JPH0710239B2 - Diagnostic Polynucleotide Reagent Complexes, Their Use Including Displacement And Affinity Separation From Reagent Complexes In Solution, And Their Production - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to a diagnostic assay method for detecting the presence of a target nucleotide sequence (DNA or RNA) in a biological sample, and a polynucleotide reagent complex therefor.

A common method for detecting the presence of a particular polynucleotide in a biological sample generally involves immobilizing the sample nucleic acid on a surface as a first step. Once the sample is immobilized, a probe polynucleotide chain (usually labeled with a detectable label, eg, a radioactive phosphorus atom) is incubated with the immobilized sample,
Thereby, when the immobilized sample contains the target nucleotide sequence, it is bound to the immobilized sample by purine / pyrimidine base sequence-specific complementary base pairs. After washing away the labeled probe that has not hybridized, the presence or absence of the label on the support is measured. This measurement technique includes photographic film exposure, liquid scintillation counting, and fluorescence microscopy. See U.S. Pat. No. 4,358,535 (Falkow et al., 1982).

Ward et al. (European Patent Application No. 63,879, 1982) describe a modification of this method. In this method, rather than directly labeling the probe with a detectable label, the probe is labeled with a non-isotopic substituent, such as biotin on a particular nucleotide. In this case, the unhybridized probe is washed away and the support is then contacted with a reagent such as avidin bound to the enzyme.
The avidin-enzyme complex selectively binds biotin due to its high avidin-biotin binding affinity, which causes the enzyme to selectively anchor to the site where the target nucleotide sequence is immobilized on the support. Substrate for the enzyme is then added to detect the enzymatic reaction product, resulting in an amplified signal that is functionally dependent on the initial concentration of the target nucleotide sequence on the support. See also European Patent Application No. 97,373 (Enzo Biochem, January 4, 1984).

The above non-isotopic variants are also described in other European patent applications (Standard Oil, Illinois, European patent application 70,687, 1983). In that case, in one form (see pages 8-10 thereof) two probes specific for the target nucleotide sequence are used. A first probe capable of hybridizing to the first portion of the target nucleotide sequence is attached to a solid support. A second probe, carrying various labels and capable of selectively hybridizing to a distinct second portion of the target nucleotide sequence, is then used to contact the support. Second, if the target nucleotide sequence is present in the biological sample
A combinatorial structure (or Sangerti) will be formed in which the probe is attached to the support via the first probe and the target nucleotide sequence. By separating the support from the unbound solution components at each step, the presence of the label in the support-containing phase after the third separation is a function of the presence and concentration of the target nucleotide sequence in the sample. Will. See also WO 83/01459 [Orion-Yhtma Oy, April 29, 1983].

Another diagnostic method for a specific target nucleotide sequence (sickle cell anemia gene) by detecting double-stranded sample nucleic acid in solution with a restriction enzyme and then detecting fragments of a specific size on paper is US Pat. No. 4,395,486 Book (Wilson et al.).

Although the above methods will often detect the presence of nucleotide sequences in biological samples, each of these involves multiple steps or, inevitably, a long incubation period, which makes these Method is not practical for easy adoption in clinical laboratories. In addition, many of these methods have limited selectivity or sensitivity with respect to interfering polynucleotide sequences or with respect to reliably detecting low levels of target nucleotide sequences relative to background signals. In particular, non-specific binding of the labeled probe to the support matrix results in a substantial background signal in each method.

Apart from analyzing biological samples for target nucleotide sequences, the physicochemistry of hybrid formation (formation of double helices between complementary polynucleotide sequences) has been studied from various perspectives. These studies include studies of the phenomena of nucleic acid chain migration and displacement both in vivo and in vitro. However, the present inventors do not admit that the phenomena of chain transfer and substitution have any apparent applicability in diagnosis and detection even with reference to these studies. Sea Green and Sea Chivets
Assisted Research ", Volume 9, No.8, pp. 1905-18 (1981
, A single-stranded DNA polynucleotide of 6.1 Kb (chain length of 6100 bases) was converted into an end-labeled complementary DNA polynucleotide of 1.6 Kb in length (1.7-3.3 Kb distance) near its center. It is described to form a hybridized complex (hybrid). 6.1 Kb in solution in this complex
The labeled polynucleotides were rapidly displaced upon the addition of the complementary chain of (see Figure 19 of the same reference, page 1910). This was followed by taking aliquots of the reaction mixture, separating the polynucleotides by gel chromatography and analyzing them by autoradiography. The signal from the displaced 1.6 Kb polynucleotides increased regularly from less than 10% (depending on the concentration of nucleic acid) to more than 90% radioactive signal over a period of 85 minutes. In this case, the 1.6 / 6.1 Kb hybrid contributed to most of the residual radioactivity. Total mass corresponding to 13.8 Kb of DNA (both long and partially substituted short chains)
A putative partially substituted intermediate believed to have was clearly undetected. Literature Authors: Initial hybrid formation of two 6.1 Kb polynucleotides (forming branched chains)
Is the rate limiting step; 1.6 Kb of labeled polynucleotide
We conclude that the substitution along the pairing region is quite rapid, which is in agreement with the calculated average lifetime of 0.8 minutes for the branched chain (corresponding to a mass of 13.8 Kb). They show potential for both single-branched intermediates or double-nucleated (D-looped) intermediates (shown on page 1912 of the same reference). To better study branch migration phenomena, they delay the displacement process by using drugs that appear to delay migration phenomena and / or by using complexes containing hybrid base pairs greater than 1.6 Kb. (See pages 1913-1914 of the same reference). For the 1.6 / 6.1 Kb linkage, Green et al. Purified 6.1 Kb by removing any non-specific linkage.
It should be noted that this was only attempted with the complement of.

The above three patent applications (Diamond et al., Williams et al., And Collins et al.) Describe various types of polynucleotide substitution assays. In these methods, a reagent complex that includes a probe polynucleotide (described herein) and a labeled polynucleotide (described therein) is contacted with a sample. If the sample contains the target nucleotide sequence, this will displace the labeled polynucleotide from the reagent complex. In some forms the substitution is from an immobilized probe (as described herein); in other forms the substitution is made in solution. For some forms involving displacement in solution, the post-displacement separation of the displaced labeled polynucleotide from the intact reagent complex is then performed (see U.S. Patent Application No. 607,885, claims 17-19). ). Although all examples of this post-displacement separation are size-based, the specification states that it can be performed by affinity chromatography. The biotin-avidin affinity linkage has been described in detail in connection with probe immobilization and is also shown in claim 44 of the same 607,885 as a means for post-substitution affinity separation. The description shows biotin or the like as the immobilizable group on the probe or nitrophenol as the hapten. As shown elsewhere in U.S. Pat. No. 607,885, the biotin-avidin pair would have a very high Kass, and it would be assumed that binding was essentially irreversible.

Immobilized homopolynucleotide segments have been used for reversible binding of specific polynucleotides (eg, poly (rA) -terminated RNA reversibly bound to oligo- (dT) -cellulose). For example, Etc Aviv and P. Radar's Proc.Nat.Acad.Sci.USA, 69, 1408-
Pp. 1412 (1972), and P. T. Gilham and J. Am. Chem. Soc., J. Am. Chem. Soc., 86, 4985-4989 (1964)
Year).

SUMMARY OF THE INVENTION The present invention is a reagent complex (a probe polynucleotide containing a labeled polynucleotide bound to a portion of a target binding region).
The labeled polynucleotide is rapidly replaced in solution with the target nucleotide sequence of the sample, and then affinity-separated based on the unique characteristics of the probe polynucleotide. Based on the ability to measure directly or indirectly. This label is used as a reliable quantitative measure that is functionally related to the presence and concentration of the target nucleotide sequence in the sample. Elements of a probe polynucleotide useful for post-substitution affinity separation include:
Due to its characteristics, the binding of the probe polynucleotide can be easily and conveniently reversed, whereby the reagent complex can be separated from the specific labeled polynucleotide during the production, and thus the reagent complex can also be produced. It is useful. The labeled polynucleotide that is conveniently separated from the reagent complex by this method is one that has not hybridized to the probe polynucleotide or that has not hybridized in a sufficient and stable state. This purification step helps to avoid the occurrence of non-specific or pseudo-dissociations that would be misinterpreted in the nucleic acid strand displacement assays. This purification treatment is useful for the removal of excess reagents introduced during the production of the polynucleotide-reagent complex, specifically those that carry a signal-generating moiety but are not chemically or physically associated with the labeled polynucleotide. is there.

Accordingly, the present invention provides (i) a probe polynucleotide capable of base pairing to a target nucleotide sequence via a purine / pyrimidine base pair hydrogen bond, and (ii) a purine / pyrimidine base pair hydrogen bond,
A labeled polynucleotide bound by base pairing to a probe polynucleotide in a region of the probe polynucleotide that is at least partially coextensive with the region in which the probe polynucleotide can base pair with the target nucleotide sequence , A diagnostic reagent complex for determining the presence of a predetermined target nucleotide sequence in a nucleic acid of a biological sample, wherein a possible base pairing between the target nucleotide sequence and the probe polynucleotide is a reagent complex From the probe polynucleotide containing a binding segment distal to (ie, spaced from) the region where the probe polynucleotide can base pair with the target nucleotide sequence; Nukure This coupling segment of tides, labeled polynucleotide under conditions to maintain the state of being bound to the probe polynucleotide, Afuinitei reagent to provide a diagnostic reagent complex, wherein capable of reversibly binding.

The invention provides (a) (i) a probe polynucleotide capable of base pairing to a target nucleotide sequence via a purine / pyrimidine base pair hydrogen bond, and (ii) a purine / pyrimidine base pair hydrogen bond, Providing a reagent complex of labeled polynucleotide bound by base pairing to the probe polynucleotide in a region of the probe polynucleotide that has at least a portion in common with the region in which the probe polynucleotide can bind to the target nucleotide sequence. (B) contacting the reagent complex with a sample in solution under conditions which bind the probe polynucleotide when the target nucleotide sequence is present and displace the labeled polynucleotide from the complex; (c1) contacting step (b ) After which the remaining reagent complex is separated from a portion of the reaction solution, and (C2) A method for determining the presence of a predetermined target nucleotide sequence in the nucleic acid of a biological sample, which comprises the step of determining the presence of the substituted labeled polynucleotide in the solution phase after separation. The probe polynucleotide comprises a binding segment distal to the region where the probe polynucleotide can base pair with the target nucleotide sequence; the separating step (c1) comprises the solution product of the contacting step (b) above To the immobilized affinity reagent in contact with, to recover a solution phase substantially free of binding segments from the immobilized affinity reagent, the measurement step (c2) is performed on this solution phase, In this case, the method includes a method in which the affinity that has been immobilized and the binding between the reagent and the reagent complex are reversible.

