NO882593L - IMPROVED KEY ACID HYBRIDIZATION TECHNIQUE AND KIT FOR THIS. - Google Patents

Description

The present invention relates to a nucleic acid hybridization method, and a diagnostic kit for use with the method.

All articles and references identified in this specification are incorporated herein by reference.

In traditional microbial diagnostics, the presence of a microbe in a sample is determined by isolating the microbe. After enrichment cultures, the microbe is determined either on the basis of the biochemical characteristics or the immunological characteristics. Both methods of identification require that the microbe in the sample has the ability to multiply. Such identification can be difficult and very time-consuming.

With the development of genetic research, it has been discovered that microbes, even those no longer living, can be determined by the specific nucleic acids they contain. The nucleic acid can be deoxyribonucleic acid (DNA), which is usually double-stranded, or ribonucleic acid (RNA), which is usually single-stranded. Organisms such as bacteria, fungi, viruses, yeast etc. can all be determined in this way. In general, the identification method involves artificially induced lysis, whereby the cell wall of the organism is destroyed and secretes nucleic acid which can thus be determined.

The hydrogen-bonded structure of the double helix of a nucleic acid molecule (eg DNA) can be destroyed by heating and/or treatment with alkali. Since there are no covalent bonds linking the partner strands together, the two polynucleotide chains of a duplex nucleic acid molecule separate completely when all the hydrogen bonds are broken. This process of strand separation is called denaturation. A very useful property of denatured nucleic acids is that the reaction can be reversed under suitable conditions, . so that two separated complementary strands from the same source can rejoin to form a double helix.

This is called renaturation. Renaturation involves the reaction of two complementary nucleotide sequences that were separated by denaturation. This technique can also be extended to include any complementary nucleotide sequences fusing together to form a duplex structure. This is referred to as hybridization when nucleotide sequences from different nucleic acid constituents are involved; e.g. the reaction between single-stranded DNA and RNA, or between two single-stranded DNAs from different sources.

Nucleic acid hybridization is a well-known method for the identification of specific nucleic acids. This ability that two single-stranded nucleic acid preparations have to hybridize forms the basis for nucleic acid analysis. The principle of such analyzes is to expose two single-stranded nucleic acid preparations to each other, and then to measure the amount of double-stranded material that has been formed.

Hybridization assays typically involve the use of polynucleotide hybridization probes. A probe generally includes a single-stranded fragment of a nucleic acid, or a double-stranded fragment that has been denatured. It is characterized by having a specific nucleotide sequence that is complementary to a corresponding nucleotide sequence on a target nucleic acid (the nucleic acid to be determined). The probe and the target polynucleotide (single-stranded), form duplex molecules when they are brought together by base pairing of the complementary sequences. The probe is generally prepared in purified reagent form, and an easily detectable label can be incorporated into the molecular structure of the probe. Presence of the target nucleotide can thereby be confirmed by the formation of duplex hybrid molecules carrying the label.

Polynucleotide hybridization probes provide an inexpensive, efficient, and rapid method for the detection, localization, and isolation of the "target" nucleotide sequences. Klausner et al., "Biotechnology", August 1984: 471-478, provides interesting background on polynucleotide hybridization probes, including a discussion of preparation and use. Known methods for the preparation of polynucleotide hybridization probes and for the use of such probes are well documented in the literature See, eg, Southern, J. Moi. Biol. 98: 503-517 (1975); Falkow et al., US Patent 4,385,535; Leary et al., Proe. Nati. Acad. Sci. 80: 4045-4049 (1938); Langer-Safer et al., Proe. Nat. Acad. Sci. 79: 4381-4385

(1982); and Langer et al., Proe. Nati. Acad. Pollock. 78:6633-6637

(1981). As described in these other references, such known methods for preparing probes include cloning a probe region into a double-stranded DNA plasmid. The plasmid carrying the probe region is labeled, usually by enzymatic polymerization techniques. Such techniques include, e.g. nick translation (Rigby et al., J. Mol. Biol. 113:237 (1977)); gap filling (Bourguignon et al., J. Virol. 20:290 (1976)); and terminal addition, where the techniques are performed in the presence of modified nucleotide triphosphates. The probe nucleic acid can also be labeled chemically with haptens or biotin (Tchen et al., P.N.A.S. 81:3466 (1984), Forster et al., Nucl. Acids Res. 13:745 (1985), Viscidi et al., J. Clin. Microbiol.23:311 (1986).

There are two principal ways of performing hybridization assays: solution (or liquid) hybridization and solid support hybridization. In solution hybridization, single-stranded nucleic acid preparations are mixed together in solution. In the case of larger amounts of material, the reaction can be followed by changes in optical density. When smaller amounts material is included, the probe may carry an easily detectable label, such as a radioactive label. The unreacted single strands are separated from the double strands, and double-stranded nucleic acid can be determined by detecting the presence of the label in the double -stranded material. Hydroxyapatite (HA) has been used as a standard method for separating hybridized probe from non-hybridized probe in solution. Under appropriate conditions, HA can selectively bind hybridized DNA probe, but it does not bind non-hybridized probe. Others methods are therefore available for the separation of hybridized probe from non-hybridizers t probe. Such a method includes the use of a specific enzyme, e.g. S^nuclease, to selectively degrade unhybridized probe into smaller fragments. Double-stranded molecules are not affected by the enzyme. The degraded probe can then be separated using well-known size separation techniques, such as polyethylene glycol precipitation, chromatography, electrophoresis and ultracentrifugation, etc. Britten et al., Methods in Enzymology, XXIX, p. 363, Eds., Grossman & Moldave , Academic Press, New York, 1974.

Solution hybridization has advantages that include speed and reaction efficiency. But it also has serious drawbacks. Separation of hybridized non-hybridized probe is generally labor intensive, time consuming, cannot be performed by automation, and can be otherwise limiting. The size separation techniques described above, for example, are relatively non-specific, inefficient and show poor reproducibility. Although the HA separation method is more specific, it is only generally useful if the target polynucleotide is present in large excess relative to the probe, or if the target is RNA, or both. Effective separation can be difficult with impure samples, e.g. plant and animal tissue homogenates, blood, faeces, nasal and urethral mucus, etc.

In solid carrier hybridization, one of the single-stranded nucleic acid preparations is immobilized by being attached to a solid carrier. Multiple methods exist for connecting one of the. the terminal ends of a polynucleotide to a given support matrix. See, e.g. Weissback, A. and Poonian, M., Methods in Enzymology, Vol. XXXIV, Part B, 463-475, 1974. Derived forms of polynucleotides, e.g. one that harbors a terminal aminohexyl nucleotide can be readily attached to various matrices. Mosbach, K., et al., Methods in Enzymology, Vol. XLIV, 859-886, 1976. Igoribonucleotides can be immobilized on boronate derivatives of various matrices. Schott, H., et al., Biochemistry, 12, 932, 1973.

As an example, nitrocellulose adsorbs single-stranded DNA but not RNA. Further adsorption of DNA on the nitrocellulose can be prevented by well-known treatments. So if another denatured DNA or RNA preparation is added, it will be attached to the solid support only if it has the ability to base pair with the DNA that was originally adsorbed. Typically, the nucleic acid preparation that is not initially bound to the solid support is labeled. After the hybridization reaction is complete, the solid support is separated from the reaction mixture, and the degree of hybridization can be determined by measuring the label attached to the solid support.

An improvement on solid support hybridization is sandwich hybridization, as discussed in Ranki, US Patent 4,486,539. The sample is exposed to conditions that make the target polynucleotide (the nucleic acid to be determined) single-stranded. The sample is then mixed with two purified nucleic acid reagents. The first nucleic acid reagent comprises a single-stranded nucleic acid fragment, having a nucleotide sequence of at least 10 bases, and which is attached to a solid support. The second nucleic acid reagent includes a single-stranded nucleic acid fragment, which has a nucleotide sequence of at least 10 bases, and which is labeled with a radioisotope. The nucleic acid reagent has the ability to form hybrid molecules by complementary base pairing with the target polynucleotide. The two nucleic acid reagents. do not have the ability to hybridize with each other. The solid support is thereby washed to remove the label that is not incorporated into hybrid molecules. Presence of target nucleic acid is then determined by measuring the label on the washed solid support. Sandwich hybridization also provides improved specificity over methods using a single nucleic acid reagent because it involves two specific hybridization processes.

