Preparation of nucleic acid from mononuclear cells

Abstract: Method and kit for the preparation of nucleic acid, such as for amplification. The nucleic acid may be prepared by centrifuging a sample of whole cells, particularly mononuclear cells, with an appropriate centrifugation medium so as to separate desired cells from non-desired cells, then removing and lysing said desired cells. A complexing agent able to complex ferric ion may be added to the lysed cells. The nucleic acid associated with the lysed cells can be amplified, and the nucleic acid can also be screened for a certain nucleic acid sequence, such as a viral nucleic acid sequence.

Description: BACKGROUND OF THE INVENTION : The field of the present invention is the preparation of nucleic acid for study, research and investigation. It is common to require nucleic acid to be isolated and purified ( i.e. , prepared) from various tissues in order to detect the presence of a particular nucleic acid -- for example, the presence of HIV-1 DNA or RNA in a blood cell of a human. For this purpose, the nucleic acid is generally extracted after extensive purification of appropriate blood cells, lysis of these cells and purification of the released nucleic acids to remove substances that might inhibit later analytical procedures. In particular, it is important to produce nucleic acid of a quality and purity to allow its amplification. "PCR Protocols" (Academic Press 1990), Chapter 18, pages 146-152 discusses the separation of mononuclear cells from whole blood by use of density gradient centrifugation and release from such separated cells of DNA or RNA for amplification. Biotechniques (1991) Vol.10, No. 4,506-513 discusses extraction of DNA for amplification from forensic-type samples, e.g. bloodstains and other blood samples, at high temperature and in the presence of a metal ion-chelating resin to prevent DNA degradation. Two common methods which allow amplification of a specified sequence of nucleic acid (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) are one termed the "polymerase chain reaction" (where two primers are used to synthesize nucleic acid lying between the regions where the primers hybridize), and one which uses RNAse H, reverse transcriptase and RNA polymerase. These methods are described respectively in U.S. Patent 4,683,202 (Mullis et al) and in PCT/US90/03907 (Publication No. WO 91/01384; Kacian et al.) SUMMARY OF THE INVENTION : The present invention is directed in one aspect to a method for amplifying a target sequence in nucleic acid derived from cells such as mononuclear cells (e.g. T-lymphocytes and/or monocytes) wherein a ferric ion complexing agent, more particularly deferoxamine, sideramine, sideramycin or ferrimycin, is employed in the amplification mixture to improve amplification of any target sequence present. Nucleic acid so amplified maybe used for various purposes, including screening the nucleic acid for the presence of viral nucleic acid sequences, using a probe which is complementary to a selected nucleic acid sequence of the virus. It is also useful for detection of genetic anomalies or defects in the nucleic acid. Accordingly, methods and kits of the invention are designed to allow rapid and easy preparation of nucleic acid without the need for extensive purification procedures. Thus, in a further aspect, the present invention features a method for preparing nucleic acid from cells and incorporating it into an amplification mixture as discussed above. A sample containing various cells ( e.g. , whole blood) and an appropriate centrifugation medium are centrifuged to cause a population of mononuclear cells to gather in a discrete layer. This layer is separate and distinct from the remainder of the cells and detritus in the sample, except for a small amount of platelets and/or lipids or other low density components, or other soluble and suspended constituents. Surprisingly, the presence of platelets and other components does not prevent amplification of nucleic acid purified in this method. Preferably, the population of cells includes mononuclear cells from whole blood. However, the cells may be derived from other sources, such as pleural fluid, synovial fluid, or an in vitro source of cells. The cells or sample of cells need only be available to be centrifuged for separation, or in a state to be lysed (as discussed below), and then effectively used in later reactions according to the present invention. The centrifugation medium may be isotonic and/or isopycnic to the cells, and is preferably of a density intermediate between that of the desired cells and of the other cells in the sample. The cells are removed from the centrifugation composition, for example by suction using a pipette, and lysed. Such lysis makes the nucleic acid associated with the cells available for the amplification. For this purpose, a lysing agent may be employed. The use of a strong alkali such as KOH, NaOH or LiOH is preferred. Such a lysis also denatures any double-stranded nucleic acid from the cells, thereby facilitating amplification. Alternatively, sonic disruption may be employed to lyse the cells. The method includes adding a complexing agent that complexes with, and effectively removes, ferric ions (Fe +++ ) from the solution before amplifying the nucleic acid. The presence of ferric ions interferes with certain enzymatic procedures, such as those used for nucleic acid amplification. The complexing agent is preferably selected so that it does not complex with zinc ions (Zn ++ ), manganese ions (Mn ++ ) or magnesium ions (Mg ++ ), as these ions are beneficial and tend to facilitate these enzymatic reactions. Such an agent is selected from the chelating agents deferoxiamine (also known as desferrioxamine and desferrioxamine B), sideramine, sideramycin and forrimycin. Preferably, deferoxiamine is employed. In other preferred embodiments, a second or different complexing agent may be added that complexes with calcium ions (Ca ++ ), or other ions that interfere with an amplification reaction or other reactions performed with the lysate of the present invention. Calcium ions may also interfere with