The present invention comprises (a) (i) a probe polynucleotide having a region capable of base pairing to a target nucleotide sequence via a purine / pyrimidine base pair hydrogen bond, and (ii) a purine / pyrimidine base pair hydrogen bond. hand,
Hybridizing a labeled polynucleotide capable of base pairing to a probe polynucleotide in a region of the probe polynucleotide having at least a portion in common with a region capable of base pairing to the target nucleotide sequence; Includes a binding segment distal to a region where the probe polynucleotide can base pair with the target nucleotide sequence; (b) binding the reagent complex to the probe polynucleotide by selective reversible adsorption at the binding segment. Separation from the unlabeled polynucleotide; the conditions for adsorbing the reagent complex on the binding segment and desorbing the reagent complex are as follows: the labeled nuclei hybridized to the probe polynucleotide in the hybrid formation step (a). Useful for determining the presence of a predetermined target nucleotide sequence in the nucleic acid of a biological sample, which comprises a step in which the tide is sufficiently relaxed to maintain a hybridized state during the separation step (b). It also includes the method of manufacturing diagnostic reagents.

DETAILED DESCRIPTION OF THE INVENTION In this specification the following terms are used according to their commonly accepted meaning in the field of molecular biology.

A polynucleotide or a polynucleotide chain is bound to each other by a 3 ', 5'-phosphodiester bond and is a pendant group by a carbon-nitrogen bond at the carbon 1-position of the sugar, such as a purine base or a pyrimidine base, for example (provided that Uracil (naturally bound only to ribosomes, rU), thymine naturally bound only to deoxyribose (dT),
By linear polymer structure of a pentose sugar (generally ribone or deoxyribose) linked to cytosine (dC or rC), adenine (dA or rA), and guanine (dG or rG). Thus, polynucleotides include deoxyribonucleic acid (DMA) and ribonucleic acid (RNA) chains, as well as complex DMA-RNA chains.

The ends of these polynucleotide chains are pentose 5
The carbon atom at position 5 is not attached to another pentose (but may carry a hydroxyl group, a monophosphate group, or other natural or synthetic moiety), and the carbon atom at position 3 of the pentose is Not attached to other pentoses (although they may also carry hydroxyl groups, monophosphate groups, or other natural or synthetic moieties) 3 '
Called the end.

Complementary base pairs or purine / pyrimidine base pairs are 2
By hydrogen bonds between opposing bases that form the pendants of an antiparallel polynucleotide strand of a book. This is the most energetically favorable for natural DNA when dG faces dC and dA faces dT. Bases other than the five naturally occurring bases also have preferential pairs. For example, 5-methylcytosine preferentially binds guanine. For illustration purposes, this pair is shown in many figures by parallel straight lines (in the sense of 5'vs 3 ') with the opposite chains pointing in antiparallel directions. However, the actual geometry of the double-stranded segment is described in US patent application Ser. No. 607,885, first
One should recognize the points that are usually helical in various pitches (the well-known double helix), as shown schematically in Figure D.

Hybridization here means mixing two polynucleotides under conditions that lead to the formation of a double-stranded structure to form a double-stranded structure in which complementary base pairs have a complementary sequence or a nearly complementary sequence. Is used to mean.

The basic components of the invention are probe polynucleotides (sometimes referred to herein as probes), labeled (sometimes referred to herein as tagged) polynucleotides, nucleic acids (some of which are sometimes referred to herein as target polynucleotides or target nucleotide sequences). Is a biological sample containing. The sample may or may not contain the labeled nucleotide sequence. A support (immobilization) affinity reagent is also provided on which the probe is immobilized as part of the diagnostic, manufacturing or both separation steps. When performing a diagnostic method, additional reagents or devices are often required for reading. The term read directs the labeled polynucleotide found in the product liquid phase by being displaced from the reagent complex and then separating the displaced labeled polynucleotide in solution from the probe polynucleotide and the reagent complex. Or it means indirect detection.

In practicing the present invention, the probe polynucleotide is a linear or circular polynucleotide capable of specifically binding to a particular target nucleotide sequence of a sample by complementary base pairs in at least one region of the purine / pyrimidine base sequence. is there. This bond may be between DNA and RNA, DNA and DNA, or RNA and RNA. Therefore, the probe may be either DNA or RNA. As will be discussed in more detail below, it is generally only a particular region of the probe that selectively binds to the target nucleotide sequence.
Other regions of the probe do not participate in hybrid formation but play an important role in the present invention (eg, by acting as attachment sites for affinity ligands).
It may be a variety of natural or synthetic sequences. The fact that there is some separation between the attachment site and the region where the target nucleotide sequence binds will be described in more detail below.

For the region of the probe to which the target nucleotide specifically binds (referred to herein as the target binding region; TBR in the figure), each nucleotide within this sequence is bound to its proper complementary binding partner (eg, for dT) within the target nucleotide sequence. This combination may be perfect in terms of finding dA), or may include some inconsistencies. At least part of the target binding region of the probe is preferably single-stranded in the reagent complex, ie not complementary to the labeled polynucleotide sequence. This single-stranded region is sometimes referred to herein as the early binding region (IBR in the figure). This is because the target nucleotide sequence can bind to this base region without replacing the labeled polynucleotide. This type of initial binding region of the probe is at least 15 bases in length, preferably at least 50 bases in length. The overall target binding region is the initial binding region,
And most or (preferably) all labeled polynucleotide binding regions (in the drawings and the discussion below).
LBR) is included. The overall target binding region (TBR) length is not determined independently, but rather can be considered as a function of the preferred length of the IBR and LBR portions, or a more preferred length sum. Lengths of more than 500 bases per probe initial binding region are generally not required and in most cases are not a significant disadvantage. A suitable lower limit for the base pair length of this region for clinical laboratories depends somewhat on the base sequence and base composition of the target binding region of the probe polynucleotide, and other physical factors that follow, with particular objectives Hybridization, post-substitution affinity separation, hybridization kinetics, and the reading system employed. Another constraint is that when the binding segment comprises a homopolymeric nucleotide sequence (eg, poly (dC)), the same sequence may not be present in the labeled polynucleotide, as described for some embodiments below. This is a preferable point.
As further described below, in each of these cases, the base paired LBR region will usually be substantially longer (and have a higher melting point) than the base paired binding segment (BS) if each is in bound form. (The former complexed with a labeled polynucleotide and the latter complexed with a homopolynucleotide affinity reagent (eg, immobilized poly (dG))).

In certain embodiments, the solid phase to which the affinity reagent is immobilized is of almost any type commonly used, including in particular polymeric materials, ceramic materials, walls of test tubes and other containers, paper, nitrocellulose or Includes glass. In one form of the invention, the solid phase comprises particles or beads made from materials such as proteins, polystyrene, latex or glass. The solid phase is present in a liquid slurry and may be pressed into a thin film or a relatively thick disc, or as a packed bed in a column (illustrated in FIGS. 1 and 2C),
Alternatively, it may be present as a lining on the inner wall of the capillary.

The means for attaching the affinity reagent to the solid support may be simple adsorption, but is preferably in the form of a specific covalent bond, an ionic bond or a hydrogen bond.

In the case of the present invention, the probe polynucleotide is not immobilized on a support in the complex, but rather the reagent is mixed with the biological sample, whereby hybridization (if any) occurs in solution. , The entire reagent complex is in solution. As will be described later, the probe actually contains substituents which can be immobilized or separated after formation of the hybrid, for example by conduction through a porous bed or filter containing the affinity reagent on the support. Such immobilization will leave only the displaced labeled polynucleotide in the liquid phase for subsequent measurements.

Probe polynucleotides of this type (in some cases the attachable groups are not yet present) can be isolated in various ways, for example from a suitable plasmid, isolated from bacteria or other suitable microorganisms as part of the genetic material of the microorganism or It can be reproducibly produced in, or as part of, a viral vector or obtained from other suitable biological sources. Generally, a small region of nucleic acid containing the probe polynucleotide sequence, a portion of which forms the target binding region, will be recombinantly inserted into any such cloning vector. The remainder of the cloning insert, which is not the probe polynucleotide sequence, can be conveniently selected from any of the polynucleotide sequences that are heterologous to the target nucleotide sequence (if any). In certain embodiments of the invention, such heterologous sequences include sequences carefully selected for particular properties (eg, the presence of unique restriction enzyme recognition sites). Under certain conditions, the entire gene, or a sequence containing the entire gene, can be used as an insert (vector plus inserted nucleotide sequence is circular or linear). If the probe polynucleotide is double-stranded at the time of manufacture, it is usually isolated and then denatured (by heat, pH adjustment, or by base pair disruption by other conventional methods). Cleavage (especially by restriction enzymes or by site-directed chemical cleavage) is commonly used to form the desired linear double-stranded segment, and if the double-stranded cyclic form is growing. Cleavage will occur prior to denaturation. In some cases, it is preferred to purify individual chains from double-stranded structures and individually use them as probe polynucleotides (or one as a probe polynucleotide and one as a labeled polynucleotide). This purification can be done by standard methods such as denaturing gel electrophoresis or affinity chromatography. In some cases, probe polynucleotides are produced as single-stranded molecules by replication with a single-stranded vector (eg, M13 bacteriophage).