An advantage of solid support hybridization is how easily the hybrid molecule formed from the target nucleic acid and the probe(s) can be separated from the reaction mixture. Previous types of solid support hybridization techniques, including the sandwich hybridization method of Ranki discussed above, have serious drawbacks. The kinetics result in much slower reaction rates in solid carrier hybridization than in solution hybridization, as not all the hybridization components can diffuse. A reaction that can take minutes in resolution can take hours and days to complete when a solid carrier is involved. Hybridization efficiency is thus much lower, since some nucleic acids are not available for base pairing. A lot of preparation is also necessary. Typically, several hours are required to prepare the sample for hybridization, and one to two hours are required for washing. Relatively large quantities of probes are also required.

It is therefore desirable to have a method for analyzing nucleic acids that combines the advantages of both solution hybridization and solid carrier hybridization.

Diagnosis of genetic disease, according to the state of the art, generally involves time-consuming and labor-intensive techniques. In an accepted diagnostic method for determining genetic disease, the method is known as restriction fragment length polymorphism (RFLP). The RFLP technology generally uses an extensive separation step based on the size of the genetic patches that are made after treating the sample DNA with a restriction enzyme. The specialized case of sickle cell anemia presents the most easily understood variation of RFLP technology. For example, The New England Journal of Medicine, July, 1982: pp. 30-32 and pp. 32-36, contains two articles illustrating how the current prenatal test for sickle cell anemia is performed. In general, test sample DNA is treated with a restriction enzyme (Mst II) that provides two differently sized pieces of the globin gene for the normal and sickle cell alleles. The pieces are separated by size, e.g. an agarose electrophoresis gel. The pieces are then single-stranded, transferred to a sheet of filter paper, and the exact pieces are determined in the amount of equal-sized pieces by a hybridization probe. Both gel electrophoresis and the transfer steps in the test require the use of trained personnel, and are time-consuming.

There is therefore a need for a simplified method for diagnosing the presence of gene mutations due to genetic diseases.

The present invention satisfies the above requirements. It specifically covers a method for detecting, either qualitatively or quantitatively, a single-stranded target nucleotide in a liquid sample. The method comprises the following steps: a) combining the sample with at least two different probes, which are a first probe and a second probe, to form a reaction mixture, each probe consisting of a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the target polynucleotide, wherein the probes together form hybrid molecules by complementary base pairing with the target polynucleotide, neither probe is attached to a solid support, and the probes do not hybridize with each other, and the other probe has a detectable label;

b) then contacting the reaction mixture with a solid support which binds the first probe but not the second

the probe, and not the target nucleotide; and

c) then determining whether any of the brand is attached to the fixed carrier.

Single-stranded target polynucleotide can be formed by denaturing a double-stranded polynucleotide. The two probes preferably bind different parts of the target polynucleotide to be determined, so that the two probes do not compete with each other. The two different parts are preferably next to each other or close to each other.

The method according to the present invention is suitable for microbial diagnostics. It is also suitable for genetic disease diagnosis such as for sickle cell anemia, and for cancer diagnosis, among other applications. With regard to the latter, the method of the present invention incorporates a separation step, such as using a restriction enzyme, along with a sandwich hybridization method. For example, the method can be used to detect a target polynucleotide in a sample which can also contain a standard polynucleotide, where the nucleotide sequence of the two polynucleotides is substantially similar to each other, and the standard polynucleotide. is a portion A and a portion B, and the target polynucleotide has a portion A' and a portion B', and wherein the standard and target polynucleotides have at least one nucleotide that is different and which is located between portions A and B of the standard polynucleotide and between the portions A' and B' on the target polynucleotide, the method comprising the following steps: a) separation of both the standard and the target polynucleotides present into single-stranded segments, such that none of the segments of the standard polynucleotide contain both parts A and B, while at least one of the segments of the target polynucleotide contains both parts A' and B';

b) combining treated sample from (a) with at least two probes, being a first probe and a second probe, to

form a reaction mixture, each probe includes a single-stranded polynucleotide, the probes cannot hybridize with each other, the first probe base pairs complementary with part A, and with part A', the second probe base pairs complementary with part B, and with part B', the two probes together form hybrid molecules with complementary base pairing with the single-stranded one

the segment of the target polynucleotide containing the parts A' and B'; and c) subsequent determination of the presence of hybrid molecules containing both probes.

Neither probe is attached to a solid support, and the other probe has a detectable label, where the steps for determining the presence of hybrid molecules containing both probes further include the following steps: a) contacting the reaction mixture with a solid support that binds the first the probe but not the other probe, and not segments containing only part B or only part B' ; and b) subsequently determining whether any of the label is bound to the solid support.

DRAWINGS

These other features, aspects and advantages according to the present invention will be better understood with reference to the following description, attached claims and attached drawings: Fig. 1 is a schematic representation of the steps of the analysis method according to the present invention. Fig. 2 is a schematic representation of a sickle cell assay using the method according to the present invention. Fig. 3 and fig. 4 is a plot of the relative signal for mark detection vs. target DNA concentration. Fig. 5 is a plot of the binding characteristics of an avidin cellulose solid support and a biotin-labeled probe.

DESCRIPTION

A method and a kit that includes the features according to the present invention can be used to detect the presence of polynucleotide (such as DNA or RNA) containing organisms, such as viruses, bacteria, fungi, yeast and other microorganisms and other infectious agents. The process and the kit can be used, for example, in food hygiene investigations, medical diagnostic applications and any microbial diagnostics. Suitable samples Including animal and plant tissue homogenates, blood, serum, feces, nasal and urethral mucus, water, dust, soil, etc. Solid samples are first mashed or homogenized in a liquid medium to prepare a test sample. The method is also suitable for diagnosing genetic disease when normal genes have been mutated. The procedure is also suitable for cancer diagnostics.

In the method according to the present invention, a sample containing cells presumed to contain polynucleotides to be detected (the target polynucleotide) is pre-treated, if necessary, to release the target polynucleotide from the cells of the organism into solution, or to make the cell wall permeable to the reagents used for detection of the target polynucleotide. The target polynucleotide is usually a DNA or an RNA, or derivatives or fragments thereof.

Hybridization takes place between single-stranded polynucleotides. If the target nucleotide is thus double-stranded, the test sample is exposed to conditions which can denature the target nucleotide present, e.g. heat, or heat plus high pH, such as 100°C for 5 min, or treatment, with 0.5 molar NaOH for 5 min, at 20-40°C. Conditions for denaturing polynucleotides are well known.

The method according to the present invention, as illustrated in fig. 1, includes a solution sandwich hybridization step and a harvesting step. The solution sandwich hybridization step involves combining the test sample with at least two different specific nucleotide hybridization probes to form a reaction mixture. For example, two probes can be used, being a first probe, a binding probe, a second probe and an identification probe. Each probe includes a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the target single-stranded polynucleotide. Each probe thus has the capability of complementary base pairing with the corresponding nucleotide sequence on the target polynucleotide. The two probes preferably bind different parts of the target polynucleotide, so that the two probes do not compete with each other; the parts are preferably close to each other (no more than about 300 nucleotides apart), it is more preferred that the parts are right next to each other (no more than 10 nucleotides apart). The probes cannot hybridize with each other. In the reaction mixture, the two probes and single-stranded target polynucleotide hybridize together to form complex double-stranded hybrid molecules. To ensure fast and efficient hybridization, none of the probes is attached to a solid support. The second probe, the identification probe, is further characterized by having an easily detectable label. The method is termed sandwich hybridization because the target polynucleotide is "sandwiched" between the two probes in the resulting hybrid molecule.

The probes may be in separate reagents. Because the probes do not hybridize with each other, it is also possible to have all the probes in a single reagent.

The order of combining the probes with the test sample is generally not important. The probes can be added to the test sample in seriatum (one after the other), all simultaneously, or in any other combination.

The reaction mixture is maintained at conditions that result in hybridization. The conditions depend on, and are generally known for, the particular polynucleotide constituents involved. The hybridization is essentially carried out to completion. For most polynucleotides this takes more than about an hour. For example, at a salt concentration of about 0.15 molar, a temperature of 65°C and a plasmid probe concentration of 1.0 µg/ml, the time required to reach 1/2 completion is less than 20 minutes.

In the harvesting step, the reaction mixture is contacted with a solid support that has the ability to bind the first probe (binding probe), but not the second probe (identification probe), and not the target polynucleotide. The terms "binding" and "binding" herein refer to strong binding via specific functional groups, as opposed to low levels of non-specific binding. The hybrid molecules that are formed by binding the target polynucleotide to the two probes are thus bound to the solid support via the binding probe.

The second probe is chosen so that there is little non-specific binding of the second probe itself to the solid support. Presence of target polynucleotide in the test sample is thus confirmed by determining whether any of the label is bound to the solid support. Quantitative determination of the amount of the target polynucleotide in the test sample is also possible by measuring the actual amount of label bound to the solid support.