enzymatic reactions used in amplification procedures. The second complexing agent is most preferably selected so as to fail to complex with zinc ions or magnesium ions, or to have more affinity for undesirable ion species (such as Fe +++ and Ca ++ ) than for desirable ion species (such as Zn ++ , Mn ++ , and Mg ++ ). In yet more preferred embodiments, zinc ions, manganese ions or magnesium ions may be added to the solution to facilitate enzymatic reactions. Thus, the invention features a method of amplifying nucleic acid in which the nucleic acid to be amplified is produced in a solution with ferric ions, and those ferric ions are effectively removed from the mixture by use of a complexing agent selected from deferoxamine, sideramine, sideramycin and ferrimycin such that amplification can occur. The solution may also contain calcium ions, which are preferably also removed from the solution by a complexing agent, so that amplification may occur. Once the nucleic acid has been amplified, methods are known in the art for screening the amplified nucleic acid using known nucleic acid hybridization and detection techniques. For example, by using a nucleic acid probe complementary to a target sequence (which preferably includes the sequence targeted for amplification), hybridizing the probe to the target nucleic acid sequence, and detecting the complex of probe and target by well known methods, such as Southern or northern blots or by the homogeneous solution phase procedure ("HPA") described in Arnold et al, Clin. Chem. , 35: 1588 (1989), and PCT/US88/02746. By the use of such a probe detection method, it can be determined whether viral nucleic acid, such as that from HIV or hepatitis B virus (HBV), is associated with the nucleic acid present in the sample. Such a screening may also target any other sort of nucleic acid sequence, such as a genetic anomaly or defect. The use of such amplification procedures allows even a single copy of the targeted nucleic acid in the portion of the sample recovered after centrifugation to be detected using the present invention. It is a further feature of the invention to provide kits for carrying out methods in accordance with the invention. Such a kit will include a ferric ion complexing agent which is deferoxiamine, sideramine, sideramycin or ferrimycin and one or more further components selected from: (a) a centrifugation medium (and an osmotic agent, if necessary), (b) a lysing agent sufficient to lyse cells separated from the sample, and (c) amplification materials sufficient to amplify the target nucleic acid sequence. Such a kit may also include a calcium ion complexing agent and a supply of the probe for a desired nucleic acid target, or a known genetic anomaly. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS : Fig. 1 is a graph depicting the red blood cell counts, hematocrits, and plasma density for 66 different individuals. Fig. 2 is a chart depicting the effects on amplification of zinc titration in concert with deferoxamine. Fig. 3 is a chart depicting the effects of polyethyleneimine (PEI) and deferoxamine on amplification. DESCRIPTION OF THE PREFERRED EMBODIMENTS : In one aspect, the present invention provides a method for amplifying a target nucleic acid sequence in an amplification mixture wherein said mixture includes a first complexing agent able to complex ferric ions, said first complexing agent being selected from deferoxamine, sideramine, sideramycin and ferrimycin, so that amplification of the target sequence in said mixture yields more amplified nucleic acid than when said first complexing agent is absent from said mixture. In a further aspect, the present invention provides a method of preparing an amplification mixture for nucleic acid amplification comprising the steps of: (a) centrifuging a composition comprising a centrifugation medium and a sample of blood cells comprising mononuclear cells under centrifugation conditions such that a substantial portion of said mononuclear cells in said composition gather in a discrete band; (b) separating said discrete band from said composition; (c) lysing said mononuclear cells in said discrete band by (i) providing to said band a lysing agent, or (ii) exposing said band to sonic disruption, so that said mononuclear cells in said band lyse, thereby rendering said nucleic acid available for amplification; (d) providing to said band a first complexing agent able to complex ferric ions, said first complexing agent being selected from deferoxamine, sideramine, sideramycin and ferrimycin; and (e) subjecting said nucleic acid to conditions sufficient to amplify a target nucleic acid sequence. Obtaining A Sample Of Cells A sample of cells may be provided from any suitable source, such as from whole blood, or from synovial fluid, pleural fluid, or other fluids containing cells of interest. Preferably, the sample comprises mononuclear cells in anticoagulant treated whole blood, which may be obtained from any animal, including a human being. The blood may be pretreated if desired, e.g. , to remove red blood cells and/or fibrin, provided that the mononuclear cells (or other cells of interest) remain in the fluid. Other blood fractions or any other fluids containing cells of interest can be used. The blood, or other source of a sample of cells, may be obtained using any available method, including those known in the art. The sample can be obtained and stored, and even subjected to centrifugation, in the same device. If the sample is blood, it may be desirable to treat the sample with an anti-coagulant, such as EDTA, citrate, oxalate or heparin, so that the blood does not coagulate prior to performance of the rest of the method, or during use of the rest of the materials and apparatus in the kit. Preparing The Composition It is useful to make a composition of the sample and a centrifugation medium such that, upon centrifugation, the mononuclear cells gather in a discrete band. By "gather in a discrete band," it is meant that the desired cells effectively move through the composition to a particular density level, and are sufficiently separated from the remainder of the cells and other