In other cases, if the nucleotide sequence is short enough to be chemically synthesized (currently 100 nucleotides or less in length), organic chemical synthesis may be used to Some or all can be manufactured. This chemically synthesized DNA can then be used directly as a probe polynucleotide, or it can be inserted into the genome of a suitable plasmid or other vector and biologically replicated. The replicated DNA (or RNA transcribed therefrom) can be used as a probe polynucleotide after suitable manipulations such as restriction enzyme cleavage and chain purification. The chemically synthesized DNA corresponds to or is complementary to the final probe polynucleotide or a portion thereof.

If the binding segment is a homopolynucleotide sequence (eg poly (dC)), this sequence is chemically synthesized,
It can be inserted into the same genome and finally appear within the probe polynucleotide sequence. Alternatively (as shown in the examples below), joining the homopolynucleotide sequence to the probe polynucleotide (at the 3'end of the terminal deoxynucleotidyl transferase, in the case of terminal deoxynucleotidyl transferase) (eg, by terminal deoxynucleotidyl transferase). You can

Labeled polynucleotides used in the reagents and methods of the invention are generally smaller DN than probe polynucleotides.
A piece or RNA piece, which has two important features. That is, (a) stable, but reversible binding at a probe-specific locus, and (b) the label being sensitive to detection, especially after displacement. These features are discussed separately here, and then the effect of particular types of labels on stability and substitution.

The pairing of the labeled polynucleotide and the probe polynucleotide is performed over a smaller number of bases than the pairing of the target nucleotide sequence of the sample and the probe. In most cases, the bases of the probe polynucleotide to which the labeled polynucleotide specifically binds are a subgroup of the bases of the probe that subsequently bind to the nucleotide sequence of the sample, and thus in the above target binding region of the probe. Form part of what is called.

The term labeled polynucleotide binding region (LBR) herein means the base sequence in the probe to which the labeled polynucleotide binds in the complex. In a preferred embodiment (as shown in all figures) the labeled polynucleotide binding region is wholly part of the target binding region. In other embodiments (see Figure 1G of U.S. Patent Application No. 607,885) only part of the label binding region, but preferably most, is part of the target binding region. . When the length of the labeled polynucleotide binding region outside the target binding region of the probe polynucleotide is about 15 bases or more, the labeled polynucleotide is associated with the probe when the only attachment is through the pairing in this region. It is not preferable because it may be difficult to cancel. In certain preferred embodiments, the region of the probe polynucleotide to which the labeled polynucleotide binds is a subgroup of this relatively large region of the probe polynucleotide, which is at one end of the target nucleotide sequence of the sample to which it subsequently binds or It is in the vicinity.

Other forms of probe attachment to the labeled polynucleotide are possible, but generally not preferred. Such other deposits must be removed before, during or after the replacement (in any case before the measuring step of the method). Multiple labeled pronucleotides on single-stranded probes (US Patent Application No. 607,885
(Shown in FIG. 6 of the specification and the accompanying description) is one form of the invention that can be utilized particularly when a higher number of displacement labels per displacement reaction is desired.

The size of the labeled polynucleotide binding region of the probe itself is not critical to the invention. This variety of sizes can be corrected, for example, by changing the temperature of the displacement step (contact step (b) above) and the size of the probe region to which the sample target nucleotide sequence binds. A generally preferred size for the labeled polynucleotide binding region (and corresponding nucleotide sequence of the labeled polynucleotide) is at least about 15 bases, preferably at least about 15 bases.
20 bases, more preferably at least about 25 bases. A preferred range is from about 20 to about 500 bases (up to 1000 bases are also preferred), especially from about 25 to about 200 bases.
Labeled polynucleotides with unusually short pairing segments (noting factors such as GC base content) are too hot during the substitution process (also taking into account factors such as salt concentration that affect the melting point). , Will dissociate non-specifically from the probe. See Dye Fleif Elder, et al., "Biopolymers," Vol. 7, pp. 81-93 (1969). Abnormally long pairing segments (eg, over 1000 bases) have no intrinsic advantage. Such long mating segments are sometimes less preferred. In this case, the overall target nucleotide region of the probe is lengthened and a longer target nucleotide sequence is necessarily required to effectively displace the labeled polynucleotide. The binding portion of the target polynucleotide is also used for small polynucleotides if it is too long, or can no longer be produced by the particular technique of choice (eg organic chemical synthesis of the entire labeled polynucleotide). Is. Organic chemical synthesis is currently less than about 100, especially about 6
While labeled polynucleotide binding regions of 0 or less are generally relatively easy, improvements in synthetic techniques of this kind will facilitate the production of longer sequences.

The minimum length of the labeled polynucleotide (and the labeled polynucleotide binding region) is primarily related to the stability of the reagent complex. Factors that affect this stability other than length include GC base content (the effect of which on melting point is well known) and internal base pair mismatch. Melting point measurements are a useful method for achieving stability and comparable effective length for sequences with one or more internal base pair mismatches. For example, a 30-base sequence with an internal mismatch (and therefore only exactly 29 pairs) would have similar potency to a shorter, exactly matched sequence (at least the stability of the reagent complex). Regarding). base
The degree of difference from the 29 pair behavior will depend on the location of the base pair mismatch and the base exchanges made. Effective length is
It can be expected that the mismatched pair will be different depending on whether it is a purine / purine, purine / pyrimidine, or pyrimidine / pyrimidine. However, it can be determined empirically by measuring the melting point. In this method, one or a series of probes is used, as shown in the report such as Earl El Ornstein and J. A. Fresco, "Biopolymers", Vol. 22, 1979-2000 (1983). The / labeled polynucleotide complex is subjected to various modes of temperature. The melting point of the complex between the probe and the labeled polynucleotide containing the mismatch is measured and this value is compared to a slightly shorter pairing on the same probe polynucleotide (although the match within the pairing region is perfect). The effective pairing length of the labeled polynucleotide containing the mismatch can be estimated by comparing it with the melting point of the complex of, and it can be applied to the above preferable range and more preferable range. However, the above correlations based on melting points are primarily intended as a simple estimation means. The practical advantage with respect to certain labeled polynucleotide binding regions is in part to the extent to which the labeled polynucleotide is actually retained in the complex during storage of the reagent or contact with non-homologous nucleic acids under the conditions of use. Or not (but still replaced by the nucleic acid containing the target nucleotide sequence). Mismatches are tolerable but generally not preferred and, if present, generally do not constitute more than 2/5 of the pairing region, preferably not more than 1/10 (eg, labeled polynucleotide / probe polynucleotide pair). If the combined region is 30 base pairs long, the maximum mismatch is 3 and the match is 27).

The labeled polynucleotide may include, in addition to the pairing region, a region of nucleotides that does not specifically bind to the probe polynucleotide. These regions serve to attach the pairing region to a detectable label, or are themselves labeled (eg, by radioactive labeling, or by covalent attachment to indirect markers such as avidin or biotin), or have any special function. Exists without. The labeled polynucleotide itself may be linear or circular, and the region other than the pairing region may be double-stranded (provided that it is preferably not double-stranded). The labeled polynucleotide is preferably not circular when the probe polynucleotide is circular.

The one or more detectable labels are generally at one or several points along the labeled polynucleotide (particularly where the label is a radionuclide or biotin), or at one end, or at the internal It may be located only at one specific position (for example, at a purine or pyrimidine base that is not involved in base pairing with the probe polynucleotide) (by a standard method). If there is at least one unpaired labeled polynucleotide region in the reagent complex, the label is preferably present or concentrated on or within such an unpaired region. . Directly detectable labels that can be used are radionuclides (especially ³² P, ³⁵ S, ¹⁴ C or ¹²⁵ I labeled nucleotides), fluorescent molecules (eg at the free end of labeled polynucleotides or unpaired bases of labeled polynucleotides). Fluorescein or rhodamine derivatives attached to one or more) or moieties that can be detected by other means (which may include cleaving), such as the haptenic moiety nitrophenol. Indirectly detectable labels include antigenic determinants, affinity reagents, antigens or antibodies that can be recognized by immunochemistry or other affinity reactions, such as EP 63,879, WO 83/02277, and EP 97,373. Described above, as well as those exemplified by biotinylated nucleotides present in or added to labeled polynucleotides (e.g., multiple nucleotides in the absence of template linkage to labeled polynucleotides). By the enzyme terminal deoxynucleotidyl transferase which inserts at the 3'end of. Other indirect labels include an enzyme attached to the labeled polynucleotide, particularly at the free end away from the probe-paired region, the presence of which results in a substrate for the enzyme after the replacement and separation steps of the method. It can be measured by adding and quantifying the enzyme substrate or preferably the enzyme product.
Similarly, the label may be an apoenzyme, a coenzyme, an enzyme-based modifier, etc., and the other necessary reagents are usually added with the appropriate enzyme substrate after substitution and affinity separation. Of course, if the enzymatic reaction cannot occur in the presence of all but one (eg the substrate) components, these other reagents may be present in solution during the displacement step (b) above. A number of labels can be added during the preparation of the labeled polynucleotide (eg, by the terminal deoxynucleotidyl transferase enzyme). Multi-labeled polynucleotides, such as those containing enzymes (or apoenzymes) and those containing coenzymes can also be used. One mode of attachment of the enzyme to the labeled polynucleotide is by affinity reagents such as streptavidin /
It is via biotin. As shown in FIG. 2B, this format produces a reagent complex by hybridizing a biotin-labeled polynucleotide to the probe and then coupling a streptavidin-enzyme conjugate to the biotinylated polynucleotide, followed by affinity separation ( Part of the method of the invention) may be used in embodiments in which the reagent complex is purified from unbound conjugate and then the displacement step (b) of the method is performed. Alternatively, for example, streptavidin-enzyme is bound to a biotin-labeled polynucleotide, then this streptavidin-enzyme-labeled polynucleotide is hybridized to a probe polynucleotide, and then the reagent complex is bound to unbound streptavidin by the above affinity separation method. -Can be purified from enzyme conjugates. In addition, the third of U.S. Patent Application No. 607,885
There may be a separate detectable moiety on the probe that interacts with the label of the labeled polynucleotide in the reagent complex, as shown in the A-3D diagram.

In addition to the label of the labeled polynucleotide, the section of the labeled polynucleotide may act as the detectable label. In the application of Sea Barry et al. (Pending examination), the 3'terminal ribonucleotide segment of the labeled polynucleotide (signal strand) is digested to form ribonucleoside phosphate (containing ADP or optionally AMP), It can be phosphorylated to ATP and then detected by a luciferase catalyzed reaction with luciferin. The relevant description of the above specification is incorporated herein by reference.