There are at least two main methods for determining whether any of the label is bound to the solid support, both measuring the distribution of the second probe in the solid phases of the liquid phase of the reaction mixture after the harvesting step. In the first method, the excess identification probe that is not incorporated into the hybrid molecules is removed by conventional methods such as washing, evaporation, decantation, etc. The amount of - the label bound to the solid support - is then measured. Presence of the label on the solid support indicates the presence of target polynucleotide in the sample. In the second method, the solid support may be separated from the liquid phase of the reaction mixture, or it may remain mixed with the liquid phase. The amount of unbound second probe in solution in the liquid phase is measured and is compared to the total amount of other probes added to the reaction mixture. A difference in the two amounts indicates that some of the second probe is bound to the solid support, which in turn indicates the presence of target polynucleotide in the sample.

Preferably, the first probe (binding probe) includes a first binding group and the solid support includes a second binding group, where the two binding groups bind quickly to each other by forming strong bonds. The two bonding groups together form a bonding pair. One of the binding groups of the binding pair is attached to the base material of the solid support, and the other binding group is part of the first probe. The blind pair can e.g. be any of the following pairs: avidin-biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ion-metal ion binding substances, enzyme substrate, boronate cis-diols, staph A proteins-antibodies, enzyme inhibitors, etc. The binding pairs also include pairs of the derivatives to the above-mentioned components. The criteria for selecting particular binding pairs include (1) the rate at which the binding groups of the pair react by binding to each other, (2) the strength of the bond between the binding groups of the pair, (3) minimal interference of the binding group on the first solution sandwich probe the hybridization step, (4) the ease with which the binding groups can be attached to the matrix material of the solid support and be used to form the first probe.

The matrix material of the solid support can be a material selected from e.g. agarose, cellulose, glass, latex, polyacrylamide, polycarbonate, polyamide (e.g. nylon)/polyethylene, polypropylene, polystyrene, silicon dioxide, silicon and derivatives thereof. This list is by no means complete. Any solid substance on which one of the binding groups of the binding pair can be attached is suitable.

The solid carrier can be in different forms. For example, it can be in the form of microparticles with particle sizes less than 100 mp (eg microcrystalline cellulose), macroparticles with particle sizes of 0.1 to 2.0 mm, sheets, tubes, pipette tips, plates, filters and spheres etc.

Methods for attaching the second binding group to the solid support are well known. Methods for changing the polynucleotide portion of the first probe to contain the first binding group are also well known. (Tchen et al., P.N.A.S. 81:3466 (1084), Forster et al., Nucl. Acids Res. 13:745 (1985), VIscidi et al., J. Clin. Microbiol. 23:311 (1986). Biotechnology, Aug. 1983:471-478, J. Mol. Biol. 98:503-517 (1975); Falkow et al., US-PS 4,385,535; Leary et al., Proe. Nat. Acad. Sci. 80 :4045-4049 (1983); Langer-Safer et al., Proe. Nat. Acad. Sci. 79:4381-4385 (1982); and Langer et al., Proe. Nat. Acad. Sci. 79:6633 -6637 (1981), Rigby et al., J. Mol. Biol. 113:237 1977), Bourguignon et al"., J. Virol. 20:290 (1976).

The second probe (the identification probe) contains an easily detectable label. Various methods for labeling specific polynucleotide probes are known. Any label that has the ability to be easily detected and does not interfere with the solution hybridization step can be used. Suitable labels include radioisotopes, light labels, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins', metal chelators, etc. and their derivatives. Detection can be direct, as with radioisotopes, or indirect, as with a hapten followed by an enzyme-labeled antibody.

Radioisotopes, such as<32>p,<125>I, etc., may be used to label the probe. The radioactive labels can be detected using well-known methods, such as gamma counting or scintillation counting. But using radioisotope labels can be expensive and dangerous. Detection of radioactivity usually requires expensive equipment. Specially trained personnel and safety measures are necessary for handling radioactive material. Radioactive isotopes have limited half-lives; and thus the labeled polynucleotide probe usually has a relatively short shelf life (usually a few weeks).

For the reasons mentioned above, other labeling systems were developed, such as light labels and enzyme-based labels. As discussed in Heller, et al., EP-PS 82303701.5, luminescent labels may include chemiluminescent, bioluminescent, fluorescent, or phosphorescent, and under appropriate conditions may provide sensitivities comparable to that of the radioisotopes. The fluorescent label can be attached to any point on the single-stranded polynucleotide segments of the probe; but terminal positions are known to be more desirable.

Some examples of light marks are as follows; (1) chemiluminescent: peroxidase and functionalized iron porphyrin derivatives, (2) bioluminescent: bacterial luciferase, firefly luciferase, flavin mononucleotide (FMN), adenosine triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and various long-chain aldehydes (decylaldehyde, etc. );

(3) fluorescent: fluorescent nucleotides such as adenosine nucleotides, ethenocytidine nucleotides, etc., or functionalized nucleotides (aminohexaneadenosine nucleotides, mur-curinucleotides, etc.) which can first be covalently labeled, and then covalently attached to the single-stranded polynucleotide segment to the probe; (4) phosphorus 2-diketones such as 2,3 butadione, 1-[carboxyphenyl]-1,2-pentadione and 1-phenyl-1,2-propanedione. Procedures for attaching the light label to the probe are well documented in the art. The label is measured by exciting the label and then measuring the light response with photodetectors. Fluorescent or phosphorescent labels can be excited by irradiation with light of the appropriate wavelength. Chemiluminescent or bioluminescent tags can be chemically excited using methods well known in the art.

The identification probe can also be labeled with an enzyme. The hybridization product is detected by the action of the enzyme on a substrate for the enzyme. For example, an enzyme that has the ability to act on a chromogenic substrate may be selected. The conversion ratio of the substrate can be calculated by optical analysis. The ratio is then correlated with the presence or absence of the target polynucleotide. It is well known that avidin and biotin can be used to bind an enzyme to a specific nucleic acid probe. Examples of suitable enzymes include e.g. beta-galactosidase, alkaline phosphatase, horseradish peroxidase, and luciferase.

One of the main advantages of the light-labeled and enzyme-based markers is that their qualitative detection can be visualized without the need for expensive detection equipment. Quantitative determinations of such marks can also be made using photometric equipment.

It is preferred that the first and second probes are each complementary to substantially mutually exclusive parts of the target polynucleotide. In other words, the first and second probes should not compete for the same base sequence to the extent that sandwich hybridization is prevented.

The probes can be obtained from appropriately restriction endonuclease-treated polynucleotides from the organism of interest, or from double-stranded polynucleotides by enzymatic methods such as Exo III cleavage or RNA polymerase transcription. In these cases the probes are RNA or DNA fragments. In other cases where the base sequence of a unique moiety is known, the probes can be synthesized by organic synthetic techniques (Stawinski, J. et al., Nuc. Acids Res. 4, 353, 1977; Gough, G. R. et al., Nuc. Acids Res. 6, 1557, 1979; Gough, G. R. et al., Nuc. Acids Res. 7, 1955, 1979; Narang, S. A., Methods in Enzymology, Vol 65, Part I, 610-620, 1980). It is also possible to prepare oligodeoxyribonucleotides of defined sequence using polynucleotide phosphorlase (E. Coll) under appropriate conditions (Gillam, S., and Smith, M., Methods in Enzymology, Vol. 65, Part I, p .687-701, 1980). It is also possible to produce large quantities of the probe by cloning; e.g. by recombining the determined sequence into a plasmid or M13 bacteriophage vector, and then cloning the recombinant component using standard procedures. The cloning method is preferred.

The size of the probes can be from 10 nucleotides to 100,000 nucleotides in length. Below 10 nucleotides, the hybridization system is not stable and will begin to denature above 20°C. A complementary polynucleotide sequence of 12 is approximately the minimum length required for appropriate binding specificity. The generally used minimum value is about 15. Above 100,000 nucleotides, hybridization (renaturation) becomes a much slower and incomplete process, see Molecular Genetics, Stent, G. S. and R. Calender, pp. 213-219, 1971. It is not necessary that the entire probe is complementary to the target polynucleotide. on a base'by base scale. Preferably, the probes should be from about 15 to about 50,000 nucleotides in length, preferably from about 15 to about 10,000 nucleotides in length. The number of complementary (base pairing) nucleotides on the probe is preferably between about 200 to about 5,000. The complementary nucleotides are preferably next to each other, especially when the number of complementary nucleotides is low (below 200). However, one-to-one complementation of all base-pairing nucleotides on target and probe is not required. Smaller probes (15-100) can be prepared by automated organic synthetic techniques. Probes of size from 100-10,000 nucleotides can be provided from appropriate enzymatic methods, or by recombinant DNA methods.