detritus in the composition that later enzyme reactions, such as amplification, can proceed. Such centrifugation media are known in the art. See , e.g. , Percoll Methodology and Applications Density Marker Beads for Calibration of Gradients of PERCOLL®, published by Pharmacia, Laboratory Separation Division, Uppsala, Sweden; Sepracell-MN® Separation Manual, published by Sepratech Corp., Oklahoma City, Oklahoma; NYCOMED® Density Gradient Media, published by Nycomed, Oslo, Norway. The centrifugation medium may be isotonic to the cells of the sample, and may also be generally isopycnic to the cells from the sample or, preferably, of a density intermediate the cell population of interest and other cell populations in the sample. The centrifugation medium and the sample of cells may be mixed prior to centrifugation such that there is no density gradient prior to centrifugation. By "isopycnic," it is meant that the centrifugation medium is approximately the same density as the cells in the sample, such that, upon centrifugation, desired cells from the sample will separate from the remainder of the mixture, which remainder, in the case of a sample from whole blood, will contain red blood cells and other white blood cells. Alternatively, the composition can be formed as a density gradient prior to or during centrifugation. In a preferred embodiment using differential cut-off density centrifugation, the sample of cells is mixed with a centrifugation medium such that the resulting density is intermediate between the buoyant density of the cell population of interest and other cell populations in the sample. For example, the density of the mixture is greater than the buoyant density of mononuclear cells and less than the buoyant density of red blood cells and granulocytes in a sample of whole blood. In this embodiment, the centrifugation mixture is subjected to centrifugation such that the mononuclear cells "float" to the meniscus in a relatively narrow band. Centrifugation of such a mixture for longer than such time, for example until isopycnic equilibrium is reached, will result in a broader band (or bands) of desired cells. Differential cut-off centrifugation or other centrifugation procedures within the scope of the invention can be adapted without undue experimentation in light of the present specification for cells other than mononuclear cells from whole blood. A centrifugation medium having a density of between 1.105 and 1.115 grams per milliliter may, for example, be employed. Generally, the final desired density of the centrifugation mixture is about 1.077 grams per milliliter of fluid to separate mononuclear cells. However, the density of the centrifugation medium and the mixture will vary according to the osmolarity, type and volume of the cells in the sample. By "isotonic," it is meant that the composition, once it is made, is approximately isotonic to the mononuclear cells from the sample, such that less than 20% of the mononuclear cells rupture pursuant to the effects of an osmotic difference. Generally, this isotonic state is achieved through the use of an osmotic agent, such as sucrose, sorbitol or mannitol or an appropriate salt, e.g. sodium chloride or sodium glutamate. An appropriate salt is a salt that does not otherwise interfere with the performance of the method; accordingly, a salt which effectively prohibits the amplification of the nucleic acids is not desirable. It is sometimes advantageous if the osmotic agent does not add to the ionic concentration in the mixture, e.g. , such as when the osmotic agent is sucrose. The centrifugation medium preferably is able to be used directly in the sample, such as blood, and does not lyse the cells in the sample. The centrifugation medium may comprise colloidal silica or colloidal silica coated with polyvinyl pyrrolidone. A centrifugation medium prepared with a compound having iodinated benzoic acid derivatives may be employed. Some examples of useful centrifugation media include PERCOLL, SEPRACELL-MN, and NYCO-DENZ. Other useful media, including Ficoll-hypaque or Ficoll-isopaque, are typically used by layering rather than mixing. For example, if the centrifugation medium lyses white blood cells, then nucleic acid may be prematurely released. If red blood cells are lysed they release hemoglobin, and therefore a source of ferric ion, which interferes with the later amplification reactions. It is preferred that the centrifugation medium allow more than or equal to about 50%, desirably at least 80%, of mononuclear cells present in the sample of whole blood to be recovered after centrifugation. Centrifugation The mixture is then subjected to centrifugation such that the desired cells effectively move to form a discrete band of isolated cells. In the case of isopycnic centrifugation or centrifugation using centrifugation media intermediate the desired cell population and other cells in the sample, the desired cells effectively move toward the end of the centrifuge tube that is toward the center of rotation, rising to the top of the centrifugation medium to form an upper portion containing isolated cells, along with some platelets and/or lipids and other constituents. In one example, the mixture is centrifuged at about 2,900 rpm for about 20 minutes (about 1,500xg). The centrifugation is gentle enough that the centrifugation does not cause a substantial part, or significant percentage, of the cells to lyse, which means that sufficiently few cells lyse that the amplification of nucleic acid recovered from intact cells may proceed. The centrifugation may be performed in a centrifugation tube wherein the part of the tube near the discrete band ( e.g. , the part of the tube near the center of rotation in isopycnic centrifugation), is formed so as to accentuate that portion of the composition containing the desired cells, such as mononuclear cells. This may be done, for example, by narrowing the interior diameter of the tube. Further, the centrifugation may be performed using a device that will inhibit turbulence, and therefore mixing, while making the composition, while performing centrifugation, and/or while removing the isolated, desired