Most forms of label have little or no decrease in the melting point of the reagent complex, especially if they are far from the pairing region of the labeled polynucleotide for the probe,
A more important point is that, as evidenced by the negligible effect on the hybridization reaction between the target nucleotide sequence and the probe polynucleotide (for the purpose of testing), the bases of the labeled polynucleotide and the probe polynucleotide are It will have little effect on the strength of the mating. Some form of label, such as biotin covalently attached by specific chemical methods onto the nucleotides of the paired region of the labeled polynucleotide, and a large enzyme molecule or fluorescent moiety attached, for example, to or near the paired region of the nucleotides , Has a noticeable effect on the stability of the reagent complex. Such an effect will generally reduce the stability of the binding of the labeled polynucleotide / probe polynucleotide (and thus its melting point). This effect may be slightly beneficial in enhancing the rate of displacement, but may increase non-specific dissociation or shedding of the labeled polynucleotide. However, such non-specific shedding, by increasing the temperature during the displacement step, increases the length of the labeled polynucleotide binding region and thereby stabilizes double helix formation with reagents such as Mg ⁺⁺ ions or It can usually be reduced by adding spermidine or by other physical means of this kind.

The pre-separation of reagent complexes from various impurities is often only size if the labeled polynucleotide is significantly smaller than the probe polynucleotide or complex, such as size exclusion chromatography (si).
ze exclusion chromatography). This is based on the double-stranded portion of the complex (in the labeled polynucleotide binding region of the probe) which is considered to be absent in the labeled polynucleotide or probe polynucleotide except for the small internal binding region. Such separation can also be performed. This property allows the reagent complex to be separated from the unbound labeled polynucleotide or probe strand, for example, by affinity chromatography using double-strand specific anti-nucleic acid antibodies, or hydroxylapatite chromatography.

An excess molar amount of labeled polynucleotide may be required (for reasons described below) in the process step of forming the reagent complex by hybridizing the probe polynucleotide and the labeled polynucleotide. Under these conditions, it is particularly desirable to remove from the reagent complex solution after the hybridization reaction, the labeled polynucleotide molecule that does not participate in the hybridization reaction and is thus free in the solution or does not form a hybrid. .

To this end, the reagent complex is conducted onto the immobilized affinity reagent for the binding segment of the probe polynucleotide in the reversible binding step of the present invention. Following formation of the reagent complex, which is typically immobilized by an affinity reagent, unbound labeled polynucleotides, as well as other labels used to bind the detectable label or other labeled moiety to the nucleotides of the labeled polynucleotides. The chemical reagent (eg streptavidin-enzyme conjugate) is washed away. Such washes are bound slightly (eg, by fewer complementary bases at other positions on the probe rather than by more than 15 complementary bases at the binding site of interest). , Or designed to also remove the support-bound labeled polynucleotide in a non-specific manner. The probe polynucleotide and the complex of probe polynucleotide and labeled polynucleotide, which is barely attached to the support, will also be removed during this washing step. Washing is non-specific based on a labeled polynucleotide (with or without probe polynucleotide) that is then separated from or separated from the probe in the replacement step (b) of the method of the invention regardless of the specific replacement. It is preferable to do so under a condition sufficient for substantially removing the active background signal and for a sufficient period of time. Reagents (eg proteins) for blocking non-specific binding sites on the support may be used. After such washing, for example, a low concentration of ionic species (eg 10 mM · Tris HCl, 1.0 mM · EDT
The reagent complex is released from the immobilized affinity reagent by washing the affinity matrix with a buffer containing A, pH 7.5). Such low ionic strength results in short segments of complementary base pairs (eg, 3-10 bases of poly (dG) on the support versus 3-10 bases of poly (dC) in the probe polynucleotide binding segment). Is effective for decoupling.

However, as shown in Example 2 below, in some cases, even if an affinity column, which is not thoroughly washed, is used, the reagent complex containing no unbound labeled polynucleotide is appropriately purified.

In use, the actual contacting or displacement step with the sample material (which may contain nucleic acids containing the target nucleotide sequence) is usually at a temperature less stringent than the washing step in the manufacturing process described above, It will be carried out under conditions of ionic strength and time, and thus less likely non-specific dissociation or debinding of the labeled polynucleotide. The desired temperature range in the contacting step is about 15 to about 90 depending on the ionic strength of the solution and other additives that affect the melting point.
℃. The most effective temperature will be the temperature at which hybridization of the sample to the probe occurs at the highest or near highest rate. However, in certain cases, more convenient (generally lower than the above) temperatures may be employed, for example around room temperature (15-25 ° C). Numerous documents [eg JAG
Wettomul and N. Davidson, “Jay.
J. Mol. Biol., 31: 349-370 (1968) and C. Minson and G. Derby, "New Developments in Practical Virology," Vol. 18, 18.
The rate of hybrid formation is also a function of pH and the concentration of nucleotides in the sample, as described on pages 5-229 (1982, Alan Ris). Further, at least in some cases, the US patent application of Williams et al.
As described in more detail in 684,308, a volume excluding p
Olymer) is preferably present. Other reagents such as dextran sulfate can also be used. Enzymes and other proteins that affect substitution reactions, such as E. coli AT
The P-dependent recA protein is also extremely useful in this method (as described in Collins et al., US Pat. No. 6,84,305, cited above).

The proportions, amounts and concentrations of reagent complexes are not individually critical and it is generally desirable that the entire hybridisation mixture of sample and reagent complexes be as concentrated as feasible. In most cases, it is preferred that the probe polynucleotide carrying the binding site for the target nucleotide sequence also be present in the sample in a 10-fold molar excess or more (preferably 100-fold molar excess) or more of the expected level of the target nucleotide sequence. Ah The sample itself may contain nucleic acid, and it is preferable that the nucleic acid is completely or partially in solution (separated from cell membrane, protein, etc.) and single-stranded for the hybridization process of Atssei. . Presence of complement of target nucleotide sequence (double-stranded sample DN
(Due to the degeneration of A) can be a hindrance. This interference appears to be low, at least in the preferred form of the invention. In hybrid formation (after displacement), which selectively involves immobilization of the probe, the displaced labeled polynucleotide remains and remains in solution phase, and this type of displaced labeled polynucleotide then becomes It is measured whether it has rehybridized with the complementary segment. This is because the newly formed hybrid does not contain a binding segment and thus does not show affinity for the immobilized reagent. In many solution hybrid formations this interference will be minimized by kinetic effects.

Generally, the time required for the substitution reaction to take place to a sufficient extent should be less than 2 hours, generally less than 1 hour and often less than 15 minutes. Conditions can be adjusted to substantially complete within these times. But longer times are not necessarily at a disadvantage. The rate limiting step is considered to be the step in which the sample or target nucleotide sequence finds the complementary sequence of the probe polynucleotide (See Green and See Tibets, "Nucleic Acid Research", Vol. 9, pp. 1905-18 ( 1981
Year)). Once the target / probe hybrid formation is initiated, displacement of the labeled polynucleotide is expected to occur within 1 minute, often within 1 second.

The displacement reaction mixture is quenched (eg, by cooling below 20 ° C.) and then passed through an affinity column (or other form of immobilized affinity reagent as described above). A series of fractions are obtained by passing a series of solutions through the column, the displaced labeled polynucleotide being present in the initial liquid fraction. Remaining reagent complex is either allowed to be found in later fractions (with sufficient solution to remove the binding segment from the immobilized affinity reagent) or is left on the column.

The dimensions of the column used for post-displacing separation can be an important parameter in certain cases. When small scale samples are used for individual displacement (catalysis) reactions, it is generally desirable that the various fractions collected from the column are also small scale. As shown in Example 4 below, about 25 mg of oligo (dG)
It would be preferable to use such a small amount of the affinity reagent because cellulose provides excellent capture of the reagent complex (rather than the 250 mg affinity material used in Examples 1-3). This allows the concentrated displacement reaction mixture to be loaded onto the column prior to loading or with a large amount of wash solution (eg, solutions 134, 136 described below in connection with FIG. 2C).
And no need to dilute with 138). The effect of the small volume of eluate is that the signal (eg enzyme product or bioluminescence) can be measured for all or a larger portion of the eluate.

After the post-displacement separation has taken place, the presence and (often) measurement of the labeled polynucleotide can be determined for the early liquid fraction or for the late liquid fraction or solid phase. Preferably the measurement is performed on the liquid phase (initial liquid fraction). Measuring the presence and amount of displaced labeled polynucleotide in the liquid phase has the advantage of a relatively low background signal. The background signal may be mostly due to non-specific dissociation or shedding from the solid support, as described above, or to incomplete separation. Furthermore, in the absence of the target sequence in the sample, there is no signal from the labeled polynucleotide in the liquid phase.
For comparison, if the detection is in the solid phase or in the late eluate from the affinity column, the highest level of labeled polynucleotide will be detected for negative results. Especially when a high molar excess of reagent complex is used, the measurement of the label remaining on the solid support is less sensitive.

As mentioned above, the binding segment of the probe polynucleotide may be a homopolynucleotide sequence, in which case the immobilized affinity reagent used in both production and use is a complementary homopolynucleotide sequence. Reversibility differences can be attributed to long binding segments on the probe (eg, 20 nucleotides or more), short complementary polynucleotide segments (eg, about 4-12 nucleotides) for immobilized affinity reagents used to make reagent complexes, diagnostics. Immobilized affinity reagents for use in specific applications are obtained by using longer complementary polynucleotide segments (eg, 20 or more nucleotides). Other linking segments that can be used in the method of preparation or separation after substitution (and are also reversible) include sugar side groups (eg maltose segments) or chemically linked polysaccharides (eg poly (glucose)). . Immobilized affinity reagents for this type of glycoform-binding segment include lectins and boronates. See "Solid Phase Biochemistry, Analytical and Synthetic Perspectives", edited by Davryeu Etsch Scouten, pp. 17-78 and 149-188 (Zyon Weiry & Sons, New York; 1983).

