stranded nucleic acid binding protein for detection to take place.
The reaction mixture for the polymerase chain extension step may optionally include additional ingredients or components if desired. Thus, such additional components can include other substances or molecules which bind to DNA. Thus, amines such as spermidine may be used. It was observed that improved results may be obtained using spermidine in run-off extension reactions.
Alternative additional components include DNA binding proteins such as RecA. In particular, it has been observed that a synergy occurs between RecA and a single-stranded nucleic acid binding protein, leading to improved results. Accordingly, the combination of RecA with a single-stranded nucleic acid binding protein represents a preferred embodiment according to the present invention. Other DNA binding proteins involved in DNA replication, recombination, or structural organisation may also be used in similar manner.
Other components which may be included, particularly when a double stranded substrate is used, include DMSO and formamide, and other agents which may destabilise or assist in destabilising double-strand formation, for example accessory proteins involved in DNA replication such as helicase.
When a single strand nucleic acid binding protein is used in accordance with the methods of the present invention, the methods are robust and results are readily reproducible. The read-length, i.e. the length of nucleic acid which can be successfully sequenced, has been increased by four times as compared to previous sequencing-by-synthesis methods. The methods of the invention are suitable for midi-sequencing, ie. the sequencing of nucleic acid molecules having 9-50 bases, mini-sequencing, the detection of single bases such as SNPs (single nucleotide polymorphisms) responsible for genetic diseases and the sequencing of nucleic acid molecules of 100 bases or more. It is in the successful sequencing of larger molecules that the benefits of a single strand nucleic acid binding protein in, particularly PPi based, sequencing methods are observed. The problems of maintaining a constant signal intensity for incorporation of one nucleotide, two nucleotides and so on, throughout the whole sequencing run are overcome.
In particular, the present invention may advantageously be used in the sequencing of 25 or more, advantageously 30 or more, 50 or more, or 60 or more bases.
As well as increasing the read length, the use of a single stranded nucleic acid binding protein enables the use of longer template molecules. Thus, sequence information from e.g. a 50 base region within a template molecule of 400 or more bases can be obtained. Template molecules of 800 or more, even 1200 or 1500 or more bases can be used in the methods of the present invention.
The amount of sequencing template accessible for the sequencing reaction may be reduced due to specific and/or unspecific interactions between the template and components of the reaction mixture and/or the surface of the vessel holding the reaction mixture. Such interaction may result in the reduction of the detected signal and/or generation of unspecific sequencing signal. SSB is believed to protect the template from such undesirable interactions and thus improve signal intensity and specificity. Furthermore, the protein may assist in xe2x80x9copening-upxe2x80x9d and/or maintaining or stabilising the xe2x80x9copenxe2x80x9d structure of a double-stranded template.
When using the sequencing methods of the invention to detect mutations in a nucleic acid molecule, the sequencing reaction may advantageously be run bidirectionally to confirm the mutation.
Moreover, a further beneficial feature of the present invention is the stimulation of Klenow polymerase which is observable using the single-stranded nucleic acid binding protein according to the methods described herein.
Thus in a further aspect, the present invention provides a method of maintaining a constant signal intensity during a method of nucleic acid sequencing-by-synthesis comprising the use of a single-stranded nucleic acid binding protein. More particularly, the methods comprise including or adding a single-stranded nucleic acid binding protein to the sequencing reaction mixture (i.e. the template, primer, polymerase and/or nucleotide (e.g. dNTP/ddNTP) mix).
It is to be understood that xe2x80x98constant signal intensityxe2x80x99 in this context means that the strength of the signal, however measured, which indicates incorporation of the correct base-pair nucleotide remains substantially the same throughout the sequencing reaction, whether it is the first nucleotide incorporated, the twentieth or the sixtieth etc. Similarly, the strength of signal indicating incorporation of two nucleotides (ie. two adjacent bases are the same in the molecule to be sequenced) remains constant throughout the whole sequencing reaction and so on for three bases, four bases etc.
The single-stranded nucleic acid binding protein is present in the xe2x80x98reaction mixturexe2x80x99, i.e. together with the reagents, enzymes, buffers, primer, sample etc. which may include a solid support and is the site of polymerisation and also, where appropriate, detection.
A further benefit of the use of a single-stranded nucleic acid binding protein in accordance with the present invention is the relatively small amount of sample nucleic acid which is required for generation of useful sequence information. Approximately 0.05 pmol DNA in a 50 xcexcl reaction is sufficient to obtain sequence information which means the quantity of enzymes needed for carrying out the extension reactions is less per cycle of nucleotide additions and per full sequencing reaction.
In a further aspect, the present invention provides a kit for use. in a method of sequencing-by-synthesis which comprises nucleotides for incorporation, a polymerase, means (e.g. any reagents and enzymes needed) for detection of incorporation and a single-stranded nucleic acid binding protein.