cells. An example of such a device is a porous nylon filter that will allow desired cells to pass through it. Preferably, the band of desired cells formed will comprise 20% or less, more preferably 10% or less, of the total volume of the composition. Preferably, it will comprise at least 20%, more desirably at least 30%, of the mononuclear cells present in the composition. Removal Of The Portion Containing The Desired Cells After centrifugation, the portion of fluid containing the desired cells (the discrete band) can be removed from the rest of the mixture using any means known in the art, such as by suctioning the portion using a pipette. Generally, less than about 20% of the total original volume is removed, e.g. , about 500µl of 6ml, and preferably only about the top 100µl at or adjacent the meniscus. Lysing The Desired Cells After removing the isolated desired cells, the cells are lysed to release the nucleic acid from, or associated with, the cells. This means any nucleic acid either attached to or found within or on the desired cells. The cells may be lysed by any desired method, including, for example, the use of a strong alkali, use of an enzymatic agent, use of a detergent, use of osmotic shock, use of chaotropic concentrations of solutes, or use of sonic disruption. In a preferred embodiment, the cells may be lysed by use of potassium hydroxide. This alkali, or an equivalent alkali, e.g., NaOH, (CH 3 ) 4 NOH or LiOH, is particularly useful since it also inactivates cellular nucleases and thus prevents inhibition of later enzymatic reactions using the alkali-induced lysate, and may also denature double stranded nucleic acid Addition of A Chelating Agent An agent that complexes with ferric ion and/or calcium ion and/or other undesirable ions may be added to the density centrifugation medium or to the solution during or after lysis. "Complexes" means that the agent effectively attaches itself to the ion, whether through chelation, coordination, covalent bonding or some other form of bonding or attachment, such that the ion may no longer prevent the later amplification reaction of the invention. These ions are removed from the solution resulting from the lysis because they may inhibit enzymes that may be used in other steps of the method, such as amplifying the nucleic acid from mononuclear cells. Preferably, the complexing agent is a chelating agent that removes such ions from solution, but does not bind significantly with zinc ions nor magnesium ions, as these ions are helpful to some reagents that may be used in the later amplification reaction. See generally Zinc in DNA Replication and Transcription, Wu, F.Y.H., and Wu, C.W., Ann. Rev. Nutr. 7:251 (1987). Examples of chelating agents that may be used are deferoxamine and transferrin. Deferoxamine has a 10 31 binding constant for the ferric ion, see Antioxidant Capacity of Desferrioxamine and Ferrioxamine in the Chemically-Initiated Lipid Peroxidation of Rat Erythrocyte Ghost Membranes, Videla, C.A., et al., Biochem. Int'l, 16, 799 (May 1988), yet does not specifically bind zinc nor magnesium ions. Examples of calcium ion complexing agents are oxalate and citrate. Oxalate and citrate also bind Fe +++ with greater stability compared to Mg ++ , especially at neutral-alkaline pH. Other agents, such as EDTA, are not preferred chelating agents, as they bind zinc ion and magnesium ion, along with other divalent cations. Some useful chelating agents may have the potential to bind desirable ions (e.g., Mg ++ , Zn ++ ), but may be beneficial if they bind undesirable ions preferentially. For example, EGTA binds Ca ++ in preference to Mg ++ . Thus, if the concentration of EGTA is less than the concentration of Mg ++ , there will still be a desirable and predictable activity of free Mg ++ but trace levels of Ca ++ will be effectively sequestered. Furthermore, if such a chelator is mixed with a desirable species, such as Mg ++ , prior to adding it to the reaction of interest, the free activity of Mg ++ contributed to the reaction from other sources will not be reduced by addition of the complexed chelator, but the chelator may effectively sequester preferentially bound species such as Ca ++ , especially if the association/dissociation rates are relatively rapid. The use of a chelating agent is particularly helpful when an anticoagulant has been used previously in the method, as the use of such an anticoagulant may cause or allow the release of ferric ions. Later reactions, such as amplification, using the lysate may also be inhibited by a variety of polyanions. Provision of an effective amount of a polycation such as polyethyleneimine (PEI) can neutralize such polyanions. An effective amount of such a polycation is that amount necessary to reduce inhibition by a polyanion. It is surprising that a polycation such as PEI would have such a beneficial effect because nucleic acids themselves are densely charged anions. Amplification Of Nucleic Acid From Desired Cells The nucleic acid may now be amplified using any method known in the art, such as a polymerase chain reaction procedure, or a procedure described by Kacian, supra , using RNAse H, reverse transcriptase and RNA polymerase. Preferably, the amplification will target the same sequence of nucleic acid that will be the target of the probe, discussed below. The nucleic acid amplification may be performed on the nucleic acid from the desired cells without first removing the debris from the centrifugation and lysis, nor otherwise "cleaning up" the portion containing the isolated desired cells, other than appropriately preparing the chemical balance of the solution to be subjected to amplification, for example, neutralizing the lysis agent such as potassium hydroxide. Screening For Desired Nucleic Acid The amplified nucleic acid is now ready to be screened for a desired sequence of nucleic acid using any procedure known in the art or other related procedures, such as hybridization using a strand of DNA or RNA complementary to the desired nucleic acid sequence, which complementary strand is known as a "probe." The probe may be hybridized to the nucleic acid and detected