The methods and reagents of the present invention can be used to detect and measure various concentrations of various target nucleotide sequences. Viruses, bacteria and fungi especially pathogenic to microorganisms containing infectious agents whose nucleic acids (genome and others) can be targeted; eg cytomegalovirus or gonococcus (Neisseria gono)
rrhea) is included. Genetic disorders or conditions that may be targeted include β-thalassemia, α ₁ -thalassemia, cat sickness, and certain retinoblastomas. The methods and reagents of the present invention are primarily used when multinucleotide nucleotide deletions, insertions, substitutions or rearrangements are involved in distinguishing a target nucleotide sequence from the closest sequence present in a sample to be tested negative for the target sequence. It is applicable to detect genetic disorders or mutations. To the extent that the present invention is applicable to genetic disorders due to single base mutations, complete complementarity of substituted bases or other mutated and adjacent nucleotides is part of the target binding region of the probe polynucleotide. It is desirable that the position of that base within this region influences the selectivity of the method. Of the alterations in structural or regulatory genes, alterations or differences in gene expression, activation or transposition can be detected by the method of the invention, especially for specific situations by targeting mRNA. Other perturbations in the expression of structural genes can also be detected. The method can also be used for HLA typing for tissue transplantation, determination of antibody resistance genes in microorganisms, detection of altered tumorigenic sequences or altered tumorigenic expression levels, as well as specific infections of food, drug and water samples. It can also be applied to screening for sex substances or other microorganisms.

Selection of the target sequence for a particular test depends on determining the sequence that is unique or relatively unique to the target organism or condition. This target sequence is used to create a target binding region complementary thereto, and then a labeled polynucleotide of an appropriate length is created to bind to a part of the target binding region. In some cases, to provide improved specificity when the individual target sequences are only relatively unique,
Multiple reagent complexes that target different parts of the same organism or condition of nucleic acid may be used.

One form of the production method and the diagnostic reagent complex of the present invention is shown in FIG. The probe polynucleotide chain P is indicated by the numeral 10 in unmodified form. It contains a target binding region TBR capable of hydrogen bonding to the target nucleotide sequence for detection via purine / pyrimidine base pairs. The polynucleotide sequence of the subgroup of the target binding region TBR directs the labeled polynucleotide binding region LBR and has the function described above and below in connection with the hybridization of the labeled polynucleotide L.

The probes used in the methods, reagents and uses of the present invention are modified as indicated by the structure identified by the number 12 (and the designation P ') and, as shown, preferably bind away from the target binding region TBR. Includes segment BS. The binding segment BS is shown at one end (3 'end) of the modified probe P'and the labeled binding region TBR is shown as being at the opposite end, but no specific position is required. . In particular, the target binding region TBR may be in any position separate from the binding segment, preferably spaced from the binding segment BS. In each case the target binding region is intermediate the two ends; the probe polynucleotide of each example contains at least 205 nucleotides between the 5'end of the TBR and the 5'end of the probe polynucleotide, It contains at least 348 nucleotides between the 3'end and the binding segment (3'-end poly (dC) sequence). Some of the probe polynucleotides in the group of probe strands in each example had incomplete digestion of the probe polynucleotide precursor strands by the restriction enzymes used therein, so that a large number of nucleotides were added to the 5'or 3'side of TBR. Will include.

A preferred means for linking the binding segment is a natural or modified poly (ene) containing repeating natural or modified purine or pyrimidine nucleoside (e.g. dA, rA, dC, dG, dT or biotinylated dU). If it is a segment of nucleotides, an enzyme, such as terminal deoxynucleotidyl transferase (TdT) (G.R.Deng and R.U.,
(R.Wu), “Nukuraykku Aids Research”, 9
Vol. 4, pp. 4173-4188 (1981)), the unmodified probe P is extended in the presence of an appropriate nucleoside-5'-triphosphate used for enzyme addition. In such a case, the binding segment would be the modified probe 12 (first
It may be a homopolymer segment at the 3'end (indicated by P'in the figure). Such TdT extensions should minimize the number of 3'ends in the reaction mixture to avoid or minimize decorated probes 12 containing homopolymer segments or polynucleotides other than P '. Is considered desirable.

The modified probe 12 is then provided with a polynucleotide chain L, at least a portion of which is complementary to the labeled polynucleotide binding region LBR of the probe (both P and P ').
The target complex 16 (described in detail below) is formed by hybridizing with a free labeled polynucleotide 14 consisting of It is preferable to use an excess of labeled polynucleotide in this manufacturing operation. To the extent that some unmodified probe strand 10 remains after, for example, a reaction mediated by deoxynucleotidyl terminal transferase, these are unmodified probes hybridized to labeled polynucleotide L with a detectable label T. It should be appreciated that it is also possible to form reagent complexes, each of which contains P (further shown as numeral 24 below FIG. 1).

Reaction mixture containing the desired complex 16, but also containing the remaining probe polynucleotides 10 and 12, as well as excess unhybridized labeled polynucleotide 14 and reagent complex 24 containing unmodified probe P. Is then purified before use. Purification means for the above embodiments include some type of size chromatography (eg, on a size exclusion resin column) that separates most of the unhybridized labeled polynucleotide 14 from the fraction containing reagent complex 16 of interest. To conduct). Then, the target fraction is brought into conduction in contact with the affinity reagent immobilized on the binding segment BS, which is shown as a chromatographic column 17 in FIG. As shown in FIG. 1, the fraction of interest is passed through the column first, and then a series of solutions is passed through the column.
The first solution 18 is a washing solution designed to wash away the substance not containing the binding segment BS from the column. Therefore, this is where the affinity reagent binds the binding segment BS tightly. The washed-out substance includes a non-hybridized labeled polynucleotide 14, an unmodified probe polynucleotide 10, and a reagent complex 24 each containing an unmodified probe strand P. Other excess reagents that are introduced during the manufacturing process of the reagent complex and are not chemically or physically associated with the desired reagent complex will also be eluted in the first liquid 18. Therefore, these substances will elute from column 17 into the initial fraction and they will not be recycled to the terminal deoxynucleotidyl transferase reaction step for the reasons described below, but rather will be discarded. The second liquid 20 supplied to the column is
Designed so that the affinity bond between the affinity reagent and the binding segment BS on the column 17 is destroyed, and the hydrogen bond between the labeled polynucleotide binding region LBR and the labeled polynucleotide L (particularly in the target reagent complex 16) is not destroyed. It was done. Therefore, if the binding segment BS is attached to the column material via a purine / pyrimidine base pair hydrogen bond, as is the case when the binding segment BS is a homopolynucleotide segment, this latter binding is labeled. It is desirable that the cumulative binding strength be lower than the binding between polynucleotide L and the labeled polynucleotide binding region LBR (related to the lower melting point under standard conditions). Generally, the homopolynucleotide segment is 20 nucleotides or less in length, while the labeled polynucleotide binding region is 25-50 bases or more in length.

Under certain conditions it may be considered to use a homopolynucleotide segment of 20 nucleotides or more in length, in which case the standard polynucleotide binding region will remain labeled with the labeled polynucleotide in the reagent complex. When the reagent complex can disassociate from the column, the lower limit will be so tightly limited. The first limiting factor is the shorter length of the BS or homopolymer segment attached to the solid support.

As a result of this affinity chromatography step, the fraction containing the desired reagent complex 16 and the unhybridized modified probe polynucleotide 12 as the only polynucleotide-based impurity that appears to be present in significant amounts. Is obtained. But labeled polynucleotide
14 was used in a large excess in the hybrid formation step, and for a sufficient period (and other conditions
(For strength and temperature, for example)
The ratio of modified probe 12 to reagent complex 16 of interest will be small. Furthermore, when the complex is used in a substitution reaction (the method of the present invention), the modified probe 12 does not give a false signal, and the result is that it hybridizes with the target polynucleotide sequence without producing a substituted labeled polynucleotide. It just becomes The only effect would therefore be to reduce the maximum expected signal by a factor approximately equal to P '/ (P'L + P'). In this formula, P'is the product fraction 25
Is the amount of modified probe strand 12 in the product, and P'L is the amount of the desired reagent complex 16 in the product fraction 25. In the unusual case where the reagent complex contains unacceptably high levels of unhybridized probe 12 on labeled strand 14, further purification may be required to remove this material. This is for example a hydroxyapatite chromatographic separation (which will delay the reagent complex on the basis of its double-stranded region), or an immobilization which is complementary to the labeled polynucleotide binding region LBR or part thereof. Chromatography on the generated polynucleotide sequence, which retards the free probe polynucleotide 12. As a result, the product fraction 25 is specifically fractionated, and the reagent complex 16 shown in FIG.
An enriched product solution is obtained.

On the other hand, when the substitution is carried out in a solution and the separation after the substitution is carried out by using an affinity reagent specific to the binding segment BS, the labeled polynucleotide which is not hybridized and is mixed in the product fraction 25. A false (non-specific) signal will be emitted by 14 or reagent complex 24.

The product fraction 25 need not be further separated before storage and use, but other steps that can be performed include dialysis (lower ionic strength), evaporative concentration, and / or lyophilization. Care should be taken in these subsequent steps not to result in a combination of ionic strength and temperature under which the labeled polynucleotide L is dissociated (melted) from the reagent complex 16.

After passing the first liquid 18 and the second liquid 20, it is desirable to pass the regeneration liquid 22 to move the residual substances other than the immobilized affinity reagent from the column 17. The column is now ready for re-equilibration to conditions (eg ionic strength) suitable for injecting the hybridisation reaction mixture.

Many variations on the method shown in FIG. 1 are considered.
For example, prior to hybrid formation of the labeled polynucleotide L to the probe P, a homopolymer segment (HS,
This is a form of binding sequence BS) can be added to probe P. However, in this case, a similar HS tail could be added to many labeled polynucleotides. Due to such an HS tail on the bound labeled polynucleotides, even when these labeled polynucleotides are in unbound form, the It seems that these may attach to the affinity matrix in the separation step after chain substitution. This resulting reduction or loss of detectable signal can be prevented by pre-blocking the appropriate ends of the labeled polynucleotide (eg 3 of the labeled polynucleotide prior to adding the HS tail to the reagent complex). 'By phosphorylating the terminal with terminal deoxynucleotidyl transferase).