Labeling of smaller polynucleotide segments with relatively bulky labeling portions (e.g. chemiluminescence labels) can in some cases interfere with the hybridization process. It is therefore important to choose important brands. Some of the criteria for selecting labels are: ease of incorporation of the label into the polynucleotide without inhibition of hybridization, ease and sensitivity of detection of the label, low non-specific binding of the labeled probe to the solid support, and high stability.

Correct hybridization conditions in the solution-hybridization step are determined by the nature of the first binding group on the first probe (the binding probe), and by the label attached to the second probe (the identification probe), the size of the two probes, [G] + [ C] (guanine plus cytosine) the content of the probes and the complementary nucleotide sequences on the target nucleotide and how the test sample is prepared. The label can affect the temperature and salt concentration used to carry out the hybridization reaction. For example, chemiluminescence catalysts can become sensitive to temperatures and salt concentrations that the absorbing/emitting components can tolerate. The size of the probes affects the temperature and time of the hybridization reaction. Assuming similar salt and reagent concentrations, hybridizations involving reagent polynucleotide sequences in the range of 10,000 to 100,000 nucleotides require from 40 to 80 minutes to occur at 67°C, while hybridizations involving 14 to 100 nucleotides require from 5 to 30 minutes at 25° C. Sequences with a high [G] + [C] content will hybridize at higher temperatures than polynucleotide sequences with a low [G] + [C] content.The conditions used to prepare the test sample and to maintain the target polynucleotide 1 single-stranded shape can affect the temperature, time and salt concentration used in the hybridization reaction. The conditions for preparation are that the test sample is affected by the polynucleotide length required and the [G] + [C] content. In general, the longer the sequence or the higher the [G] + [C] content, the higher temperature and/or lower salt concentration required for denaturation The concentration of probe or target polynucleotide in the mixture gene also determines the time required for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time required. The degree of hybridization was also affected by the type of salt present in the incubation mixture, the concentration of the salt, and the incubation temperature. Sodium chloride, sodium phosphate and sodium citrate are the salts that are usually used for hybridization and the salt concentration that is used can be as high as 1.5 - 2M. The salts mentioned above give comparable rates of polynucleotide hybridization when used at the same concentrations and temperatures as do the comparable calcium, lithium, rubidium and cesium salts. Britten et al. (1974) (Methods in Enzymology, Volume XXIX, Part E., ed. Grossman and Moldave; Academic Press, New York, page 364) and Wetmur and Davidson

(1968) (J. Molecular Biology, Vol. 31, page 349) present data illustrating the usual hybridization rates obtained with the salts commonly used. The hybridization rates of DNA with RNA differ somewhat from the hybridization rates of DNA hybridizing with DNA. The size of the variation is rarely more than tenfold, and varies depending, for example, on whether an excess of DNA or RNA is used. See Galau et al. (1977) (Proe. Nati. Acad. Sei. USA, Vol 74, #6, p. 2306).

There are preferred general conditions that are not optimal, but where hybridization occurs for almost all probe and target combinations of 100 nucleotides or more. These conditions are approximately 0.075 molar sodium chloride, 0.025 molar sodium phosphate (pH 6.5), 65°C, and polynucleotide concentrations of 0.001 to 1.0 pg/ml. There are also known methods for achieving similar conditions at low temperatures, e.g. by adding a denaturing agent such as formamide in the reaction (Casey et al., Nucl. Acids Res. 4: 1539

(1977)). Addition of other reagents in the hybridization reaction can also increase the reaction rate, for example dextran sulfate (wahl et al., P.N.A.S. 76:3683 (1979), polyethylene glycol (Amasino, Anal, Biochem. 152:304 (1986)) and phenol (Kohne et al. , Biochemistry 16:5329 (1977)).

A kit suitable for detecting a single-stranded target polynucleotide in a test sample by the analysis method according to the present invention can include a liquid polynucleotide reagent or reagents including at least two different probes, being a first probe, a binding probe, a second probe and an identification probe. Each probe includes a single-stranded polynucleotide containing "a nucleotide sequence complementary to a portion of the target polynucleotide. Neither probe is attached to a solid support. The probes cannot hybridize with each other. The other probe has a detectable label. The kit also includes a solid carrier which has the ability to bind the first probe, but not the second probe and not the target polynucleotide. The other characteristics of the probes and of the solid carrier are generally as described above in the description of the method according to the present invention. The kit may also further include ways for qualitative and/or quantitative determination of the label. There may be more than one probe in each polynucleotide reagent. As the probes do not hybridize with each other, they may advantageously be combined in a single reagent.

It is also preferred that one or more of the following components are included as parts of the kit: (a) the detergent that can dissolve the various components such as sodium dodecyl sulfate, or sodium lauryl sarcosinate; (b) protease, such as proteinase K and pronase; (c) reagents to facilitate denaturation of the target polynucleotide, including salts and solvents; (d) reagents used to detect the label on the second probe. The components of (a), (b), (c) and the probe reagent may also be combined into a single reagent or a number of reagents, as required.

The method mentioned above and the kit can be adapted for use in testing a series of target nucleotides. A separate set of probes is provided for each target polynucleotide, each set being specific for the particular target polynucleotide. Each set of probes includes at least two different probes, carrying the binding and identification probes (first and second probes), which are characterized above. The solid support used has a binding group that has the ability to bind the binding groups on each of the binding probes that are specific for the majority of target polynucleotides, but none of the identification probes, and none of the target polynucleotides. Therefore, a single universal solid support can be used with all the different binding probes. In the kit, each set of probes is preferably held in a separate liquid reagent.

The method and kit described above can also be adapted for testing multiple target polynucleotides in a test sample. A separate set of probes is provided for each target polynucleotide, each set being specific for the corresponding polynucleotide. None of the probes have the ability to hybridize with each other. Each set of probes consists of at least two different probes, which are a first probe (a binding probe) and a second probe (an identification probe), where the probes are as characterized previously. All the first probes carry an identical binding group, so that all the first probes can be harvested on the same solid support. The solid support binds none of the identification probes and none of the target polynucleotides. The other probes contain different tags, each tag corresponding to a specific target polynucleotide. After the harvesting step, detection of the respective labels on the solid support provides information on the presence and/or quantities of the corresponding target polynucleotide in the sample. In the kit, all the probes are preferably stored in a single liquid reagent.

The advantages of the analysis method and the kit according to the present invention are many. The method provides reaction speed and reaction efficiency corresponding to solution hybridization, and ease of separation of the hybridization products from the test sample corresponding to solid support hybridization. The assay procedure is very specific, as it needs the complementation of at least two probes with the target polynucleotide. The solution sandwich hybridization step can generally be completed in less than an hour, and the harvesting step takes no longer than about 5 minutes (optimally less than about 0.5 min). This provides significant time savings.

The kit according to the present invention is easy to use. The user only needs to keep a single, solid carrier reagent, which can be used for analyzing all the target polynucleotides. Only one liquid reagent is required for each target polynucleotide. A third reagent for detecting the label may in some cases be necessary.

Another considerable advantage of the method according to the present invention is that no extensive preparation of the test sample is necessary. Prior resolution hybridization methods detect target nucleic acids that have been purified from other cellular components. The nucleic acids in the cells or viruses are usually in tight complexes with other cell components, usually protein, and in this form they are not available for hybridization. Simply destroying the cell or virus to release its contents does not necessarily mean that the polynucleotides are available for hybridization. The polynucleotides remain in complex with other cell or virus components, even if they are released from the cell, and may even be extensively degraded by nucleases which may also be released. A probe added to such a mixture may additionally form a complex with "sticky" cell or viral components and thus become unavailable for hybridization, or the probe may be degraded by the action of nuclease. A variety of methods exist for the purification of polynucleotides. These methods are all time-consuming - one that takes an hour is considered very fast - and require multiple manipulations. It was unexpectedly discovered that the analytical techniques according to the invention can be used with impure samples, thereby avoiding the necessity of elaborate and time-consuming purification steps. The analysis can easily be automated, which in turn will simplify the consumer's work. The analysis results can be either qualitative or quantitative.

Another aspect according to the method and test kit of the present invention is aimed at genetic disease diagnosis and cancer diagnosis.