using any known detection technique, such as traditional Southern or Northern blotting techniques, or the homogeneous protection assay ("HPA") described by Arnold et al., supra . In a preferred embodiment, the probe will detect the nucleic acid associated with an HIV virus. Kit For Effecting The Above Method A simple kit for performing the above method may be prepared from readily available materials and reagents. The kit may be designed so that the reagents complement each other, for example, the osmotic agent may be sucrose and the centrifugation medium PERCOLL®. EXAMPLES : Preparing Density Centrifugation Medium PERCOLL® is provided by the manufacturer at a density in the range 1.130 ± 0.005 g/ml. The precise density is supplied with each lot. To make a 1 liter (1000 ml) suspension of PERCOLL® in 0.25 M sucrose at a specified density, ρ Desired , the volume (in ml) of PERCOLL® stock required can be estimated from the following equations: ρ Desired = V Percoll ρ Percoll + V Osmot ρ Osmot +(1000- V Percoll - V Osmot ) 1000 ml or, V Percoll = 1000(ρ Desired -1)- V Osmot (ρ Osmot -1) (ρ Percoll -1) In this example, sucrose is the osmoticum and is introduced into the mixture as a 5X (1.25 M) stock; thus for a final concentration of 0.25 M, the volume ( V Osmot ) to be added = 200.0 ml. The density of 1.25 M sucrose is 1.1607 g/ml. The final term in the numerator of the first equation (1000- V Osmot - V PERCOLL® ) represents the volume of water added to bring the final volume up to one liter and assumes a nominal density of 1.000 g/ml for water. Thus, 1.) To make a suspension with a density of 1.110 g/ml using a stock solution of PERCOLL® supplied at a density of 1.131 g/ml, mix: 594.4 ml PERCOLL® 200.0 ml 1.25 M Sucrose 205.6 ml H 2 O 2.) Accurately measure the refractive index (RI) of the resulting suspension. Calculate the density using the relationship (described by Pharmacia) appropriate for density centrifugation medium (DCM) comprising PERCOLL® in 0.25 M sucrose (DCM 1 ): ρ DCM 1 =(6.6145× RI DCM 1 )-7.8620 The RI expected for the desired 1.110 g/ml suspension would thus be 1.3564. 3.) If the desired density is difficult to obtain, or sufficiently accurate volumetric glassware is not available, use the formula above as a guide to estimate volumes required to make 2 suspensions with densities which bracket the desired final density (e.g., 1.105 g/ml and 1.115 g/ml). Measure the RI of each and calculate the true density of each. Calculate the volumes of each to mix (e.g., V low and V high (= V Tot - V low )) to obtain the desired density and total volume: ρ Desired = ρ low V low +ρ high ( V Tot - V low ) V Tot 4.) A similar strategy can be used for making PERCOLL® suspensions in other osmotica such as 0.15 M NaCl. The density of NaCl solutions is also available in the literature and can be calculated as a function of concentration. For example, 1.50 M NaCl, which can be used as a 10X stock instead of the 5X sucrose in the formulation above, has a density of 1.058 g/ml. The density can be calculated from the RI of such a PERCOLL®/saline DCM 2 by the following equation: ρ DCM 2 =(6.8166× RI DCM 2 )-8.0923 A refractive index of 1.350 would correspond to the desired density of 1.110 g/ml for DCM 2 . 5.) Agents other than PERCOLL® can be used to generate the desired density. (As outlined in the Pharmacia and Nycomed product literature, the desired properties of a density reagent include: sufficient solubility and specific gravity to yield solutions of at least the desired density, little osmotic activity of its own, and low viscosity in solution.) For example, NYCODENZ® stock solutions can be made by dissolving it in water at a density greater than desired as the final DCM density (e.g. ≥0.35 M). The density of an aqueous NYCODENZ® solution can be predicted from its molarity: ρ Nycodenz =(0.43914[ Nycodenz ])+0.99828 and confirmed by its RI: ρ Nycodenz =(3.242× RI Nycodenz )-3.323 Such a NYCODENZ® stock solution of known density can be used in conjunction with a suitable osmoticum to yield an iso-osmotic DCM of the desired density using the same procedure described for PERCOLL® above. The densities of the resulting mixtures can be calculated from the refractive indices using the following constants (from Nycomed): ρ DCM 3 =(3.553× RI DCM 3 )-3.751 for NYCODENZ® in sucrose (DCM 3 ), and: ρ DCM 4 =(3.287× RI DCM 4 )-3.383 for NYCODENZ® in saline (DCM 4 ). The buoyant density of the cells present in the sample, such as blood, can change if the osmotic strength is changed. Typically, the buoyant density of the cells decreases as the osmolarity is decreased and increases as the osmolarity is increased. While various combinations of osmolarity and medium density can be used to achieve similar results, the near-physiological osmolarities described here are suited to the purpose of this method (preparing cells for subsequent amplification reactions). Further, Red Blood Cells (RBCs) are typically more sensitive to osmotic shock than White Blood Cells (WBCs) and the most suitable sample processing conditions are those which minimize the possibility of RBC lysis. As described herein, a density of 1.110 g/ml is preferred for the DCM to mix with whole blood in order to obtain a final mixture density of 1.077 g/ml to separate mononuclear cells from whole blood. This density for the DCM was chosen empirically based on the results of several experiments including examinations of the number of cells recovered, of the purity of the desired fraction, i.e. low granulocyte contamination as determined by Wright staining, and by observing a typical ratio of lymphocytes-to-monocytes, as determined by Wright staining, among the cells recovered. This DCM density is also a preferable density based on the typical buoyant density of mononuclear cells (usually considered to be <1.077 g/ml) and the normal range of blood composition. The packed volume of RBCs for most adult individuals is usually at least 35% of the total blood volume, although this can be lower for individuals with significant anemia. When the DCM and whole blood are mixed, the density of the resulting liquid phase should be a combination