Another variation on the method of FIG. 1 is the target binding region TBR and binding segment B within probe polynucleotide 12.
Various changes in the position of S are included.

Another variation of the first embodiment of Figure 1 is that of 2A, 2B,
Represented by the second embodiment shown in Figures 2C and 2D. This is shown in FIG. 2A as intermediate structure 1 containing modified probe strand P ′ and labeled polynucleotide strand 114.
Starts at 15. As in the embodiment shown in FIG. 1, the modified probe polynucleotide P'has a target binding region TBR (here about 1,100 nucleotides (1.1 Kb)).
And the 3'end binding region BS (shown here as a sequence of deoxycytidylate residues,
The binding segment BS thus comprises in this embodiment designated as homopolymer segment HS). In each of the examples below, a modified probe polynucleotide is used in which TBR was derived as part of plasmid pBR322 cloned into Bacteriophage M13mp9. TB
R is at least 205 nucleotides from the 5'end and at least 205 nucleotides from the homopolymer-type binding segment HS
Space 348 pieces. Unlike the labeled polynucleotide 14 of the first embodiment (FIG. 1), the labeled polynucleotide chain 114 of the second embodiment comprises an indirect label. That is, the biotin moiety B is pendant to a short segment of deoxyuridine nucleotide U at the 3'end of the labeled polynucleotide chain. This segment can be attached to the labeled polynucleotide chain (prior to hybridizing to the probe polynucleotide P ') by chain extension mediated by terminal deoxynucleotidyl transferase (see, for example, Ward et al., Supra). See description). The production of the modified probe P ′ and the hybridization of the probe to the labeled polynucleotide chain 114 are carried out by the procedure described in the text relating to FIG. 1 and the text of each example relating to the corresponding steps. You can Purification can be accomplished by size exclusion chromatography, which removes unhybridized labeled polynucleotide strand 114 or (optionally) unhybridized probe strand, in particular. Then a conjugate of enzyme E to avidin or streptavidin (this conjugate is shown as AE in FIG. 2)
Is added. This binds to the pendant biotin at the 3'end of the labeled polynucleotide chain 114 due to the affinity of biotin-avidin or biotin-streptavidin for pendant biotin. The combination of labeled polynucleotide chain 114 and enzyme conjugates AE is referred to as labeled polynucleotide L for this second embodiment. In a variation of the method shown here, the enzyme conjugate is reacted with the labeled polynucleotide chain 114 to allow the labeled polynucleotide L to hybridize to the probe strand P'to form the reagent complex 116. The nucleotide L can be formed.

As in the case of the reagent complex 16 shown in FIG. 1, the reagent complex 116 can be purified by conducting it to the column 17 containing the affinity ligand for the homopolymer segment attached to the probe P ′. This is shown schematically at the beginning of Figure 2C. In general, the description of the operation of such a column with respect to the first embodiment applies here as well. However, the slightly sublime complex of biotin, streptavidin (or avidin) and the enzyme is labeled polynucleotide L
Due to the above, certain precautions are required when operating the column 17. First, in order to prevent the unhybridized labeled polynucleotide L from finally entering the chromatographic product 25, one or more components of the labeled polynucleotide are attracted (and the chromatographic operation is performed). Any site on the column that causes a delay) should be eliminated as much as possible. Example 2
One way to eliminate this type of site, as shown in, is to use biotin saturated deoxyuridine, before using the column normally.
Alternatively, it may be saturated with its mono-, di-, or triphosphates or other modified forms. In the above example, biotin was less potent in the absence of uridine. The affinity column 17 can remove the probe chain P not containing the homopolymer-type binding segment HS and the unbound AE complex.

Purified reagent 25 containing reagent complex 116 and unhybridized probe strand P ', then nucleic acid (some of which (G) contains target nucleotide sequence TS)
Is added to the displacement reactor 130 along with the sample containing. This is shown schematically in Figure 2C. As shown in FIG. 1B of US Pat. Application No. 607,885, the target nucleotide sequence TS is first the target binding region T of the probe (P 'of reagent complex 116).
It will hybridize to part of the BR. As shown in Figures 1C, 1D and 1E of U.S. patent application Ser. The labeled polynucleotide is replaced from the polynucleotide P '.

Other reagents may be present in the reactor 130, as indicated for the description with respect to FIG. This is particularly the case in the specification of Williams et al. (U.S. Patent Application No. 684,30) cited above.
8) and Collins et al. (684,305).

After a suitable period, column 132 containing immobilized affinity reagent for homopolymer segment HS
Charge the displacement reaction mixture. The binding between the immobilized affinity reagent of the column 132 and the homopolymer segment HS may be irreversible, or it may be controllably reversible as described below. As with the preparative column 17 discussed in connection with FIG. 1, a series of solutions 134, 136 and 138 may be passed through the column after the substitution reaction mixture has been introduced. However, in some configurations using the disposable column 132, only the first liquid (washing liquid 134) is needed. The eluate from this single wash (or the initial eluate with a series of solutions) contains all or substantially all of the displaced labeled polynucleotide, the presence and amount of which in the sample to be analyzed. It is functionally related to the presence and amount of target nucleotide sequence TS. Quantitation may be performed, for example, by assaying the initial eluate with an enzyme reactant that produces a detectable fluorescent, absorptive or luminescent signal in a quantity that is functionally related to the presence and quantity of enzyme E in the eluate being analyzed. It can be carried out by enzymatic analysis by the method.

Column 132 with reagent complex 116 using a minimum amount of salt solution 136.
It is often desirable to wash out from the latter to obtain the late eluate. Similar enzymatic analysis of this eluate may give numerical values that quantitatively adjust and interpret the results of the previous analysis. If it is desired to reuse the column 132 without discarding it, the column 132 can be washed with the regenerant liquid 138 (and / or further with the re-equilibration liquid). A part of the sample polynucleotide G is bound (by the complementary nucleotide pairing of the target nucleotide sequence TS to the target binding region TBR), the probe P ′ is a minimum amount of salt solution 136.
Alternatively, it will elute from the column in the regenerating fluid 138 with an unchanged excess of reagent complex.

By Atsusei aliquots example per enzyme activity from the column (as shown by connexion schematically in curve 140 at the bottom of the 2D view), it is possible to observe the peak of activity P _DLP and P _RC. Here, P _DLP represents the amount of substituted labeled polynucleotide recovered from the affinity column 132 and P _RC represents the amount of reagent complex. The left end of the curve in Figure 2D corresponds to the maximum retention time. peak
P _RC and P _DLP are the elements measured at each peak (second
(Fig. D) below. It is sufficient to quantify only one of these peaks for this assay. It is preferable to quantify the peak P _DLP , since the amount of labeled polynucleotide displaced increases from 0 (or background value) in proportion to the amount of target nucleotide sequence TS present in the sample. In contrast, the peak P _RC is usually large and a relatively small amount as a function of the presence of the target nucleotide sequence TS in the sample, since a large excess of the complex is usually used above the expected level of target nucleotide sequence. It will only show a decline.

Example 1 Isotopically labeled polynucleotides and oligos (dG)
Assay for Post-Substitution Replacement Assays Using Cellulose Affinity Chromatography Probe Polynucleotides The parental cloning vehicle of the M13 series used in these examples was first constructed by Joachim Metzing et al. (Jay Metzing et al. Acade
Sai USA (Proc.Natl.Acad.Sci.USA) ”, 74
Vol., 3642 (1977), J. Metzing, "Methods in Enzymology", 101, 20-78 (198).
3 years)). These single-stranded bacteriophages were genetically engineered to contain genetic markers and various restriction sites that allow for the selective insertion of foreign DNA of interest. The use of the M13 cloning vehicle is particularly useful in the following examples, as infection of E. coli with M13 phage particles releases virions containing single-stranded phage DNA into the growth medium. The various clone numbers used herein (eg, M13mp9 clone II-16) are the identifying points for bacteriophage clones that adopted the meshing nomenclature for the parental forms of these virus strains.
The probe polynucleotide used in this example and each of the following examples was derived from recombinant DNA M13mp9 clone II-16. This M13mp9 clone II-16 DNA was produced by inserting a 1.1 Kb fragment obtained by digesting pBR322 DNA with Pst I / Bam HI restriction enzyme into replication double-stranded M13mp9. The 1.1 Kb insert is a fully mature (single-stranded) form of M13mp9 clone II-16 DNA in which the 5'end of the selected labeled polynucleotide (when hybridized) is circular or linear. Nucleotide inserts are placed adjacent to the Bam HI restriction site (or remaining portion) in the DNA. Cloned pBR322 DNA
A large amount of single-stranded M13mp9 DNA was obtained after transformation of recipient bacterial cells with M13mp9 clone II-16 containing the insert.
This single-stranded circular DNA was then exhaustively digested with the restriction enzyme Hinf I, and about 25 fragments were obtained at the time of complete digestion. Only one of these contained the sequence of interest (pBR insert). Hinf I per circular viral DNA segment
The number of fragments is estimated based on the number of Hinf I restriction enzyme recognition sites in M13mp9 DNA. pBR322Bam HI-PstI fragment is internal
It has no Hinf I recognition site. The complete digestion product fragment containing the pBR322 DNA insert is about 1.6 Kb in length, in addition to about 205 bases from the original M13mp9 DNA extending in the 5'direction of the molecule and about 348 bases extending in the 3'direction. , PBR322DNA
It consists of (1.1Kb). Hinf I of M13mp9 clone II-16 DNA
For digestion products (including fragments containing the pBR322 insert),
A homopolymer addition (“tailing”) reaction with terminal deoxynucleotidyl transferase (TdT) was then performed. This enzyme mediates the addition of deoxynucleotides to the 3'-OH end of DNA molecules. In this example, DNA (Hinf I digestion product) 60-7
By mixing 0 pmol with TdT472 units and 7.4 nmol dCTP (114-fold molar excess of dCTP over the 3'-OH end)
-"Tailed" DNA was produced. The appropriate tail forming buffer was then added and the mixture was incubated for 2 hours at 37 ° C. After incubation, EDTA was added (final concentration 10 mM) and the mixture was added. The enzyme activity was destroyed by boiling for 3 minutes and this mixture was used directly without purification to form a hybrid with the labeled polynucleotide.