Genetic disease is the result of mutations in a polynucleotide's nucleotide sequence. Such mutation is also a factor in the development of cancer. As a consequence or such genetic disease or cancer, a sample taken from the patient, which contains a normal polynucleotide (standard polynucleotide), may also contain an abnormal polynucleotide (target polynucleotide) which is a variant of the normal polynucleotide. The sample may also contain only the abnormal polynucleotide. The normal and abnormal polynucleotides usually have substantially identical nucleotide sequences, with the exception of the mutation sites on the anti-polynucleotides. The normal and abnormal polynucleotides thus react differently to certain reagents.

The method according to the present invention can be used to detect such differences between the normal and abnormal polynucleotides. The method combines a separation step with a solution sandwich hybridization method. The separation step includes a segmentation step, and may also include a denaturation step if the target polynucleotide is double-stranded. The order of these two steps is not important.

Restriction reagents, e.g. restriction enzymes, are known. A restriction enzyme can, under suitable conditions, be used in a segmentation step to cleave a polynucleotide at very specific sites on the nucleotide backbone. The standard and target polynucleotides are almost identical. The standard polynucleotide has part A and part B. The target polynucleotide has part A' and part B'. The two polynucleotides differ in at least one nucleotide which is located between the polynucleotides' respective parts of A and B, and A' and B'. The restriction reagent is chosen to cleave the standard polynucleotide into segments that do not contain both parts A and B; the restriction reagents cleave the target polynucleotide into segments where at least one contains both parts A' and B'. Typically, where the target polynucleotide is a genetic variant of the standard polynucleotide, the portions A and A', and portions B and B', are identical.

Target and the standard nucleotides present, or their segments (from the segmentation step), are denatured, ie made single-stranded, if necessary, either before or after the segmentation step. The treated sample thus contains single-stranded versions of target and/or standard polynucleotides present, ready for hybridization. The treated sample is then combined with at least two probes to form a reaction formation. The two probes are a first probe and a second probe. Each probe includes a single-stranded polynucleotide. The probes do not hybridize with each other. The first probe base pairs complementary with part A on the standard polynucleotide, and with part A' on the target polynucleotide. The second probe base pairs complementary with part B of the standard polynucleotide, and with part B' of the target polynucleotide. The two probes together form hybrid molecules by base pairing with single-stranded segment(s) of the target polynucleotide, containing both parts A' and B'. With respect to the standard polynucleotide, because part A and part B are on separate segments after sequencing, no hybrid molecules incorporating both the first and second probes will be formed. The probes are attached to separate segments. Hybrid molecules having both the first and second probes will only be formed if abnormal (target) polynucleotides are present in the sample.

Presence in the reaction mixture of hybrid molecules having both probes incorporated would indicate the presence of target polynucleotide in the sample. Determination of hybrid molecules formed by this sandwich hybridization procedure can be performed using previously known methods. For example, a system similar to that described in Ranki, US Patent 4,486,539, in which one of the probes is already immobilized on a fixed carrier, be used. Not all probes need to be in solution. Other methods are also known. Alternatively, solution-sandwich hybridization, followed by a harvesting step as previously discussed, can also be used. In the latter case, further limitations of the method of this invention described above are that: neither probe is attached to a solid support, and the other probe has a detectable label. The reaction mixture is contacted with a solid support that binds the first probe but not the second probe, and neither segment contains only part B or only part B'. A subsequent determination is made to see if any of the label is bound to the solid support as previously discussed.

The following procedure is, for example, suitable for sickle cell analyses. A test sample believed to contain the mutated gene-

The DNA strands (e.g. in the sickle alleles) are treated with a restriction enzyme, e.g. Mst II. Such enzymes have been used in gene mapping, to detect structural gene deletions in the DNA strands. Direct identification of mutant genes in DNA, e.g. hemiglobinopathies sonu are caused by point mutations in the DNA, was also possible by virtue of the specificity of such restriction enzymes. A simple nucleotide change in the enzyme's cleavage site can be quickly detected if the appropriate enzyme is used. The restriction enzyme Mst II is known to cleave DNA on average to form fragments of 1.1 and 1.3 kb from the p™ and 3^ genes, respectively. Mst II cleaves a sequence (CCTNAGG) that is a fragment of the Ddel sites (CTNAG). The DNA from the sickle cell gene has a nucleotide variation in the Mst II site. The mutated DNA strand will not be cleaved with the site by the Mst II enzyme. The restriction enzyme-treated test sample was then subjected to conditions which cause the polynucleotides to become single-stranded. A single target polynucleotide single probe hybridization assay procedure recently described for detecting the presence of a target polynucleotide was then performed, with the additional restriction that the first and second probes bind sites on the DNA strand opposite the Mst II site involved in the mutation. The only way in which a complex hybrid molecule, including the target polynucleotide and both probes, will be formed is if the gene is mutated - e.g. a sickle cell. The normal gene is cleaved between the binding sites of the two probes. The sickle cell analysis is schematically represented in fig. 2. This method eliminates previous analytical steps involving gel electrophoresis and transfer to solid phase (filter paper). The simplification is absolutely essential. This analysis method can equally be used for the diagnosis of other genetic diseases in which gene mutations are included.

The cancer diagnostic aspect would take the form of a quantitative analysis for levels of messenger RNA from an oncogene, although analyzes similar to the sickle cell example would also be useful.

EXAMPLES

Example 1

This example correlates the harvested signal to the target D NA concentration.

In this example, the target polynucleotide is mpB1017; probe 1 is pHBC6 which has biotin as the first binding group; probe 2 is M13 10W marked with<32>p; the solid support is avidin-cellulose, which binds biotin on the first probe.

ABBREVIATIONS. SSC is 0.15 molar sodium chloride and 0.015 molar sodium citrate. SSC is used at different concentrations, "10X SSC" will e.g. be, 1.5 molar sodium chloride, and 0.15 molar sodium citrate. EDTA is ethylenediamine tetraacetate, SDS is sodium dodecyl sulfate, DNA is deoxyribopolynucleotide, BSA is bovine serum albumin, tris:HCL is tris-hydroxymethylaminemethane adjusted to appropriate pH with hydrochloric acid.

MATERIALS AND METHODS. Plasmid DNAs were obtained by growing transformed E. coli strain LE392, followed by lysis of the cells with lysozyme, SDS and NaOH. DNA was further purified by centrifugation in CsCl in the presence of ethidium bromide, see Mauiatis, T., Fritsch, E.F., and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, Replicative form (RF) DNA from M13 bacteriophage, strain 10w was obtained in a similar manner by culturing infected E. coli strain 71.18. Single-stranded virus - DNA from M13 clone mpB1017 was obtained from virus in the growth medium of infected E. coli 71.18 by precipitation with polyethylene glycol, centrifugation in CsCl and extraction of the purified virus with SDS, phenol and chloroform. Salmon sperm DNA was obtained from Sigma Chemical Company, St. Louis, MO (USA).

Double-stranded DNAs were labeled by nick translation (Rigby, et al., J. Mol. Biol., 113:237, 1977) essentially as described by Leary et al., Proe. Nat'l. Acad. Pollock. [USA] 80:4045, 1983. Plasmid pHBC6 contains 5,400 base pairs of the human β-globin gene, cloned in plasmid pBR322 (Fukumaki et al., Cell 28:585, 1982). M13 clone mpB1017 contains 1310 bases of the human globin gene, homologous to 1310 bases from plasmid pHBC6. M13 strain 10w contains 7,500 bases of bacteriophage genes, homologous to the same number of bases in clone mpB1017. The regions of mpB1017 DNA homologous to pHBC6 and M13 10w are non-overlapping.

The solid phase for harvesting solution sandwich product was prepared by activation of microcrystalline cellulose with N,N'-carbonyldiimidazole, followed by coupling to proteinavidin, using methods similar to those described by Paul et al., J. Org. Chem. 27:2094, 1962. The coupling ratio was 1 gram of activated cellulose to 1 mg of avidin. A 1:1 mass of the prepared solid phase of avidin:cellulose contains approximately 1 mg of cellulose in 6.0 ml of buffer.

Solution hybridizations were performed in a final solution containing 5X SSC, 0.02% (w/v) each of polyvinyl-pyrrolidone-40, ficoll-400, and BSA, as well as 25 mM NaPO4 buffer, pH 6.5, and 250 µg /ml sonicated salmon sperm DNA. All DNAs (probe 1, probe 2, target, and carrier salmon sperm) were denatured at 100°C for 3 to 5 minutes before starting the hybridization reaction.

Other details from the sample analysis are provided below.