of the partial volumes contributed from each source, i.e., the DCM and plasma, and their respective densities: ρ Plasma/DCM = V DCM ρ DCM + V Plasma ρ Plasma V DCM + V Plasma If equal volumes of DCM, with a density = 1.110 g/ml, and blood are mixed and if 65% of the blood volume is plasma, with a density of 1.026 g/ml, then the density of the resulting liquid phase will be 1.077 g/ml, which is an ideal density for separating mononuclear cells from other blood cells. In such a mixture, most of the mononuclear cells will float to the top of the mixture, along with many of the platelets; the RBCs and granulocytes will sediment. Using this approach, it is apparent that the preferred density will vary according to conditions within the blood cell sample (such as blood) and the DCM. Determining an appropriate density using the above mentioned factors does not require undue experimentation. Blood from the majority of individuals will have a packed RBC volume (as measured by hematocrit procedures) greater than 35% of the total blood volume. Thus mixing equal volumes of blood and 1.110 g/ml DCM will yield a liquid phase with a density >1.077 g/ml for most blood samples. The target mononuclear cell fraction will float even more readily to the top of such a mixture. Fig. 1 shows the RBC counts and hematocrits determined for blood taken from 66 different individuals. The 2 nd y-axis shows the corresponding density of the DCM-plasma mixture which would result from mixing a specified volume of DCM with an equal volume of blood having the hematocrit directly opposite on the primary y-axis. The example shows that the strategy described here would result in DCM/plasma mixture having a density between 1.076 g/ml - 1.082 g/ml for the majority of individuals. As the density of the mixture increases, some granulocyte contamination might be expected among the mononuclear cell (MNC) fraction. However, mixtures throughout this density range should be very effective at separating MNC and granulocytes since the buoyant density of the granulocytes typically averages 1.086 g/ml and only a small fraction have a buoyant density <1.082 g/ml. Moreover, for some aspects of the invention, such as nucleic acid target amplification and/or hybridization analysis, even substantial granulocyte contamination is not a significant problem since these techniques have been designed to be specific for desired target sequences even in the presence of vast excess levels of non-target sequences. For example, while it is advantageous to recover a cell population which is highly enriched in MNC, which include target cells for HIV infection, over granulocytes, which are not target cells for HIV infection, it is not absolutely required. It is more important in such a case to use conditions which result in recovering most of the MNC than to recover highly purified MNC. Using the procedures described above it has been possible to make DCM of the desired density ±0.002 g/ml reproducibly and reliably. It should be noted that the invention does not require that the blood and DCM be combined in equal volumes or that the DCM density is constrained to be 1.110 g/ml. It is within the skill of the art without undue experimentation to calculate the appropriate volumes to combine for a wide range of DCM densities based on the partial volumes of the components and their respective densities as described above. Conversely, the appropriate DCM density to use can be calculated to accommodate the desired volume and ultimate mixture density specifications. Example of Blood Fractionation to Recover Mononuclear Cells 1.) Mix 3.0 ml EDTA anti-coagulated whole blood with 3.0 ml isosmotic PERCOLL®/sucrose DCM (density = 1.110 g/ml) in a screw cap test tube. Cap securely and invert several times to mix thoroughly. 2.) Centrifuge in swinging bucket rotor at 1600 x g for 20 min. RBCs will pellet. Granulocytes will pellet with, and band on top of, the RBCs. MNC will float to the top of the mixture. Other centrifugation conditions may be used. For example, greater g-force can be used for a shorter time, but it is preferred that g-force not exceed a level where full isopycnic equilibrium is attained, as the MNC may then band below the meniscus. Geometry of the tube may also affect centrifugation time and force. For example, a shorter column of liquid should reach isopycnic equilibrium more quickly. Further, devices to stabilize the meniscus and/or MNC band may be used. Adapting the centrifugation conditions appropriately for such differing conditions, or for different cell types or sample sources, does not require undue experimentation for one of skill in the art. Also, the invention may include a centrifugation procedure comprising at least one wash step. Such methods are known in the art. The appearance of the MNC band will vary slightly depending on the specifics of the centrifugation and the centrifugation apparatus, such as the diameter of the tube, volume of blood used, etc., but will typically concentrate at the highest point of the meniscus. 3.) The complete MNC band may be harvested by aspirating into a pipet, manipulating the pipet so that the tip remains in a region of dense cell accumulation. Continue until most of the MNC are recovered. Typically, this will result in collection of 300-700 µl of the MNC/DCM/plasma suspension. Example of Cell Lysis Method For Mononuclear Cells 1.) Mix 250 µl of the MNC suspension with 250 µl 0.14 N KOH. Vortex to mix well. Heat at 95°C for 30 min. 2.) Adjust the pH of the resulting hydrolysate to pH 8.0 ± 0.5 (typically) by adding 50 µl of a solution comprising: 0.65 N Acetic acid (HOAc) 0.066 M Tris(hydroxymethyl amino)methane, base 0.084 M Tris·HCl 3.) Mix well, e.g., by vortexing. Other methods to cause lysis may also be used, for example, combinations of other basic solutions followed by neutralizing acid solutions, or enzymatic or mild detergent agents. Such methods, for mononuclear cells and for other cells, are known in the art. Using the Lysate in Amplification Reactions The above lysate contains a crude mixture of biologically derived molecules, many of which are low molecular weight hydrolysis products