Labeled Polynucleotides Labeled polynucleotides used in this example and each of the following examples are chemically synthesized oligonucleotides (27me) having a sequence of 5'-CACAGGACGGGTGTGGTCGCCATGATC-3 '.
derived from r). This unlabeled oligonucleotide was 5'end labeled with ³² P for use in these experiments.
After 5'-OH phosphorylation, it was not incorporated [γ-
³² P] ATP was removed by DEAE-Sephadex A-25 ion exchange chromatography and the labeled polynucleotide was used without further purification. The general specific activity obtained was about 3000 Ci / mmol (27mer), and a quantitative transfer was estimated.

Hybridize 5x molar excess of end-labeled 27mer to 5xSSC
Reagent complexes or hybrids were formed by mixing with the C-tailed probe polynucleotide in the presence of (0.75M-NaCl + 0.075M sodium citrate).
Generally, about 2.5 pmol of C-tailed probe polynucleotide (fragment containing pBR322 insert) DNA is end-labeled with about 27 mer.
Exposed to 12.5 pmol and kept the mixture at 50 ° C. for 1 hour. The hybrid mixture was gradually cooled to room temperature and then applied to a Sepharose® 4B (Farmasia) column (7 mm × 170 mm) and TE (10 mM Tris-
Eluted with HCl + 1 mM EDTA, pH 8.1) buffer. Eluate 2
Drops or 3 drop fractions were collected and radio-assayed. High molecular weight hybrids eluted early (near the porosity of the column) and unhybridized 27mer eluted with much longer retention times. Thus, the unhybridized labeled oligonucleotide was removed from the reagent solution. High molecular weight fraction is M13mp9 clone II-16
C-tailed Hinf I fragment, which contains the fragment carrying the pBR322 insert (probe polynucleotide) hybridized to the labeled polynucleotide, and which has not necessarily progressed to 100% hybridization. Therefore, it also contained a hybridized probe polynucleotide. Generally, a hybrid formation efficiency of 40 to 50% was observed. The volume of the high molecular weight fraction containing the hybrid is then adjusted from 600-700μ to about 100μ.
And used for subsequent displacement experiments.

Target Polynucleotides Target Polynucleotides (“analyte” and “competitor” DNs to demonstrate chain substitution in this and the following examples)
A) is a pBR322 DNA complementary to the pB322 insert in M13mp9II-16 DNA (ie M13mp9 clone II).
Single-stranded plasmid M13mp8 containing the DNA strand obtained by digesting two Pst I / Bam Hl of pBR322 DNA not found in −16)
It was clone 20 DNA. M13mp8 clone 20 DNA and M13mp9
The only region of significant homology in clone II-16 DNA was the region of the inserted pBR322 sequence which contained multiple flanking cloning site DNA sequences.

Displacement Chain displacement reactions were performed in solution with 0.17 pmol of reagent complex. The competitor DNA was added to the solution containing the reagent complex at the molar ratio shown in Table 1. DN of probe and competitor
The A concentration was determined by optical measurement of a homogeneous DNA preparation in electrophoresis. The displacement reaction was carried out in 2 × SSC at 50 ° C. in a reaction volume of 17.5μ for 3 hours. In addition to each of the above experiments, instead of homologous competitor DNA, heterologous DNA (bacterial ophage lambda Hind III digested fragment, reagent complex and
Experiments were also carried out with (1: 1 molar ratio).

After the solution replacement, the sample was diluted with 250 μM of 1M NaCl + TE buffer and passed through an affinity column containing oligo (dG) cellulose. Under these conditions of salt concentration and temperature, the modified (C-tailed) DNA shows an affinity for this column matrix by CG base pairing with an oligo (dG) affinity substance, while unmodified. DNA (including substituted labeled oligonucleotides) has no affinity and should therefore pass through the column rapidly. The oligo (dG) cellulose used in this experiment (obtained from PL Biochemicals®) consists of guanylic acid chemically linked to cellulose in a homopolymer chain of up to 20 nucleotides. 1 g of oligo (dG) cellulose is 10
It contains up to 0 mg guanylic acid and will reversibly bind 10-20 A ₂₆₀ units of poly (C) according to the supplier's instructions. Oligo (dG) cellulose was washed in TE buffer, equilibrated in 1M NaCl + TE buffer, and then as a slurry, Isolab® Quik-Sep disposable polypropylene column (volume 5 ml). (Loaded with glass microfiber discs). Each displacement was individually assessed using a column containing 0.25 g of oligo (dG) cellulose (column bed size 10 mm x 15 mm). After applying the sample, incubate the oligo (dG) cellulose affinity column for 30 minutes at 15 ℃, and 1 ml aliquots 4 times each for 1M NaCl + TE,
Elution with 0.2M NaCl + TE, TE, and finally with 0.1N NaOH was performed. All column fractions were collected and analyzed for radioactivity.

Results The results of the chain displacement experiments in this example using isotopically labeled polynucleotides and oligo (dG) cellulose affinity chromatography are shown in Table 1. Instead of the homologous competitor DNA, the heterologous bacterium Ophage lambda (H
The result of the experiment using indIII fragment) DNA showed that 7.2% of the radioactivity was eluted in the 1M NaCl + TE fraction. This was not significantly different from when no competitor DNA was used (Table 1). As seen in Table 1, the displacement measured by elution of radioactivity in the 1M NaCl + TE fraction increased as a function of the concentration of competitor DNA.

Example 2 Enzyme-Labeled Polynucleotides and Assay Probe Polynucleotides After Chain Displacement Using Oligo (dG) Cellulose Affinity Chromatography The probe polynucleotides used in this example were the same as those described in Example 1.

Labeled Polynucleotides In this example, horseradish peroxidase (HRP) enzymatic activity was employed as a measure of chain displacement. The HRP-labeled polynucleotide was first tailed with a biotinylated dUTP using the same 27-mer as in Example 1 in connection with terminal deoxynucleotidal transferase (TdT), and then a modified oligonucleotide containing biotin. Was conjugated with streptavidin-HRP (SA-HRP). Isotopically labeled 27mer (5'-
³² P) was used to obtain an independent measure for the presence of oligonucleotides in different product fractions. The terminal radiolabeled 27mer product from the phosphorylation reaction (described in Example 1) was first ethanol precipitated prior to tail formation to give a relatively salt free oligonucleotide. This is because salts interfere with the TdT enzyme. Then 27mer (10 ~
15 pmol) was mixed with biotinylated dUTP using a 50-fold molar excess of modified deoxynucleotides (about 50 units) over DNA and TdT. After addition of the appropriate tail forming buffer, the mixture was incubated for 1 hour at 37 ° C. The tail length was then examined by polyacrylamide gel electrophoresis using the 27 mer without tail as a size standard. In the first experiment, this biotin-dUTP
Using the tailed oligonucleotide as it was, a hybrid with a C-tailed probe polynucleotide similar to that described in Example 1 was prepared. Unhybridized biotinylated oligonucleotide and excess biotin-dUTP were then removed by Sepharose 4B size exclusion chromatography as described in Example 1. The high molecular weight fraction (containing hybrids) obtained from the Sepharose 4B column was then added to a 50-fold molar excess of SA.
-HRP (relative to the amount of hybrids present). The binding reaction was performed in phosphate buffered saline (pH 7.4) 3
It was performed for 30 minutes at 7 ° C. Bound material then 25
diluted to a final NaCl concentration of 1M. The mixture containing bound hybrid, unhybridized C-tailed probe polynucleotide, and unbound SA-HRP was then passed through an oligo (dG) cellulose affinity column to remove unbound enzyme. . The column protocol was essentially the same as that described in Example 1, except that HRP activity in all column fractions was measured (see below). Unbound SA-HRP and untailed probe polynucleotide are both 1M NaCl
Expected to elute in the + TE buffer fraction. The TE buffer only fractions (containing bound complex) were pooled and used in the displacement experiments described below.

In later experiments, first SA-HRP was added to biotin-dUTP.
Oligonucleotide with tail (Sephad
ex.RTM.), purified on G-25 (Falmatia)) and then prepared a hybrid with the C-tailed probe polynucleotide. Biotin-dUTP tailed oligonucleotides were prepared as above and exposed to SA-HRP at a total molar ratio of 1: 1 (total biotin: SA-HRP) without purification. The mixture was incubated at 37 ° C for 1 hour and then at room temperature overnight. The bound oligonucleotide was then mixed with the C-tailed probe polynucleotide using a 3-fold molar excess of labeled oligonucleotide. These DNAs were incubated at 37 ° C for 1 hour and then at room temperature for 3 hours.
Hybridized for a time. Na after hybrid formation
The Cl solution concentration was increased to 1 M and the reagent complex was purified by passing through an oligo (dG) cellulose column as described above. Radioactively analyze all column fractions,
HRP activity was also measured. The TE buffer only fractions (containing bound hybrid complex) were pooled and then used in displacement experiments.

Displacement Experiment The chain displacement reaction was conducted essentially in the same solution as described in Example 1. However, competitor DNA is
Molar ratios of probe polynucleotide were added at values of 0, 1, 2.5, 10 and 20. The concentrations of competitor and probe DNA were calculated as described in Example 1. 2x substitution reaction
It was carried out in SSC at 37 ° C. for 3 hours. Due to the presence of the heat-labile HRP enzyme, lower reaction temperatures and higher levels of competitor DNA were required. After the solution displacement reaction, all the samples were diluted to 250μ and the salt concentration was adjusted to 1
The reaction solution was passed through an oligo (dG) cellulose column after being increased to M.NaCl. Column conditions and buffers are in Example 1
It was the same as that described in. However, prior to use, the column was treated with biotin-dUTP to reduce non-specific binding of biotinylated DNA to the Affinity Matrix in polypropylene columns. All column fractions were collected and aliquots of each fraction were radioactivity analyzed as described in Example 1. Ortho-phenylenediamine (OPD) solution (0.1M for measuring HRP activity in column fractions)
150 μl in sodium citrate) was 150 μl 0.012% H ₂ O _{2 in} 0.1 M sodium citrate and up to 250 μl HRP-containing sample solution was added to this mixture. After gently shaking the assay solution at room temperature for 30 minutes, 2M ・ H ₂ SO ₄ 150μ
Was added to stop the reaction. The contents of the assay tubes were then analyzed for A ₄₉₀ values under undiluted or diluted conditions. If dilution was required, it was done in 0.1 M sodium citrate buffer.