APPROACH. The following assay was performed to examine the feasibility of the assay of the present invention, and to determine the range of target DNA concentration in which the assay is effective. Plasmid pHBC6 was labeled with the ligand biotin by nick translation with biotin-11-deoxyuridine triphosphate (Bethesda Research Laboratories, Gaithersburg, MD, USA). This constitutes probe 1 in the analysis. Replicative form DNA from bacteriophage M13 10w was labeled with a radioactive indicator, <32>p, by nickel translation with cx-32P-deoxytidine triphosphate (Amersham, Chicago, IL, USA). This constitutes probe 2 in the analysis. Probe one contained approximately b% biotin nucleotides, and probe 2 had specific radioactivities of 0.7 to 7 X 10<7>cpm/pg. The target DNA was M13 mpB1017, and samples containing varying amounts of target DNA from 20 picograms to 1.0 micrograms were analyzed. All assays contained 100 ng probe 1 (biotin) and 50 ng probe 2 (<32>P). The probe and sample DNAs were mixed together in a solution consisting of 10 mM tris:CCl, pH 7.5, 1.0 mM EDTA, heat denatured and adjusted to the solution hybridization conditions given in the above procedure and incubated in a total volume of 0 .2 ml overnight at 68°C. This incubation time is unusually long, and was only used because it was expedient and to ensure that the reaction was completed. Control assays contained probe 1 or probe 2 and 20 ng target, or probe 1 & 2 without target. Separation of product sandwich from unhybridized probes was accomplished by adding 0.2 ml of 1:1 mass of avidin:cellulose to the reactions, incubating at 22°C for 30 min, and filtering the cellulose on a 13 mm glass fiber filter. A wash of the reaction vessel with 0.1 ml tris:HCl, pH 7.5, 1 mM EDTA, was also filtered on the same filter as the primary reaction. The filtered solid phase was further washed 2x with 2X SSC and 0.1% SDS, and 2x with 0.2X SSC and 0.1% SDS, and 2x with 0.1X SSC and 0.1% SDS. The last two washes were carried out at 50° C, all others at room temperature. All washes were with 0.5 ml solution. These washes were adapted from those used for Southern blot hybridization analysis by Leary et al., P.N.A.S. 80:4045 (1983)). The amount of probe 2 (<32>P) bound to the solid phase was determined by liquid scintillation counting, with the resin in 0.5 ml of solution in a mini container and 4.5 ml of Beckman Ready SoIv^mMP scintillation fluid. All the washing waters were also collected and counted in a similar way.

RESULTS. Results from a run are given in table I where the amount of probe 2 bound is directly related to the amount of measuring DNa in the analysis. The data represent the mean of duplicate samples.

Data from multiple runs demonstrated that the assay, as performed in this example, has a peak 1 harvested signal (probe 2) at approximately 30 ng of target DNA. At amounts above this level, the harvested signal is reduced in a logarithmic manner. Below 30 ng of target DNA, the assay is limited by b.ak binding of probe 2. to the solid phase and the specific radioactivity of the probe, but increases exponentially from about 0.5 ng to 10" ng of target DNA.' The non-linear nature of the signal as a function of the target quantity is graphically plotted in Fig. 3. Fig. 4 is an expanded version of Fig. 3 around the peak signal. The non-linear relationship may be a function of the nick translation reaction used to label the probes, since probes labeled in this way are known to show elements of cooperative binding (Meinkoth and Wahl, Anal. Biochem. 138:267, 1984).

EXAMPLE 2

This example demonstrates the binding characteristics of an avidin cellulose solid support and a biotin-labeled probe.

The kinetics and specificity of harvesting a biotin-labeled probe by binding it out of an avidin-cellulose hybridization buffer was investigated using DNAs labeled with both biotin-II-dUTP and <3>H-dATP or only <3>H -dATP (control). The DNA was plasmid pHBC6 tagged with nick translation (Rigby et al., J. Md. Biol., 113:237 (1977)).

Avidin cellulose (0.05 ml packed volume) suspended in 0.2 ml lOmM Tris:HCl plus 1.0 mM EDTA (pH 7.5) was added to each of 30 microcentrifuge tubes, a mixture (0.2 ml) containing 100 nanograms of biotin-labeled or control DNA, 50 micrograms carrier salmon sperm DNA, 20 micrograms yeast RNA, 0.02$ (w/v) each of bovine serum albumin, ficoll, and polyvinyl cryolidone in 0.075 M sodium citrate, 0.75 M sodium chloride, and 0.025 M sodium phosphate (pH 6.5) was added to appropriate tubes. The tubes containing the solid phase and hybridization mixture were incubated at room temperature on a shaking platform for times ranging from 1 min. to 300 min. After the incubation, the solid phase was collected by filtration on a Teflon membrane filter. Fraction of DNA bound was determined by subtracting the radioactivity in the filtrate and washing from the total radioactivity. which is set for each sample. The result is presented in fig. 5. Within 30 min. approximately 92% of the biotin-labeled DNA was bound to avidin cellulose, whereas an average of less than 2% of the control DNA was bound to the solid support. Binding is fast, efficient and specific for the biotin label on the probe.

The requirement of the avidin component of the solid phase for binding to biotin-labeled DNA was investigated using the same

DNA is as above, with different pretreatments of the solid support. Free biotin should compete with biotin-DNA for the avidin sites on the solid phase and pretreatment of the solid phase with 20 times the half capacity of free biotin reduced biotin-DNA binding by 50$. A further pretreatment with 10 times more free biotin reduced biotin-DNA binding by 30% more. Pretreatment of the solid phase with pronase, 1%

(w/v) sodium dodecyl sulfate and 0.1% (v/v) ε-mercaptoethanol also reduced biotin-DNA binding by 50%, and further treatment of this cleaved resin with free biotin abolished biotin-DNA binding. A slightly different approach also indicated that the major biotin-DNA binding to the solid support was not only biotin-dependent but also avidin-dependent. A control solid support was prepared by treating cellulose in the same manner as that used for coupling to avidin, with the exception that avidin was omitted from the reaction and replaced with TrisrHCl. This control resin bound 5 to b% of control DNA probe and 15 to 16% of biotin-labeled DNA probe under the conditions used above for the kinetic studies. The majority of biotin-labeled probe binding (more than 85%) to the solid support is caused by interaction with the specific ligand avidin.

EXAMPLE 3

This example demonstrates the construction of M13 vectors containing sickle mutation MstII fragments.

To analyze the major genetic form of sickle cell anemia using the assay method of the present invention, very specific M13 constructs are necessary. The DNA fragments from both sides of the Mst1 restriction site identifying the 6th codon error in a sickle cell unit were inserted into M13 phage. The two fragments that are used for this were derived from plasmid pHBC6 (Fukumaki et al., Cell 28; 585 (1982)) and purified by acrylamide gel electrophoresis. Approximately 10 µg of 200 base pair Sau 1 (-Mst II isoschizomes) located immediately downstream of the diagnostic Mst II site was treated with Klenow DNA polymerase in the presence of 250 pM dXTPer to "fill in" the Mst II site. This "blunt" DNA fragment was then inserted into the Hc II site of pUC 18. Analysis of 24 pUC 18-B-200 isolates indicated the presence of the 200 base insert in 2 isolates, pUC 18-B-200-6 and pUC 18-B-200-9. After CsCl preparation, approximately 200 µg pUC 18-B-200-9 was digested with Eco RI and Hd III before purification by gel electrophoresis. This Eco RI, Hd III-ended 200 bp fragment was then inserted into an M13 mp 18 to provide single-stranded DNA. Several constructs were made using this 200 base fragment to allow easy production of large quantities of specific RNA sequences. This fragment was inserted into transcription vector pT7-1 and pT7-2 (United States Biochemical Corporation, P.O. Box 22406, Cleveland, Ohio, 44122). These constructs allow in vitro synthesis of labeled RNA for use as a probe in sickle cell analysis according to the present invention. The other recombinant vectors required for the sickle cell trait assay utilize the 800 base pair Mst II Hpa 1 fragment located upstream of the diagnostic Mst II restriction site. This fragment was similarly cloned again using Klenow DNA polymerase to "round off" the 5' overhangs present at the Mst II end. This 800 base pair fragment was inserted into four vectors to provide single-stranded RNA and DNA. These are:

Although the present invention has been described in detail with regard to certain versions thereof, other versions are also possible. The scope of the attached requirements should not necessarily be limiting to the description of the versions included herein.

Claims (80)

1. Analysis method for detecting a single-stranded target polynucleotide in a liquid sample, characterized in that the method includes the following steps: a) combining the sample with at least two different probes, carrying a first probe and a second probe, for the formation of a reaction mixture, where each probe includes a single-stranded polynucleotide containing a nucleotide sequence complementary to part of the target polynucleotide, where the probes together form hybrid molecules by complementary base pairing with the target polynucleotide, neither probe is attached to a solid support, and the probes do not hybridize with each other, and the other probe has a detectable mark; b) then contacting the reaction mixture with a solid support which binds the first probe but not the second probe and not the target polynucleotide; and c) then determining whether any of the mark is attached to the fixed carrier. 2. Method according to claim 1, characterized in that the first probe is an RNA or DNA fragment. 3. Method according to claim 1, characterized in that the second probe is an RNA or DNA fragment. 4. Method according to claim 1, characterized in that the solid carrier is formed from a substance selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silicon oxide gel, silicon oxide and derivatives thereof. 5. Method according to claim 1, characterized in that the solid carrier is in a form selected from the group consisting of microparticulates, macroparticulates, sheets, spheres, tubes, pipette tips, plates and filters. 6. Method according to claim 1, characterized in that the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators and derivatives thereof. 7. Method according to claim 1, characterized in that the first probe has a first binding group and the solid support has a second binding group, where the two binding groups have the ability to bind to each other, and the two binding groups form a binding pair. 8. Method according to claim 7, characterized in that the binding pair is selected from the group consisting of avidin biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ion-metal ion binding substances, enzyme substrates, boronates-cis-diols, staff A protein antibodies, enzyme inhibitors and derivatives thereof. 9. Method according to claim 8, characterized in that the binding pair is biotinavidin, where the solid carrier is avidin cellulose. 10. Method According to claim 1, characterized in that each probe has a length of between 15 to about 50,000 nucleotides, the first and second probes are each complementary to different parts of the target polynucleotide, and the shortest distance between the parts is not more than about 300 nucleotides. 11. Method according to claim 10, characterized in that the parts which are complementary to the probes are lying right next to each other. 12. Method according to claim 10, characterized in that the first probe is an RNA or DNA fragment, and the second probe is an RNA or DNA fragment; the solid support is made from a substance selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica and derivatives thereof; the solid support is a form selected from the group consisting of microparticulates, macroparticulates, sheets, spheres, tubes, pipette tips, plates and filters; the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators and derivatives thereof; the first probe has a first binding group and the solid support has a second binding group, the two binding groups having the ability to bind to each other; the two binding groups form a binding pair, the binding pair is selected from the group consisting of avidin biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ion metal binding substances, enzyme substrates, boronates-cis-diols, staph A protein antibodies, enzyme inhibitors and derivatives thereof. 13. Method according to claim 1, characterized in that the detection of the target polynucleotide is qualitative. 14 . Method according to claim 1, characterized in that the presence of the label bound to the solid support is determined quantitatively, so that the detection of the target polynucleotide is quantitative. 15. Method according to claim 1, characterized in that the probes are combined with the sample in series (seriatum). 16. Method according to claim 1, characterized in that the sample is combined with all the probes at the same time. 17. Method according to claim 1, characterized in that the single-stranded target polynucleotide is formed by denaturing a double-stranded polynucleotide. 18. Method according to claim 1, characterized in that the steps for determining whether any label is bound on the solid support further include the following steps: a) separation of the second probe that is not incorporated in the hybrid molecules from the solid support; and b) then detecting the presence of the absence of the label on the solid support, the presence of which indicates the presence of target polynucleotide in the sample. 19. Method According to claim 1, characterized in that the step for determining whether any of the label is bound on the solid support further includes the following steps: a) measuring the amount of unbound second probe in solution; and b) comparing this with the total amount of second probe added to the reaction mixture to determine the presence or absence of target polynucleotide in the sample. 20. Analysis method for detecting the plurality of single-stranded target polynucleotides in a plurality of liquid samples, for each target polynucleotide, characterized in that the method includes the following steps: a) combining at least part of a liquid sample which is assumed to contain target nucleic acid with at least two different probes, being a first probe and a second probe, to form a reaction mixture, each probe consisting of a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the target polynucleotide, the first probe and the second probe together form hybrid molecules by complementary base pairing with the target polynucleotide, none of the probes are attached to a solid support, the probes do not hybridize with each other, and the other probe has a detectable label; b) then contacting the reaction mixture with a solid support which binds the first probe with the second probe and neither of the target polynucleotides; and c) then determining whether any of the marks are attached to the fixed carrier; wherein the solid support binds each of the first probes specifically for the plurality of target polynucleotides. 21. Method according to claim 20, characterized in that each of the first probes has a first binding group and the solid support has a second binding group, where the second binding group has the ability to bind each of the first binding groups on the first probes specific for the plurality of targets nucleotides. 22. Method according to claim 20, characterized in that each probe has a length that is between about 15 to about 50,000 nucleotides, for each probe pair that is specific for target polynucleotide, the first and second probes are complementary to different parts of the respective target polynucleotide, the shortest distance is between the parts of no more than about 300 nucleotides. 23. Method according to claim 22, characterized in that the parts are lying right next to each other. 24. Method according to claim 20, characterized in that at least one of the single-stranded target polynucleotides is formed by denaturing a double-stranded polynucleotide. 25. Method according to claim 20, characterized in that at least some of the probes are combined with the sample at different times. 26. Analysis method for detecting a plurality of single-stranded target polynucleotides in a liquid sample, characterized in that the method includes the following steps: a) combining the sample with a plurality of different probes to form a reaction mixture, while two of the probes are specific for each of the target polynucleotides, being a first probe and a second probe, the first probe and the second probe both consist of a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the respective target polynucleotide, the first probe and the second probe together form hybrid molecules by complementary base pairing with the target polynucleotide, the second probe has a detectable label; wherein none of the plurality of probes is attached to a solid support, the plurality of probes do not hybridize with each other, and each of the other probes specific to the plurality of target polynucleotides has a different label; b) then contacting the reaction mixture with a solid support which binds the first probes but not the second probes and none of the target polynucleotides; and c) then determining whether any of each of the different brands is bound to the fixed carrier. 27. Method according to claim 26, characterized in that each probe has a length of between about 15 to about 50,000 nucleotides, the first probe and second probe that is specific for each target polynucleotide are each complementary to different parts of the respective target polynucleotide, where the shortest distance between the parts are not more than 300 nucleotides apart. 28. Method according to claim 27, characterized in that the parts are lying right next to each other. 29. Method according to claim 26, characterized by. that at least one of the single-stranded target polynucleotides is formed by denaturing a double-stranded polynucleotide. 30. Method according to claim 26, characterized in that at least some of the plurality of probes are combined with the sample at different times. 31. Method according to claim 26, characterized in that all the probes are simultaneously combined with the samples. 32. Kit for detection of a single-stranded target polynucleotide in a sample, characterized in that the kit includes: a) polynucleotide reagent containing at least two different probes, being a first probe and a second probe, each probe including a single-stranded polynucleotide containing a nucleotide sequence complementary to part of the target polynucleotide, the probes together form hybrid molecules with complementary base pairing with the target polynucleotide, neither probe is attached to a solid support, the probes cannot hybridize with each other, the other probe has a detectable label; and b) a solid support that binds the first probe but not the second probe and not the target polynucleotide. 33. Kit according to claim 32, characterized in that the first probe is an RNA or DNA fragment. 34. Kit according to claim 32, characterized in that the second probe is an RNA or DNA fragment. 35. Kit according to claim 32, characterized in that the solid support is formed from a substance selected from the group consisting of agarose, cellulose, glass, latex, nylon, polyacrylamide, polycarbonate, polyethylene, polypropylene, polystyrene, silicon oxide gel, silicon oxide and derivatives thereof. 36. Kit according to claim 32, characterized in that the solid carrier is in a form selected from the group consisting of microparticulates, macroparticulates, sheets, spheres, tubes, pipette tips, plates and filters. 37. Kit according to claim 32, characterized in that the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators and derivatives thereof. 38. Kit according to claim 32, characterized in that the first probe has a first binding group and the solid support has a second binding group, the two binding groups have the ability to bind to each other, and the two binding groups form a binding pair. 39. Kit according to claim 38, characterized in that the binding pairs are selected from the group consisting of avidin biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzyme substrates, boronates-cis-diols, staph A protein antibodies enzyme inhibitors and derivatives thereof. 40. Kit according to claim 39, characterized in that the binding pair is biotinavidin, and where the solid carrier is avidin cellulose. 41. Kit according to claim 32, characterized in that each probe has a length of between 15 and approximately 50,000 nucleotides, the first and second probes are complementary to different parts of the target polynucleotide, and where the shortest distance between the parts is not more than 300 nucleotides. 42 . Kit according to claim 41, characterized in that the parts which are complementary to the probes are right next to "lying". 43. Kit according to claim 32, characterized in that it further includes means for qualitative detection of the mark. 44. Kit according to claim 32, characterized in that it further includes means for quantitative detection of the mark. 45. Kit according to claim 32, characterized in that at least one of the polynucleotide reagents contains more than one probe. 46. Kit according to claim 32, characterized in that all the probes are in a single reagent. 47. Kit for the detection of a plurality of single-stranded target polynucleotides, characterized in that the kit includes, for each target polynucleotide, polynucleotide reagents containing at least two different probes, being a first probe and a second probe, each probe consisting of a single-stranded polynucleotide containing a nucleotide sequence complementary to a portion of the target polynucleotide, the first probe and the second probe together form hybrid molecules by complementary base pairing with target polynucleotides, and wherein neither probe is attached to a solid support, and the probes do not hybridize with each other, and the second probe has a detectable label ; the kit further includes a solid support capable of binding each of the first probes, but none of the other probes, and none of the target polynucleotides. 48. Kit according to claim 47, characterized in that each of the first probes has a first binding group and the solid support has a second binding group, where the second binding group has the ability to bind each of the first binding groups on the first probes. 49. Kit according to claim 47, characterized in that every second probe specific for each target polynucleotide includes a different label. 50. Kit according to claim 49, characterized in that it further includes means for detecting the marks on the other probes. 51. Kit according to claim 48, characterized in that each probe includes a single-stranded polynucleotide having a length of 15 to about 50,000 nucleotides, for each probe pair specific for a target polynucleotide, wherein each of the first and second probes is complementary to a different part of the target polynucleotide , and the shortest distance between the parts is no more than about 300 nucleotides. 52. Kit according to claim 51, characterized in that the probes are complementary to immediately adjacent parts of the target nucleotide. 53. Kit according to claim 47, characterized in that at least one of the polynucleotide reagents contains more than one probe. 54. Kit according to claim 47, characterized in that all the probes which are complementary to each of the target polynucleotides are included in a single reagent, and the number of polynucleotide reagents in the kit is therefore equal to the number of target polynucleotides. 55. Kit for the detection of a plurality of target polynucleotides in a sample, characterized in that the kit includes: a) polynucleotide reagent including, for each of the target polynucleotides, at least two different probes, being a first probe and a second probe, each probe consisting of a single-stranded polynucleotide which contains a nucleotide sequence complementary to a part of the target polynucleotide, the first probe and the second probe together form hybrid molecules by complementary base pairing with the target polynucleotide, neither probe is attached to a solid support, the first probe and the second probe cannot hybridize with each other, the the second probe has a detectable label; and wherein all of the probes for the plurality of target polynucleotides do not have the ability to hybridize with each other, and wherein each of the other probes specific for the plurality of target polynucleotides has a different detectable label; and b) a solid support that binds the first probes, but none of the second probes, and none of the target polynucleotides. 56. Kit according to claim 55, characterized in that it further includes means for detection for different brands on the other probes. 57. The kit of claim 56, wherein each probe comprises a single-stranded polynucleotide having a length of about 15 to about 50,000 nucleotides, for each probe pair specific to each target polynucleotide, the first and second probes are each complementary to a different portion of the target nucleotide, where the short distance between the parts is not more than 300 nucleotides. 58. Method according to claim 57, characterized in that the first and second probes are complementary to immediately adjacent parts of the target nucleotide. 59. Kit according to claim 55, characterized in that at least one of the polynucleotide reagents contains more than one probe. 60. Kit according to claim 55, characterized in that all the probes are included in a single reagent. 61. Analysis method for detecting a target polynucleotide in a sample which may also contain a standard polynucleotide, where the nucleotide sequences of the two polynucleotides are substantially similar to each other, the standard polynucleotide has a part A and a part B, the target polynucleotide has a part A' and a part B', the standard and the target polynucleotides differ in at least one nucleotide that is located between parts A and B of the standard nucleotide and between parts A' and B' of the target polynucleotide, characterized in that the method includes the following steps: a) separation of both the standard and the target polynucleotides that are present into single-stranded segments, such that none of the segments of the standard polynucleotide contains both parts A and B, while at least one of the segments of the target polynucleotide contains both parts of A' and B'; b) combining the treated sample from step (a) with at least two probes, being a first probe and a second probe, to form a reaction mixture, each probe consisting of a single-stranded polynucleotide, the probes not having the ability to hybridize with each other, the first probe base-paired complementary with part A, and with part A', the second probe base-paired complementary with part B, and with part B', and where the two probes together form hybrid molecules by complementary base pairing with the single- the stranded segment of the target polynucleotide containing both parts A' and B'; and c) then determining the presence of hybrid molecules containing both probes. 62. Method according to claim 61, characterized in that the separation step includes treatment with a restriction enzyme. 63. Method according to claim 62, characterized in that the target and standard polynucleotides are double-stranded, and the separation step also includes denaturing the target and standard polynucleotides that are present. 64. Method according to claim 61, characterized in that part A is identical to part A' and part B is identical to part B'. 65. Method according to claim 61, characterized in that the shortest distance between part A and part B on the standard polynucleotide, and the shortest distance between part A and B on the target polynucleotide, is not more than approximately 300 nucleotides. 66. Method according to claim 65, characterized in that the shortest distance between part A and part B of the standard polynucleotide, and the shortest distance between part A' and B' of the target polynucleotide, is not more than approximately .10 nucleotides. 67. Method according to claim 61, characterized in that the target polynucleotide is a genetic variant of the standard polynucleotide, where the target nucleotide is a result of gene mutation that indicates a genetic disease. 68. Method according to claim 67, characterized in that the genetic disease is sickle cell anemia. 69. Method according to claim 61, characterized in that none of the probes is attached to a solid support, the other probe has a detectable label, and the steps for determining the presence of hybrid molecules containing both probes which further include the following steps: a) contacting the reaction mixture with a solid support that binds the first probe but not the second probe, and not segments containing only part B or part B'; and b) then determining whether any of the mark is attached to the fixed carrier. 70. Method according to claim 69, characterized in that the solid support is formed from a substance selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silicon oxide gel, silicon oxide and derivatives thereof. 71. Method according to claim 69, characterize,! in that the solid support is in a form selected from the group "consisting of microparticulates, macroparticulates, sheets, spheres, tubes, pipette tips, plates and filters. 72. Method according to claim 69, characterized in that the label is selected from the group consisting of radioisotopes, chemical luminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators and derivatives thereof .. 73. Method according to claim 69, characterized in that the first probe has a first binding group and the solid support has a second binding group, where the two binding groups have the ability to be bound to each other, and where the two binding groups form a binding pair. 74 . Method according to claim 73, characterized in that the binding pair is selected from the group consisting of avidin biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzyme substrates, boronate-cis-diols, staph A protein antibodies, enzyme inhibitors and derivatives thereof. 75 . Method according to claim 74, characterized in that the binding pair is biotinavidin, and where the solid carrier is avidin cellulose. 76. Method according to claim 69, characterized in that each probe has a length of between about 15 to about 50,000 nucleotides, where the first and second probes are each complementary to different parts of the target polynucleotide, and where the shortest distance between the parts is no more than about 300 nucleotides. 77. Method according to claim 76, characterized in that the parts which are complementary to the probes are lying right next to each other. 78. Method according to claim 76, characterized in that the first probe is an RNA or DNA fragment, and the second probe is an ENA or DNA fragment; the solid support is formed from a substance selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silicon oxide gel, silicon oxide and derivatives thereof; the solid support is in a form selected from the group consisting of microparticulates, macroparticulates, sheets, spheres, tubes, pipette tips, plates and filter; the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators and derivatives thereof; the first probe has a first binding group and the solid support has a second binding group, the two binding groups having the ability to bind es to each other; the two binding groups form a binding pair, the binding pair is selected from the group consisting of avidin biotin, hapten antibodies, antibody antigens, carbohydrate lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzyme substrates, borate-cis-diols, staph A protein antibodies, enzyme inhibitors and derivatives thereof. 79. Method according to claim 69, characterized in that the steps for determining whether any of the label is bound on the solid support further include the following steps: a) separation of the second probe that is not incorporated in the hybrid molecules from the solid support; and b) then detecting the presence or absence of the label on the solid support, the presence of which indicates the presence of the target polynucleotide in the sample. 80. Method according to claim 69, characterized in that the steps for determining whether any of the label is bound on the solid support further include the following steps: a) measuring the amount of unbound second probe in solution; and b) comparing this to the total amount of second probe added to the reaction mixture to determine the presence or absence of the target polynucleotide in the sample.