of the original sample constituents. The DNA present in these lysates, while fragmented and denatured compared to its original structure within the cells, is still a suitable reactant for a variety of chemical and biochemical reactions, including as a template for nucleic acid target amplification. To minimize the complexity and manipulations involved in practical, routine use of target amplification to analyze blood-derived nucleic acids, in one aspect of the invention the lysate is added directly to the amplification reaction as the source of the analyte, without further purification. However, it is well known that biologically-derived samples frequently contain components that are inhibitory to in vitro biochemical reactions, including enzyme reactions. For example, even nucleic acid preparations that are much more highly purified than the lysate yielded by the method described herein have been reported to contain potent inhibitors of polymerase chain reaction amplification (R. deFranchis, N.C.P. Cross, N.S. Foulkes and T.M. Cox, A Potent Inhibitor of Taq Polymerase Copurifies with Human DNA, Nucleic Acids Research 16:10355 (1988)). Many metal ions, especially polyvalent species, can inhibit enzyme activities through a variety of mechanisms, including displacement or substitution of requisite metal co-factors, or by forming stable complexes with susceptible moieties, including amino acid side chains which are necessary for catalytic activity or which result in deleterious steric changes in the enzyme or by promoting deleterious redox reactions. Chelators are sometimes employed to sequester undesirable metals but it is not obvious in any particular case that an efficacious chelator can be identified which does not itself inhibit the enzyme activity, e.g., by complexing necessary co-factors or even stripping bound metals from the enzyme. Deferoxamine, a biologically-derived natural product with extremely high affinity for Fe(III), was found to be highly effective at neutralizing the inhibition imposed by lysates of blood fractions on the amplification reactions and was found to be well tolerated in the reaction over at least the concentration range of 0.001 - 1 mM. An example of deferoxamine's use is described below and the beneficial effects demonstrated in Figs. 2 and 3. One of the agents tested and found to be effective at counteracting the lysate sample inhibition of amplification was Zn(OAc) 2 . Although reverse transcriptase has been reported to be a Zn metalloenzyme (as has T7 RNA polymerase, though this is no longer thought to be the case) it is not known if (or claimed that) the beneficial activity of Zn ++ in this context is related to the normal Zn ++ binding site of one or more enzymes in the reaction. It is possible that Zn ++ interacts with other site(s) on the enzyme(s) in a favorable way or that it interferes directly or indirectly with one of the inhibitors in reactions of this type. It was not obvious that conditions could be identified where net beneficial effects were observed. Fig. 2 shows an example of the enhancement possible from Zn ++ in the amplification reaction, especially at negligible deferoxamine levels. While the deferoxamine benefits may be slightly antagonized by high Zn ++ levels, it is apparent that mixtures can be identified over fairly broad concentration ranges of each which support a stable platform of effective amplification. Many nucleic acid reactive enzymes are inhibited by a variety of polyanions. One strategy for neutralizing polyanions is to include a polycationic species such as polyethyleneimine (PEI) in the reaction. Since nucleic acids are themselves densely charged anions, it was surprising that conditions could be identified under which enhanced amplification could be observed without detectable loss of endogenous template activity. PEI concentrations of ≤3x10 -5 % (w/v) in the amplification reaction have been consistently beneficial in these respects. An example of the enhancement possible is shown in Fig. 3. The example described below illustrates execution of an amplification that might contain one or more of these anti-inhibitory compounds. 1.) Prepare and dispense 40 µl of a solution containing the following components. The concentrations listed refer to the respective concentrations in the completed 100 µl reaction. 50 mM Tris·HCl (pH 8.0 at room temperature) 17.5 mM MgCl 2 5 mM DTT 2 mM Spermidine 6.25mM ea GTP & ATP 2.5 mM ea UTP & CTP 0.2 mM ea dATP, dGTP, dCTP, dTTP 0.3 µM ea primer "A" & primer "B" 2.) Optionally, include Deferoxamine mesylate at 0 - 1 mM final concentration. Optionally, include Zn(OAc) 2 at 0 - 0.1 mM final concentration. Optionally, include PEI at 0 - 3x10 -5 % final concentration. 3.) Add to this mixture, 40 µl of the hydrolyzed MNC suspension prepared as described above. Optionally add a known amount of purified nucleic acid containing the target sequence(s) of interest as a basis for comparing amplification performance supported by different conditions. 4.) Heat the mixture to 95°C and maintain for 5 minutes. Cool the mixture to 37°C. 5.) Add 10 µl of a solution containing 600 U Moloney MuLV reverse transcriptase (MMLV-RT), preferably in a buffered solution of composition sufficient to maintain enzyme stability during handling. Incubate the mixture for 10-15 minutes at 37°C. 6.) Heat the mixture to 95°C and maintain at this temperature for 5 minutes. Then cool the mixture to 37°C. 7.) Add 10 µl of a solution containing 600 U MMLV-RT and 400 U T7 RNA polymerase, preferably in a buffered solution of composition sufficient to maintain enzyme stability during handling. 8.) Mix briefly and incubate the mixture at 37°C for 1-2 hours. 9.) Determine the amount of amplification product formed as a copy of the intended target sequence using a specific hybridization procedure such as the hybridization protection assay. See , e.g. , Arnold et al., supra . Other forms of deferoxamine may also be used, as well as other zinc providers, such as ZnCl 2 or ZnSO 4 . Also, other polycationic polymers such as polyallyl amine or Polybrene® (hexadimethrine bromide) may also be used with or instead of PEI.

Claims: A method for amplifying a target nucleic acid sequence in an amplification mixture wherein said mixture includes a first complexing agent able to complex ferric ions, said first complexing agent being selected from deferoxamine, sideramine, sideramycin and ferrimycin, so that amplification of the target sequence in said mixture yields more amplified nucleic acid than when said first complexing agent is absent from said mixture. A method as claimed in claim 1 wherein said first complexing agent is deferoxamine. A method as claimed in claim 1 or claim 2 wherein said mixture additionally includes a second complexing agent able to complex calcium ions, so that amplification of the target sequence in said mixture yields more amplified nucleic acid than when said second complexing agent is absent from said mixture. A method as claimed in claim 3 wherein said second complexing agent has a greater affinity for undesirable ions than for desirable ions. A method as claimed in claim 4 wherein said desirable ions include at least one of zinc, manganese and magnesium ions. A method as claimed in claim 5 wherein said second complexing agent is selected from EGTA, citrate and oxalate. A method as claimed in any one of the claims 1 to 6 wherein at least one of said first and second complexing agents is provided in said mixture as a complex or salt and wherein said complex or salt comprises said complexing agent and at least one desirable ion selected from zinc, manganese and magnesium ions. A method as claimed in any one of claims 1 to 7 which further comprises preparing said amplification mixture by a method comprising the steps of: (a) centrifuging a composition comprising a centrifugation medium and a sample of blood cells comprising mononuclear cells under centrifugation conditions such that a substantial portion of said mononuclear cells in said composition gather in a discrete band; (b) separating said discrete band from said composition; (c) lysing said mononuclear cells in said discrete band by (i) providing to said band a lysing agent, or (ii) exposing said band to sonic disruption, so that said mononuclear cells in said band lyse, thereby rendering said nucleic acid available for amplification; (d) providing to said band said first complexing agent or said first and second complexing agents; and (e) subjecting said nucleic acid to conditions sufficient to amplify said target nucleic acid sequence. A method as claimed in claim 8 wherein zinc ions are provided to said band during at least one of said providing, exposing and subjecting steps. A method as claimed in claim 8 or claim 9 wherein said-composition is isotonic with said mononuclear cells such that said composition lyses less than 20% of said mononuclear cells present in said composition prior to said lysing step. A method as claimed in any one of claims 8 to 10 wherein said centrifugation medium is isopycnic to said blood cells. A method as claimed in any one of claims 8 to 11 wherein said band comprises at least 30% of said mononuclear cells present in said composition. A method as claimed in any one of claims 8 to 12 wherein said centrifugation medium contains an osmotic agent isotonic to said mononuclear cells, said osmotic agent comprising sucrose, sorbital, mannitol, sodium chloride, or sodium glutamate. A method as claimed in claim 13 wherein said osmotic agent does not alter the ionic concentration of said composition. A method as claimed in any one of claims 8 to 14 wherein said centrifugation medium comprises colloidal silica or colloidal silica coated with polyvinyl pyrrolidone. A method as claimed in any one of claims 8 to 15 wherein said centrifugation medium is prepared with a compound having iodinated benzoic acid derivatives. A method as claimed in any one of claims 8 to 16 wherein said blood cells are derived from whole blood or animal serum. A method as claimed in any one of claims 8 to 17 wherein at least 80% of said blood cells in said sample remain intact after said separating step and prior to said lysing step. A method as claimed in any one of claims 8 to 18 wherein in step(e) said conditions are sufficient to amplify said target sequence by polymerase chain reaction amplification. A method as claimed in any one of claims 8 to 19 wherein said subjecting step includes providing reverse transcriptase and RNA polymerase. A method as claimed in any one of claims 8 to 20 wherein said lysing agent is an alkali, an enzyme, or a detergent. A method as claimed in claim 21 wherein said lysing agent is KOH, NaOH, (CH 3 ) 4 NOH, or LiOH. A method as claimed in any one of claims 8 to 22 wherein said centrifugation medium has a density of between 1.105 and 1.115 grams per milliliter. A method as claimed in any one of claims 8 to 23 wherein in step (e) a polycationic polymer is provided in an amount effective to reduce inhibition of amplification by a polyanion. A method as claimed in any one of the preceding claims wherein said target sequence is present in nucleic acid of a human immunodeficiency virus or hepatitis B virus. A kit for preparing an amplification mixture as claimed in any one of claims 8 to 25 comprising: (a) said first complexing agent or said first complexing agent and said second complexing agent; and (b) one or more further components selected from: (i) a centrifugation medium; (ii) a lysing agent able to lyse mononuclear cells; and (iii) amplification materials for amplifying said target nucleic acid sequence. A kit as claimed in claim 26 further comprising said centrifugation medium wherein said centrifugation medium is selected to have a density such that at least 30% of mononuclear cells in a composition comprising said medium and a sample of blood cells containing said mononuclear cells gather in a discrete band under centrifugation conditions. A kit as claimed in claim 26 or claim 27 further comprising an osmotic agent in an amount sufficient to render said medium substantially isotonic to whole blood. A kit as claimed in any one of claims 26 to 28 further comprising said amplification materials in amounts sufficient to amplify said target nucleic acid sequence. A kit as claimed in claim 29 wherein said amplification materials are sufficient to amplify said target nucleic acid sequence using polymerase chain reaction amplification. A kit as claimed in claim 29 wherein said amplification materials include reverse transcriptase and RNA polymerase. A kit as claimed in any one of claims 26 to 31 further comprising a lysing agent able to lyse mononuclear cells. A kit as claimed in claim 32 wherein said lysing agent is KOH, (CH 3 ) 4 NOH, NaOH or LiOH. A kit as claimed in any one of claims 26 to 33 further comprising a polycationic polymer in an amount effective to reduce inhibition of nucleic acid amplification by a polyanion. A kit as claimed in claim 34 wherein said polycationic polymer is polyethyleneimine, polyallylamine or hexadimethrine bromide. A kit as claimed in any one of claims 26 to 35 further comprising a nucleic acid probe able to screen for said target nucleic acid sequence. A kit as claimed in claim 26 for screening for a virus that may be present in a sample of whole blood, said kit comprising: (a) a centrifugation medium selected to have a density such that at least 20% of mononuclear cells in a composition comprising said medium and said whole blood gather in a discrete band in said composition under centrifugation conditions; (b) a lysing agent able to lyse said mononuclear cells, thereby rendering nucleic acid from said virus available for amplification; (c) said first complexing agent or said first complexing agent and said second complexing agent; and (d) amplification materials sufficient to amplify a target nucleic acid sequence of said nucleic acid. A kit as claimed in claim 37 which includes one or more components as defined in any one of claims 28, 30, 31 and 33 to 36.

Inventors: KACIAN DANIEL LOUIS, RYDER THOMAS B, KACIAN, DANIEL LOUIS, RYDER, THOMAS B.
Applicants: GEN PROBE INC, GEN-PROBE INCORPORATED
Application year: 1993
Main classification: C12Q1
IPC codes: C12N5, C12N15, C12Q1