Results The results of a series of chain displacement experiments demonstrated that labeled polynucleotides were displaced in an amount that was directly proportional to the increase in the amount of competitor DNA. Both HRP activity and radiolabel were tracked in all column fractions. HRP activity and radioactivity in 1M NaCl + TE buffer increased in proportion to the concentration of competitor DNA when the amount of reagent complex was kept constant. At the same time, the amount of HRP activity in the TE-only buffer fraction decreased with increasing concentration of competitor DNA in the reaction samples (data not shown).

Example 3 Isotope-labeled polynucleotide in the presence of E. coli RecA protein and assay probe polynucleotide after chain displacement using oligo (dG) cellulose affinity chromatography The probe polynucleotide used in this example is M13mp9 clone 5-5. Or M13mp19
It was derived from clone 3-2. These clones were late isolates corresponding to clone M13mp9 clone II-16 described in Example 1.

Labeled Polynucleotide Labeled polynucleotide was prepared as described in Example 1.

Hybridization Hybridization reagent complex contains 2 x S of 1 molar excess of 27-mer (labeled polynucleotide) radiolabeled with end radiolabel.
It was prepared by mixing with a C-tailed probe polynucleotide in the presence of SC. The mixture was left at 50 ° C. for 1 hour and then purified as detailed in Example 1.

Target Polynucleotide The target polynucleotide used in this example was circular M13mp8 clone 20C-2. This clone was a late isolate corresponding to M13mp8 clone 20 described in Example 1.

Chain displacement reaction was carried out using 0.14 pmol of the hybridization reagent complex. The competitor DNA was added to the solution containing the hybrid reagent complex at the ratio shown in Table 2. Each reaction also contained 6.4 μg of recA protein (purified from E. coli; commercially available from Bethesda Research Laboratories (Gaitersburg, MD)). The displacement reaction is 1 × recA buffer (20 mM Tris-HCl, pH 7.5, 25
mM ・ MgCl ₂ , 2 mM dithiothreitol, 100 μg / ml BSA, 2
mM.ATP) at 37 ° C. for 1 hour in a reaction volume of about 20 μ.

The concentrations of competitor and probe DNA were calculated as described in Example 1. The amount of recA protein added to each reaction solution was about 1200: 1 (mol), and the recA protein was in excess of the probe DNA.

After replacement, each sample was treated with oligo (dG) as described in Example 1.
It was connected to a cellulose affinity column.

Results The results of the chain displacement reaction using the isotope-labeled polynucleotide in the presence of recA protein are listed in Table 2.

Similarly, heterologous DNA (circular M13mp without pBR322 insert)
8DNA) at a molar ratio of 5: 1 (M13mp8DNA: reagent complex)
9.0% radioactivity was generated in the 1M NaCl + TE buffer fraction.

Example 4 For reference, Example 2 of the application, entitled "Diagnostic Reagents, Kits and Methods for Use in Polynucleotide Substitution, Separation, Enzymatic Cleavage, and Adenosine Phosphate Detection," by Calvin P. Etch Barry et al. To quote. The experiment is summarized in Table 3.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing one embodiment of the method of the present invention for producing the reagent complex according to one aspect of the present invention. FIG. 2A is a schematic diagram showing the morphology of precursor 115 to the reagent complex of the present invention. FIG. 2B is an enlarged view of the reagent complex 116 prepared by adding the streptavidin-enzyme conjugate (AE) from the precursor 115 of FIG. 2A, compared to FIG. 2A. FIG. 2C shows the purification of the reagent complex according to one embodiment of the separation step (c1) of the method of the present invention, and shows the target nucleotide sequence (TS) in the nucleic acid sample (G) according to one embodiment of the method of the present invention. 6 is a schematic diagram showing the use of reagent complex 116 for detection. Figure 2D is a schematic graphical representation of enzymatic reaction output from an aliquot taken from column 132 of Figure 2C,
And graphically the molecular forms found in the corresponding aliquots; this figure shows the peak for the reagent complex (P _RC ) and the peak for the displaced labeled polynucleotide (P _DLP ). Therefore, FIG. 2D shows the output of one embodiment of the measuring step (c2) of the method of the present invention. The symbols in each figure represent the following. P (10): probe polynucleotide TBR: target binding region LBR: labeled polynucleotide binding region L (14,114): labeled polynucleotide P '(12): modified PBS: binding segment T: labeled HS: homopolymer segment B: biotin U: uridine nucleotide AE: avidin-enzyme conjugate TS: target nucleotide sequence 16,116: reagent complex 17,132: column 18,20,22,134,136,138: eluent 24: P + L 25: product fraction 115: P ' ＋ L 130: Displacement reactor 140: Activity curve

 ─────────────────────────────────────────────────── ——————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————— Photo: —--; Park Lane 30 (72) Inventor Calvin Purdy Full Valley New Jersey, USA 07830, Callifon, AR Earl 3, Box 54

Claims (10)

[Claims] 1. A probe nucleotide capable of base pairing to a target nucleotide sequence via a hydrogen bond of a purine / pyrimidine base pair, and (ii) via a hydrogen bond of a purine / pyrimidine base pair,
A labeled polynucleotide bound by base pairing to a probe polynucleotide in a region of the probe polynucleotide that is at least partially coextensive with the region in which the probe polynucleotide can base pair with the target nucleotide sequence , A diagnostic reagent complex for determining the presence of a predetermined target nucleotide sequence in a nucleic acid of a biological sample, wherein a possible base pairing between the target nucleotide sequence and the probe polynucleotide is a reagent complex From the region where the probe polynucleotide can base pair with the target nucleotide sequence (ie, at intervals), the probe polynucleotide comprises a binding segment; Nukure This coupling segment of tides, under conditions that keep the labeled polynucleotide is bound to the probe polynucleotide, a diagnostic reagent complex, wherein capable of reversibly bound by Afuinitei reagent. 2. The diagnostic reagent according to claim 1, wherein the binding segment is a homopolynucleotide segment. 3. The homopolynucleotide segment comprises poly (dC), poly (dG), poly (dT), poly (dU), poly (biotin-dU), poly (rC), poly (rC), and poly ( rU) selected from the group consisting of
The diagnostic reagent according to the item. 4. A probe polynucleotide comprising a target binding region capable of base pairing to a target nucleotide sequence, and a labeled polynucleotide binding region bound to a base of a labeled polynucleotide in a complex by purine / pyrimidine base pair binding, and The diagnostic reagent according to claim 2, wherein the labeled polynucleotide-binding region is contained in the target-binding region. 5. The labeled polynucleotide comprises oxygen covalently bound to an affinity moiety, and the nucleotide of the labeled polynucleotide is bound to an enzyme via an affinity moiety and a ligand moiety to which the affinity moiety is specific. The diagnostic reagent according to claim 1. 6. (a) (i) a probe polynucleotide capable of base pairing to a target nucleotide sequence via a purine / pyrimidine base pair hydrogen bond; and (ii) a purine / pyrimidine base pair hydrogen bond. ,
Providing a reagent complex of labeled polynucleotide bound by base pairing to the probe polynucleotide in a region of the probe polynucleotide that has at least a portion in common with the region in which the probe polynucleotide can bind to the target nucleotide sequence. (B) contacting the reagent complex with a sample in solution under conditions which bind the probe polynucleotide when the target nucleotide sequence is present and displace the labeled polynucleotide from the complex; (c1) contacting step (b ) After which the remaining reagent complex is separated from a portion of the reaction solution, and (c2) in the nucleic acid of the biological sample consisting of the step of measuring the presence of the substituted labeled polynucleotide in the solution phase after separation. A method for determining the presence of a predetermined target nucleotide sequence of The probe polynucleotide comprises a binding segment distal to the region where the lobe polynucleotide can base pair with the target nucleotide sequence; the separating step (c1) comprises immobilizing the solution product of the contacting step (b) to the binding segment. The immobilized affinity reagent is passed in contact with the immobilized affinity reagent, and the solution phase substantially free of the polynucleotide containing the binding segment is recovered from the immobilized affinity reagent, and the measurement step (c2) is performed on this solution phase. At that time, the binding between the immobilized affinity reagent and the reagent complex is reversible. 7. The binding segment is a homopolynucleotide segment and the affinity reagent is appended with a repeating segment of nucleotides that is complementary to the nucleotides of the homopolynucleotide segment.
A method according to claim 6 which is a matrix. 8. The labeled polynucleotide comprises an enzyme covalently bound to an affinity moiety, and the nucleotide of the labeled polynucleotide is bound to the enzyme via an affinity moiety and a ligand moiety to which the affinity moiety is specific. The method according to claim 6. 9. (a) (i) a probe polynucleotide having a region capable of base pairing to a target nucleotide sequence via a purine / pyrimidine base pair hydrogen bond, and (ii) a purine / pyrimidine base pair hydrogen bond. Through
Hybridizing a labeled polynucleotide capable of base pairing to a probe polynucleotide in a region of the probe polynucleotide having at least a portion in common with a region capable of base pairing to the target nucleotide sequence; Includes a binding segment distal to a region where the probe polynucleotide can base pair with the target nucleotide sequence; (b) binding the reagent complex to the probe polynucleotide by selective reversible adsorption at the binding segment. A method for producing a diagnostic reagent useful for determining the presence of a predetermined target nucleotide sequence in a nucleic acid of a biological sample, the method comprising separating the labeled polynucleotide from the unlabeled polynucleotide. 10. The binding segment is a homopolynucleotide segment, and the hybridization reaction mixture in the separation step (b) contacts the homopolynucleotide segment of the probe polynucleotide with a complementary immobilized homopolynucleotide segment. A method according to claim 9 comprising: