JP2003527119A - Methods for detecting nucleic acid differences - Google Patents

Abstract

(57) Abstract The present invention provides a method for detecting the presence or absence of a difference between two related nucleic acid sequences. In this method, a target nucleic acid and a reference nucleic acid are brought into contact with a movable branch structure under conditions capable of forming a 4-way nucleic acid complex. Under contacting conditions, if the reference nucleic acid and the target nucleic acid are the same, proceed until branch transfer is complete and complete strand exchange occurs. If the reference nucleic acid and the target nucleic acid are different, branch transfer does not proceed until completion, resulting in a stable 4-way complex. Detection of a stable 4-way complex determines the presence of differences between nucleic acids. Stable four-way complexes are detected by gel electrophoresis or specific isolation of stable four-way complexes using molecules that specifically bind to such complexes.

Description

Detailed Description of the Invention

1. FIELD OF THE INVENTION The present invention relates to the field of molecular biology, more particularly nucleic acid hybridization,
Holiday junction formation and branch migration (bra
nch migration). In one aspect, the invention provides methods and reagents for detecting the presence of differences between two related nucleic acid sequences. In a preferred embodiment of the invention, the difference is a mutation, eg a point mutation, deletion or insertion.
Practical applications of the invention include, but are not limited to, genotyping, discovery and detection of single nucleotide polymorphisms, characterization and quantification of polynucleotides, mutation rate detection, gene expression analysis. . Furthermore, the present invention can distinguish between homozygous and heterozygous genetic modifications.

2. BACKGROUND OF THE INVENTION The propensity of nucleic acids to selectively and specifically bind complementary nucleic acids has been exploited in the development of myriad nucleic acid hybridization techniques. Such techniques are not only useful for detecting complementarity and / or identity of nucleic acid sequences (eg, quantification of differential gene expression levels such as Northern blots, Southern blots, and gene expression chips / microarrays). In some cases, these have been used to detect differences in related nucleic acid sequences.

Current allele-specific hybridization-based genotyping techniques suffer from the inaccuracy due to their low specificity. In particular, current genotyping techniques based on polynucleotide hybridization are insufficiently specific to distinguish or identify single nucleotide polymorphisms. For example, a specific DNA strand / oligo containing one version of a specific SNP is not only a perfectly matched complementary DNA, but also a non-perfect match that contains a second version of a specific SNP. Can also hybridize. Hybridizations between two perfectly matched complementary DNA strands include those that are not perfectly matched complementary strands (those with single or multiple base pair mismatches between the two complementary strands). ), But the single base pair difference is usually too small, so the SN
Not specific enough to assess P. In contrast, hybridization between a specific cDNA and its corresponding oligo / DNA fragment immobilized on a chip / microarray is due to the fact that it is very specific and gene expression chips and Microarrays are often more specific / accurate than SNP chips / microarrays. Such hybridization does not necessarily depend on a single base pair difference between the two nucleic acids. The method disclosed herein uses highly specific allele-specific Holliday structure formation for nucleic acid hybridization techniques (
This problem is addressed by combining with, for example, a gene chip / microarray). As a result, SNP chips / microarrays can achieve the same level of specificity / accuracy as gene expression chips / microarrays.

[0004] Panyutin IG et al., 1993 paper (Panyutin IG, Hsieh P, single base mismatch interferes with spontaneous DNA branch migration, (1993) J. Mol. Biol., 230: 413-24).
In, a single-stranded oligo that is completely (or partially) complementary to a specific portion of single-stranded M13mp18 viral DNA anneals to this viral DNA and has one (or two) tails and moieties at each end. Form a target double strand. The partial double-strand formed by the oligo and M13mp18 viral DNA then forms an invasive double-strand with one (or two) complementary tails and a four-way Holliday-like structure. Can be formed.
The 4-way holiday-like structure then undergoes branch migration away from the tail (which cannot branch to the tail due to the energy barrier; existing H-bonds without forming new H-bonds). To destroy). Regarding the Holliday structure formed between the partial double strands of a single tail, oligo / M13mp18 partial single strand between the double strand portion of the double strand and the double strand portion of the invasion portion double (or Multiple)
The difference in the base pair of irrespective of presence or absence of Mg ⁺⁺ provides a sufficient energy barrier (2H-bond → 0H-bond) to prevent branch migration and release of annealed oligos. Regarding the Holliday structure formed between the partial double strands having two tails, the single base pair difference between the double-stranded portion of the oligo / M13mp18 partial double strand and the invading double strand ( Substitutions, deletions or insertions), in the presence of Mg ⁺⁺ , provide a sufficient energy barrier to prevent branch migration and release of only the annealed oligos.

One proposed method for detecting differences in related nucleic acid sequences forms a complex containing Holliday junctions between related sequences. In this method, described in US Pat. No. 6,013,439, each member of at least one pair of non-complementary strands within the complex is labeled. The two labels produce a signal depending on the labels in the vicinity of each other. Differences between related nucleic acid sequences stabilize the Holliday junction,
Thus the labels are placed in close proximity to each other and produce a signal. On the other hand, if there is no difference between the two sequences, the Holliday junction is not stable and the complex dissociates into two strands, breaking the close proximity between the two labels and weakening the signal.
Whether a stabilized complex was formed was measured and its presence indicates a difference between related sequences.

[0006] One problem with the above methods of detecting differences in related nucleic acid sequences is the use of labeled PCR primers to create the labeled nucleic acid strands necessary for the detection of Holliday junction complexes. The use of labeled primers becomes problematic, especially when the nucleic acid targeted for analysis is genomic DNA, due to the concentration of primer that must be used and subsequent interference that occurs in the presence of high levels of labeled primer. That is. This is a major drawback as one of the major practical applications of this method is the analysis of genomic DNA. Moreover, the preparation and use of labeled nucleic acids is costly and inconvenient,
Thus, it would be preferable that available methods be available to measure sequence differences that do not require the use of labeled polynucleotides or primers.

The present invention addresses the problems associated with the use of labeled polynucleotides and primers by providing new methods and reagents for the rapid and efficient identification of related nucleic acid sequence differences. Therefore, the present invention represents a highly desirable and practical addition to fields including molecular biology and medicine.

Based on the findings of Panyutin IG and Hsieh P, we have enabled the combination of genotyping assays (tens of thousands of SNPs / mutations can be measured in one assay reaction) and A genotyping method was designed that eliminated the need. As a result, our method may significantly reduce cost and improve genotyping throughput.

3. SUMMARY OF THE INVENTION In one aspect, the invention provides a method of detecting the presence or absence of a difference between two related nucleic acid sequences. The method achieves high enough sensitivity to detect the presence of any differences between nucleic acids, even single nucleotide polymorphisms. In this method, a target nucleic acid and a reference nucleic acid are contacted with a movable branch structure under conditions capable of forming a 4-way nucleic acid complex. Under the contact conditions, if the reference and target nucleic acids are identical, branch migration proceeds to completion and complete strand exchange occurs. If the reference and target nucleic acids are different, branch migration does not proceed to completion and a stable 4-way complex is obtained. Detection of stable 4-way complexes establishes the presence of differences between nucleic acids. Stable 4-way complexes are detected by gel electrophoresis or specific isolation of stable 4-way complexes with molecules that specifically bind to such complexes.

In certain embodiments, the invention provides methods for detecting the presence of differences between two related nucleic acid sequences. The method involves forming a 4-way complex that includes both double-stranded nucleic acid sequences, wherein the nucleic acid within the 4-way complex is unlabeled or forms a 4-way complex. One of the four nucleic acid strands carries a label and subject the four-way complex to conditions under which branch migration occurs, wherein if there is a difference between the two related nucleic acid sequences, the four-way complex The branch migration in it ceases and the 4-way complex continues to exist as a stable 4-way complex; and here, if there is no difference between the two related nucleic acid sequences, a complete strand exchange occurs and 4-way Branch migration in the 4-way complex continues until the complex is decomposed into two double-stranded nucleic acids, and the 4-way complex after branch migration or its degraded double-stranded product is converted into the 4-way complex. Allows specific binding of reagents to the body Subjected to conditions, and the binding of the reagent to the 4-way complex produces a detectable signal, the signal produced by the specific binding of the reagent to the 4-way complex is detected, and the signal Is associated with the presence of the difference between the nucleic acid sequences, and the undetectable signal is associated with no difference between the nucleic acid sequences.

In another embodiment, the invention provides a method of detecting the presence of a difference between two related nucleic acid sequences. The method involves forming a 4-way complex that includes both double-stranded nucleic acid sequences, wherein the nucleic acid in the 4-way complex is unlabeled or 4
One of the four nucleic acid strands forming the way complex has a label, and the four way complex is subjected to conditions under which branch migration occurs, where a difference exists between the two related nucleic acid sequences. , The branch migration in the 4-way complex ceases and the 4-way complex continues to exist as a stable 4-way complex; and where there is no difference between the two related nucleic acid sequences, the complete strand The exchange occurred and the 4-way complex decomposed into two 2
Branch migration in the 4-way complex continues until it becomes a double-stranded nucleic acid, and the 4-way complex after branch migration or its degraded double-stranded product is converted into a 4-way complex (holiday DNA) from double-stranded DNA. Subject to conditions that allow separation of the structure) and isolation of the 4-way DNA complex. The DNA fragment containing the 4-way complex can then be used as a probe for the evaluation of SNPs via hybridization with oligo / DNA fragments immobilized on a chip / microarray.

[0012] 4. 4. Brief description of the drawings (see below for description of the drawings) BRIEF DESCRIPTION OF THE TABLES Table 1 illustrates example methods for genotyping diploid genomic DNA samples using PCR.

6. Detailed Description of the Preferred Embodiments As explained in the background section, conventional methods for detecting the presence of differences between nucleic acids are imprecise, low throughput and expensive.

As detailed below, the present invention overcomes these and disadvantages by providing a novel method for detection of differences between nucleic acids. The method of the present invention exhibits improved accuracy and efficiency compared to conventional methods for detecting differences between two nucleic acid sequences.

6.1 Abbreviations As used throughout this specification, abbreviations for nucleic acids containing specific nucleobase sequences are:
The conventional one-letter abbreviation is used. That is, the naturally occurring coding nucleobases when contained in a nucleic acid are omitted as follows: adenine (A), guanine (G).
, Cytosine (C), thymine (T), and uracil (U). Also, unless otherwise stated, nucleic acid sequences shown as a sequence of one-letter abbreviations are shown in the 5 '→ 3'direction.

6.2 Definitions As used herein, the terms “nucleic acid” and “polynucleotide” are synonymous, and even deoxynucleosides or ribonucleosides, phosphodiester bonds or modified bonds such as phosphotriesters, phosphoramidites, Siloxane,
Carbonate, carboxymethyl ester, acetamidite, carbamate,
By thioether, bridged phosphoramidites, bridged methylenephosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, bridged phosphorothioates, or sulfone linkages, and combinations of such linkages are meant any nucleic acids.

The terms nucleic acid, polynucleotide, and nucleotide also specifically include nucleic acids composed of bases other than the five biological bases (adenine, guanine, thymine, cytosine, and uracil). For example, the polynucleotide of the present invention is not particularly limited, but includes 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl). Uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine (beta-D-galactosylqueosine
), Inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine , 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-
Thiouracil, β-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil, queosine, 2
-Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3
It may contain at least one modified base moiety selected from the group consisting of-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.

Furthermore, the polynucleotide of the present invention is not particularly limited, but includes arabinose,
It may comprise at least one modified sugar moiety selected from the group consisting of 2-fluoroarabinose, xylulose, and hexose.

The present invention is not limited by the source of the polynucleotide. The polynucleotide may be a human or non-human mammal, or other organism, or of recombinant origin, in vitro synthesis, or chemical synthesis. Nucleotides are DNA, RNA
, CDNA, DNA-RNA, hybrids or any mixture thereof, and may exist in double-stranded, single-stranded or partially double-stranded form. The nucleic acids of the present invention may contain the nucleic acids and fragments thereof in purified or unpurified form, including genes, chromosomes, plasmids, and biological materials (eg, bacteria, yeast, viruses, viroids, molds, fungi, etc.). Microorganisms, plants, animals, humans, etc.).

Nucleic acid can be the only minor fraction of a complex mixture, such as a biological sample. Nucleic acids can be obtained from biological samples by methods known in the art.

The polynucleotides of the invention may be derivatized or modified, for example, by biotinylation, amine modification, alkylation, or other similar modification, depending on the purpose of detection.

In some circumstances, the present invention may use nucleic acids having modified intranucleoside linkages, eg, where high nuclease stability is desired. For example, phosphonate phosphorothioate, phosphorodithioate, phosphoramidite methoxyethyl phosphoramidite, formacetal, thioformacetal, diisopropylsilyl, acetamidite, carbamate, dimethylene-sulfide,
Dimethylene-sulfoxide, dimethylene-sulfone, 2'-O-alkyl, and 2'-
Methods for synthesizing nucleic acids containing deoxy-2'-fluorophosphorothioate intranucleoside linkages are known in the art (Uhlman et al., 1990, Chem. Rev. 90: 543-584;
See Schneider et al 1990, Tetrahedron Lett. 31: 335, and references cited therein).

The term “oligonucleotide” refers to relatively short single-stranded polynucleotides, usually of synthetic origin. Oligonucleotides generally include sequences that are 8-100 nucleotides, preferably 20-80 nucleotides, and more preferably 30-60 nucleotides in length. Various methods are used to prepare the oligonucleotides used in the present invention. Such oligonucleotides can be obtained by biosynthesis or chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis is a more economical method compared to biosynthesis. Besides economics, chemical synthesis provides a convenient way to incorporate low molecular weight compounds and / or modified bases during the synthetic process. Furthermore, chemical synthesis is flexible in the choice of target polynucleotide binding sequence length and region. Oligonucleotides can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers. Chemical synthesis of DNA on a suitable modified glass or resin yields DNA covalently bound to the surface. This has advantages in washing and sample handling. For longer sequences, standard replication methods used in molecular biology, such as J. Messing (1983) Methods Enzymo
M13 for single-stranded DNA is used as described in I., 101, 20-78. Other methods of oligonucleotide synthesis include phosphotriester and phosphodiester methods (Narang et al. (1979) Meth. Enzymol. 68:90) and on-support synthesis (Beaucage et al.
1981) Tetrahedron Letters 22: 1859-1862) and phosphoramidite method (Ca
ruthers, MH et al., "Methods in Enzymology", Vol. 154, pp. 287-314 (1988),
And other methods ("Synthesis and Applications of DNA and RNA," SA Narang
Ed., Academic Press, New York, 1987, and references contained therein.

Oligonucleotide “primers” are used in chain extension (eg, nucleic acid amplification) on a polynucleotide template. Oligonucleotide primers are usually single stranded synthetic oligonucleotides and contain at their 3'ends a hybridizable sequence capable of hybridizing to a defined sequence of a target or reference polynucleotide. Generally, the hybridisable sequence of an oligonucleotide primer is at least 90%, preferably 95% of the defined sequence or primer binding site.
%, Most preferably 100% complementarity. The number of nucleotides in the hybridizable sequence of the oligonucleotide primer should be such that the stringent conditions used to hybridize the oligonucleotide primer prevent excessive random nonspecific hybridization. Is.
Generally, the number of nucleotides in the hybridizable sequence of the oligonucleotide primer is at least 10 nucleotides, preferably at least 15 nucleotides, and preferably 20-50 nucleotides. Further, the primer may have at its 5'end a sequence having 1-60 nucleotides, 5-30 nucleotides, or preferably 8-30 nucleotides, which does not hybridize to the target or reference polynucleotide.

The term “sample” means a material suspected of containing a nucleic acid of interest. Such samples include biological fluids (eg, blood, serum, plasma, saliva, lymph, semen, vaginal mucosa, stool, urine, spinal fluid, etc.); biological tissues (eg, hair and skin); and so on. Other samples include plant, food, forensic samples (eg, paper, fiber, scrap, water, sewage, drugs, etc.), such as cell cultures. If necessary, the sample is pretreated with reagents to liquefy the sample and / or release nucleic acids from the binding agent. Such pretreatments are known in the art.

The term “amplification”, when applied to nucleic acids, refers to any method that forms one or more copies of a nucleic acid, preferably the amplification is exponential. One such method of enzymatic amplification of specific sequences of DNA is described by Saiki et al. (1986) Science, 230: 1350-54.
Known as the polymerase chain reaction (PCR) as described in. The primers used for PCR vary in length from about 10 to 50 or more nucleotides, but are generally selected to be at least about 15 nucleotides to ensure sufficient specificity. It The double-stranded fragments produced are called "amplicons" and vary in length from as low as 30 nucleotides to as long as 20,000 or more.

The term “chain extension” means the extension of the 3 ′ end of a polynucleotide by the addition of nucleotides or bases. Chain extension in the context of the present invention is generally template dependent, ie the nucleotides added are determined by the sequence of the template nucleic acid to which the extended chain is hybridized. The chain extension product sequence produced is complementary to the template sequence. Generally, in the present invention, chain extension is preferably Thermis acquaticus (Taq polymerase), Thermococcus litoralis, and Pyrococcus furyosis (Pyrococcus).
catalyzed by a thermostable DNA polymerase, such as an enzyme from furiosis).

A “Holiday junction” is a branch point in a 4-way junction in a complex of two related (often identical) nucleic acid sequences and their complementary sequences. This junction undergoes branch migration to dissociate into two double-stranded sequences, with sequence identity and complementarity extending to the ends of the strands. Holiday junctions, their formation and branch migration are concepts known to those skilled in the art, for example Whitby et al.
Mol. Biol. (1996) 264: 878-90 and Davies and West, Current Biology (1998).
8: 727-27.

“Branch migration conditions” are conditions under which the migration of the Holliday junction branch proceeds along the constituent polynucleotide chains. In the practice of the present invention, conditions are generally chosen such that migration will proceed only if strand exchange does not cause a mismatch. When a single base mismatch is formed, branch migration is impeded, resulting in a stabilized Holliday junction or Holliday junction complex. Suitable conditions are, for example, Panyutin and Hsieh (1993) J. Mol. Biol., 23
0: 413-24. In some applications, conditions will need to be modified based on the nature of the particular polynucleotide involved. Such changes can be readily recognized by those of ordinary skill in the art without undue experimentation.

A “stabilized” Holliday junction is one in which the stabilized Holliday junction is detectable and is distinguishable from double-stranded DNA released by a branch migration from a Holliday junction containing the same sequence. Mismatch is a junction that has stopped branch migration, enough.

A “complex” containing a Holiday Junction means a complex of four nucleotide chains linked via a Holliday Junction, due to the difference in the two double-stranded sequences and their complements. Degradation into two double-stranded sequences can be prevented.

Two nucleic acid sequences are “related” if they are (1) identical to each other or (2) there are no differences in the sequences which distinguish the two nucleic acid sequences from each other. "
is doing. The difference may be a substitution, deletion, or insertion of any single or contiguous nucleotide in the sequence. Such a difference is referred to herein as a "difference between two related nucleic acid sequences". Related nucleic acid sequences often differ from each other by a single nucleotide. Related nucleic acid sequences generally contain at least 15 identical nucleotides to each other at each end, but may have different lengths,
Further, it may have an intervening sequence that differs depending on at least one nucleotide.

The term “mutation” means a change in the sequence of nucleotides of a normally conserved nucleic acid sequence that results in the formation of a mutant that distinguishes it from the normal (unchanged) or wild-type sequence. To do. Mutations are usually divided into two major classes,
Namely base pair substitution and frameshift mutation. The latter requires the insertion or deletion of one to a few nucleotide pairs. Single nucleotide differences can be important with respect to phenotypic normality or abnormality, eg, sickle cell anemia.

“Double-stranded” is a double-stranded nucleic acid sequence that contains two complementary sequences that have annealed to each other. A "partial duplex" is a portion of one strand that is complementary to another strand and can be annealed to form a partial duplex, but a full length strand is not. , A double-stranded nucleic acid sequence forming a single-stranded polynucleotide tail at least at one end of the partially double-stranded.

The terms “hybridization”, “binding” and “annealing” in a polynucleotide sequence are used synonymously herein. The ability of two nucleotide sequences to hybridize to each other is based on the degree of complementarity of the two nucleotide sequences, which is based on the percentage of matched complementary nucleotide pairs. The more nucleotides that are complementary to each other in a sequence, the more stringent the conditions of hybridization will be and the more specific the binding of the two sequences will be. Increased stringency is generally accomplished by increasing temperature, increasing the proportion of cosolvents, decreasing salt concentration, and other methods known in the art.

In the two sequences, one of the sequences binds to the other sequence by antiparallel sense, the 3 ′ end of each sequence binds to the 5 ′ end of the other sequence, and each A, T ( Two sequences are “complementary” if U), G, and C bind to T (U), A, C, and G, respectively, of the other sequence.

“Small organic molecule” has a molecular weight of less than about 1500, preferably 100-1000, more preferably 300-600, such as biotin, digoxigenin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding. Molecules, and haptens. Small organic molecules provide a means for attaching the nucleotide sequence to a label or support.

6.3 Methods for Detecting Differences Between Nucleic Acids The present invention is universal and whether the differences are known a priori or not.
Differences between two related nucleic acid sequences can be detected. Such differences may be any mutations within the nucleic acid sequence, such as single or multiple base substitutions or polymorphisms, deletions or insertions. The method of the present invention is quick and convenient, easy to automate, and can be performed in the same or different formats. The method of the invention is ideally suited for rapid mutation prescreening and genotyping, especially including the identification of single nucleotide polymorphisms (SNPs). The disclosed method is sensitive and quantitative and is particularly suitable for applications utilizing the polymerase chain reaction (PCR).

In general, the present invention provides methods and reagents useful for detecting differences between two related nucleic acid sequences by determining whether the construct containing the sequences is capable of forming a stabilized Holliday junction. To do. The invention provides methods and reagents for making such constructs used in the practice of the invention, and methods and reagents useful for stabilizing and detecting Holliday junctions. For example, in some embodiments of the invention, stabilized Holliday junctions are detected with one or more binding proteins capable of specifically binding to Holliday junctions. While illustrating the invention and disclosing specific embodiments of the invention that enable those skilled in the art to practice the invention, they are not intended to limit the scope of the invention.

6.3.1 Nucleic Acids The present invention is a novel method of detecting differences between two polynucleotide sequences, comprising a polynucleotide construct comprising those sequences, as described in FIG. It is provided by forming a stabilized holiday structure. For purposes of describing the invention, it will be convenient to have the two sequences being compared, the target sequence and the reference sequence. The reference sequence is generally a polynucleotide of substantially known sequence, and the target sequence is the relevant sequence for which it is preferable to detect if there is a difference compared to the reference sequence.

Sequences are related in the sense that they are identical, or can be identical if there is no difference between the two sequences. In preferred embodiments of the invention, the differences are substitutions, deletions or insertions changes or mutations, including but not limited to single nucleotide polymorphisms (SNPs). In general, the target sequence and the reference sequence are prepared as a pair of sequences capable of forming a partial duplex according to the methods detailed below.

A generic partially double-stranded A ′ is illustrated in FIG. The partially double-stranded A'comprises a complementary double-stranded region and one or more tail regions. The complementary double stranded region contains the target or reference sequence annealed to its complement. Other examples of partially double-stranded are A ″
, B ′ and B ″.

In the partially double-stranded A ′, one tail region is the oligonucleotide tail T1.
And including T2 '. Similarly, the second tail region comprises the oligonucleotide tails T3 'and T4. The tails T1, T2 ', T3', and / or T4 can be attached to the target sequence via any linkage known to those of skill in the art of polynucleotide binding. They are,
It can be attached directly via a covalent bond or a linker. The linker may be a polynucleotide or any other linker known to those of skill in the art. Preferably tails T1 and / or T3 ′ are linked directly to the target sequence via a phosphodiester bond. Similarly tails T1, T2, T3, T4, T1 ', T2', T3 'and / or T4'
Can bind to a target or reference sequence.

In one embodiment of the invention, the partially double-stranded has one tail region.
For example, if T1 is 0 bp and T2 'is 0 bp while T3'> 0 bp and T4> 0 bp (or T1> 0 bp and T2 '> 0 bp, while both T3' and T4 are 0 bp. Partially double-stranded A'has one tail region. In another embodiment of the invention, the partially double-stranded has two tail regions. For example, the partially double-stranded chain depicted in FIG. 1 has 2 when T1, T2 ′, T3 ′ and T4 are all greater than 0 bp in length.
It has two tail areas. Tail T1, T2 ', T3', and T4 are preferably 5 bp-
It is 500 bp, more preferably 5 bp to 55 bp.

All four tails consist of sequences unrelated to each other and to the template DNA,
Or a pair of polynucleotide tails (T1 / T2 'or T3) at each end of the partially double-stranded chain.
'/ T4) may be a template DNA sequence. Preferably, the tail is capable of hybridizing to a target sequence, a reference sequence or another sequence capable of complementing the tail without interference from other tails.

In order to form a 4-way structure, with the same target sequence and its corresponding reference sequence,
Two or more partially double-stranded are prepared. For example, the partially double-stranded chain illustrated in FIG.
A ′ and B ″ can form a 4-way structure under appropriate conditions. In Figure 1, the partially double-stranded A'includes tails T1, T2 ', T3', and T4. Another partially double-stranded B ″ is a pair of polynucleotide tails at each end of the partially double-stranded, including tails T1 ′, T2, T3, and T4 ′ (eg, T1 / T2 ′, T2). / T1 ', T3' / T4, T3 / T4 ') are not complementary and do not anneal to each other under applicable conditions. However, the tail T3 'at the right end of the partially double-stranded A'is complementary to the tail T3 at the right end of the partially double-stranded B ",
Therefore, it can hybridize. The tail T4 at the right end of the partially double-stranded A'is
It is complementary to the tail T4 'at the right end of the partially double-stranded B "and can therefore hybridize. The leftmost tail T1 of the partially double-stranded A'is complementary to the leftmost tail T1 "of the partially double-stranded B" and can therefore hybridize. Partial 2
The leftmost tail T2 'of the main strand A'is complementary to the leftmost tail T2 of the partially double-stranded B "and can hybridize.

6.3.2 Preparation of Nucleic Acids The partial duplexes described above can be prepared by any method known to those of skill in the art for preparing polynucleotides or nucleic acids. For example, partially double-stranded can be prepared by standard recombinant, synthetic or PCR methods, or a combination thereof. In addition, partially double-stranded or portions thereof (eg, target or reference sequences) can be isolated from natural sources. One example of a method of preparing sequences capable of forming partially double-stranded is described in US Pat. No. 6,013,439, which is incorporated herein by reference. In a preferred embodiment of the invention, the PCR method is used to create the partial duplex. FIG. 1 illustrates the preparation of partially double-stranded A ′, A ″, B ′ and B ″ by PCR. Partial duplexes such as A ′ and B ″ can be used to detect differences between the target and reference sequences.
A may be the target sequence and B may be the reference sequence or vice versa.

To determine if there is a difference between two related DNA sequences, a standard set of primers consisting of one or more forward primers and two reverse primers R1 and R2 is used to standardize Double-stranded A and B are amplified separately or together by simple PCR. R1 and R2 have the same 3'end (r ') that hybridizes to the same part of the template DNA.
= R1 '= r2') or the 3'ends of R1 and R2 can hybridize to different parts of the template DNA (r1 '≠ r2'). As illustrated in Figure 1, forward primer F1 or forward primer F2 can be used in the PCR reaction. If the forward primer F1 is used, double-stranded (with T1 / T1 'tail)
For example, A1) is created. If the forward primer F2 is used, a duplex with a T2 / T2 'tail (eg A2) will be created. The two forward primers can also be used to create a partial duplex at the end corresponding to the forward primer. For example, the use of forward primers F1 and F2 in the same PCR reaction creates a sequence that can be used to produce partially double-stranded A ′ and A ″. In addition, a tailless forward primer can be used to create a tailless duplex at the end corresponding to the forward primer.

It should be noted that, in general, the primers need not be labeled, although in some applications it may be preferable to label one or more primers. In a preferred embodiment of the invention, the distance between the binding sites of the forward and reverse primers is at least 1 base pair, preferably 1-600 base pairs, depending on the desired purpose of the polynucleotide of interest and its nature. Alternatively, it is in the range of 5 to 600 base pairs, more preferably 5 to 100 base pairs.

The PCR amplification of the present invention involves temperature cycling of double-stranded denaturation, oligonucleotide primer annealing, and primer extension with a thermostable template-dependent nucleotide polymerase. The temperature of the method for amplification by PCR is generally in the range of about 50 ° C to 100 ° C, more usually about 60 ° C to 95 ° C. The hybridization step uses relatively low temperatures of about 50-80 ° C, while
Denaturation is carried out at a temperature of about 80 ° C to 100 ° C and extension is carried out at a temperature of about 70 ° C to 80 ° C, usually about 72 ° C to 74 ° C. Generally, the process is run for about 10 seconds to 10 minutes per cycle, with up to 60 or more cycles, usually 10-50 and often 20-45. Suitable PCR protocols are known in the art, eg S
ambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press.
, New York (1989) or can be reached by one skilled in the art without undue experimentation.

PCR amplification uses convergent oligonucleotide primers that are complementary to, and designed to anneal to, the sequences flanking the region of the nucleic acid to be amplified. The primers are for the part of the strand to be amplified,
The 3'portion is designed to be at least 90%, preferably 95%, and most preferably 100% complementary. The complementary portion of the primer must be of sufficient length to selectively hybridize to the target or reference sequence under the primer annealing conditions used in the reaction. Generally, the number of nucleotides in the hybridizable sequence of the primer is at least 10, preferably at least 15 nucleotides, more preferably 20-50 nucleotides in length. In some cases, substantially all of the primers are complementary to the target sequence, such as the F primer in Figure 1. In other cases, the 5'end of the primer is not complementary to the target, in which case this non-complementary sequence will be incorporated at the end of the resulting amplicon. This non-complementary end has 1 to 60 or more nucleotides, 5
It may be ~ 30 nucleotides, or preferably 8-30 nucleotides in length. The use of such primers produces oligonucleotide tails at the ends of the partially double-stranded. Such primers can also be used to generate amplicon intermediates that are recognized by the general primers for producing the partial duplexes of the invention.

The concentration of the oligonucleotide primer used in PCR amplification is at least a desired copy number concentration, and is usually in the range of 1 nM to 1 mM, preferably 100 nM to 110 μM. For amplification of genomic DNA, about 500 nM to 1000 nM forward primer and about each 250 μM to 500 μM two reverse primers are preferred. That is,
The conditions are similar to those used to amplify STS (sequence tag site). Cloned D
When amplifying NA fragments, lower primer concentrations, eg about 25 nM to 250 nM, are sufficient.

The entire sequence of forward primer F hybridizes to the template DNA (ie both A and B). The forward primers F1 and F2 share the 3'end (f1 = f2) and hybridize to the same part of the template DNA (reference and target DNA), or the primers F1 and F2 have different 3'ends. And thereby hybridize to different parts of the template DNA (f1 ≠ f2). In addition, F1 has a 5'end portion (T1) that hybridizes or does not hybridize with the template DNA. Similarly, F2
5'end portion that hybridizes with or does not hybridize with template DNA (T2
) Has. The two reverse primers R1 and R2 have a common 3'terminal part that hybridizes with the same part of the template DNA (r '= r1' = r2 ') or
R1 and R2 have different 3'termini so that they can hybridize to different parts of the template DNA. In addition, R1 is a 5'terminal portion (T
Have 3). Similarly, R2 has a 5'terminal portion (T4) that is not complementary to the template DNA and therefore does not hybridize to it. T3 has nothing to do with T4, ie
The complementary strand of T3 (T3 ') is not complementary to T4, and the complementary strand of T4 (T4') is not complementary to T3. As a result, T4 'does not hybridize to T3 under the conditions used in this method. By multiple rounds of PCR amplification, partially double-stranded A ′, A ″, B ′, with four tails,
Many DNA products are produced, including the B ″ component strand (FIG. 1). Tailed duplexes are formed by adjusting the temperature of the solution so that the component strands hybridize to form the desired partial duplex. Also note that many other duplexes are produced. These unintended products are generally not a problem, as sufficient numbers of partial duplexes are produced under the conditions described above.

Partial double-stranded A ′ with each tail consists of a double strand of two complementary nucleic acid strands of double-stranded A, with two non-complementary oligonucleotide tails at one end of the double strand. T3 'and T4
There is. Depending on the choice of forward primer, the partially double-stranded A'has 0, 1 or 2 tails at the other end of the partially double-stranded (if T1 = 0 and T2 = 0, the tail at the left end). A partial double-strand with no tails is produced.If T1 = 0 or T2 = 0, then a partial double-strand with one tail at the left end is produced.If T1 ≠ T2 ≠ 0 then 2 at the left end. Partial duplexes with two non-complementary tails are produced). Partial double-stranded A ″ with each tail consists of a double strand of two complementary nucleic acid strands of double-stranded A, and one non-complementary oligonucleotide tail T4 'And T3. Depending on the choice of forward primer, the partially double-stranded A ″ has 0, 1 or 2 tails at the other end of the partially double-stranded (see above). Partial double-stranded B'with each tail consists of a double strand of two complementary nucleic acid strands of double-stranded B, and at one end of the double-stranded,
There are two non-complementary oligonucleotide tails T3 'and T4. Depending on the choice of forward primer, the partially double-stranded B'can have 0/1/2 tails at the other end of the partially double-stranded (see above). Partial double-stranded B '' with each tail
Consists of two complementary nucleic acid strands of double stranded B, with two non-complementary oligonucleotide tails T4 'and T3 at one end of the duplex. Depending on the choice of forward primer, the partially double-stranded B ″ can have 0, 1 or 2 tails at the other end of the partially double-stranded (see above).

When the primers F1, F2, R1 and R2 are used to generate partially double-stranded A ′, B ′, A ″, B ″ (where T1 ≠ T2 ≠ T3 ≠ T4, f1 = f2, r1 '= r2' or r1 '≠ r2'),
Usually in the reaction between A and B (target and reference DNA), primers F1 / R1 and F2 / R2
It is convenient to carry out PCR so that the partial double-stranded chains A ′, A ″, B ′, and B ″ are simultaneously produced by simultaneously containing However, it should be noted that the various replications and amplifications can be performed separately if desired. For example, as shown in FIG.
Amplification of and B can be performed in a separate reaction using primers F1 / R1 or F2 / R2. The resulting tailed amplification products can then be combined under suitable conditions to produce partially double-stranded A ′, A ″, B ′, B ″ (FIG. 1).

When the primers F1, F2, R1 and R2 are used to generate partially double-stranded A ′, B ′, A ″, B ″ (where T1 ≠ T2 ≠ T3 ≠ T4, f1 ≠ f2, r1 ′ ≠ r2 ′), PCR amplification product A1
And B1 (using F1 / R1, see FIG. 1) cover more template DNA at both the left and right ends than A2 and B2 (using F2 / R2, see FIG. 1) (ie f1 Is upstream of f2 and r1 'is downstream of r2') is important. Alternatively, PCR amplification product A1
And B1 (using F1 / R1, see FIG. 1) cover less template DNA at both the left and right ends than A2 and B2 (using F2 / R2, see FIG. 1) (ie f1 is downstream from f2 and r1 'upstream from r2') is important. PCR amplification products A1 and B1 (F1 / R1
(See FIG. 1) but does not have tails T1 and T3 if it covers more template DNA than A2 and B2 (using F2 / R2, see FIG. 1) at both the left and right ends. Note that it is meaning / irrelevant (hence preferably T1 = T3 = 0 bp). Similarly, the PCR amplification products A1 and B1 (using F1 / R1, see Figure 1) have A2 at both the left and right ends.
And B2 (using F2 / R2, see Figure 1), tails when covering less template DNA
Note that having T2 and T4 is meaningless / irrelevant (hence preferably T2 = T4 = 0 bp). As described above, in the reaction of A and B (target DNA and reference DNA), by simultaneously containing the primers F1 / R1 and F2 / R2, partial double-stranded A ′, A ″, B ′, and It is convenient to perform PCR so that B '' is produced simultaneously. However, it should be noted that the various replications and amplifications can be performed separately if desired. For example, as shown in FIG. 1, amplification of A and B is performed using the primer F1.
It can be done in a separate reaction using / R1 or F2 / R2. The resulting tailed amplification products can then be combined under suitable conditions to produce partially double-stranded A ', A ", B', B" (see Figure 1).

Usually, the primers F, R1 and R2 are used to produce partially double-stranded A ′, B ′, A ″, B ″
(Where T1 = T2 (preferably T1 = T2 = 0 bp), T3 ≠ T4, f1 = f2
= F, r1 ′ = r2 ′ or r1 ′ ≠ r2 ′), in the reaction of A and B (target and reference), the primers F, R1 and R2 are simultaneously contained to form a partial double-stranded A ′, A ″. It is convenient to perform PCR so that B, B ′, and B ″ are produced simultaneously. However, it should be noted that the various replications and amplifications can be performed separately if desired. For example, A and B
Can be performed in a separate reaction using primers F, R1 and R2, and the resulting tailed amplification products can be combined under suitable conditions to produce partial double-stranded A ′, A ″.
, B ', B''can be generated. Alternatively, only one of the F and R primers (R1
Alternatively, R2) is used to first amplify A and B, then another R primer is added in the absence or presence of the first R primer used, for an additional round of amplification.

All of these and other protocol modifications (all of which result in a partially double-stranded A ′, A ″, B ′, B ″ with the desired tail) are known to those of skill in the art. Obvious and within the scope of the invention.

6.3.3 Formation of 4-way structure In order to detect the difference between the sequences A and B, a partial double strand (A ′, A ″, B ′) containing the sequences A and B is detected.
, B ″) are contacted under conditions where the complementary tails anneal to each other, thereby initiating the formation of a four-chain complex (Holiday junction), as shown in FIG. The resulting complexes C1, C2, C3, C4 are subject to the conditions under which branch migration occurs. In their partial duplexes, the tails are not complementary to each other (eg, T1 is T2
It is not complementary to '), which limits branch movement in the direction of the tail. However, branch migration can occur in the other direction as long as the reference and target sequences are the same. If the two sequences are identical, branch migration can proceed to the end of the chain and the complex will dissociate into two duplexes, one for each strand from each of the original partial duplexes. Have. On the other hand, if the target and reference sequences are different,
Branch migration past the point of difference causes a mismatch in the newly formed duplex. Under the conditions used in the practice of the present invention, the presence of such differences actually blocks branch migration and results in a stabilized Holliday junction complex. As a result, the presence of a difference between the two sequences is reflected in the generation of a stabilized Holliday junction, which in the absence of the difference breaks into two duplexes. .

The right end of the tailed partially double-stranded A ′ has T4 and T3 ′, respectively, as the terminal portion of each chain, which T4 and T3 ′ are the rightmost tail of B ″, They are complementary to T4 'and T3, respectively, which are not complementary to each other. Partial double-stranded A'with 4 tails
, A ″, B ′, B ″ are partially double-stranded A ′ when present in the same solution under suitable conditions.
Two kinds of 4-way holiday junctions (complexes C1 and C2) consisting of B and B '' are formed. One is the hybridization of tail T1 of A'and tail T1 'of B'',
And formed as a result of the hybridization of A'tail T2 'and B''tail T2. The other is formed as a result of the hybridization of tail T3 ′ of A ′ with tail T3 of B ″ and the hybridization of tail T4 of A ′ with tail T4 ′ of B ″. Furthermore, from the partially double-stranded A ″ and B ′, two more 4
Way Holiday Junction Complexes C3 and C4 are formed. On the other hand, the T1 'of A''is
It is formed when it hybridizes to T1 of B'and when T2 of A '' hybridizes to T2 'of B'. The other is formed when the T3 of A ″ hybridizes with the T3 ′ of B ′ and when the T4 ′ of A ″ hybridizes with the T4 of B ′.

Furthermore, the four tailed partially double-stranded A ′, A ″, B ′, B ″ can form concatemers. For example, three partially double-stranded B ″, A ′ and a second partially double-stranded B ″ can form a concatemer with two Holliday junctions. However, the concatemers do not interfere with the detection of the difference between sequence A and sequence B. If sequences A and B are identical, the migration of both Holliday structural branches in B ″ -A′-B ″ proceeds to completion and the entire concatemer breaks down into two duplexes. Array
If there is a difference between A and B, both holiday structures will be stabilized. Detection of the stabilized Holliday structure is an indication of the difference between sequences A and B.

Using the teachings disclosed herein and the findings generally available to those of skill in the art, one of skill in the art will determine the appropriate conditions for hybridization of the tail to determine any particular duplex. Holiday junction formation can be performed. For example, Sambro
See ok et al. (supra), Panyutin et al. (supra), and US Pat. No. 6,013,439.

The holiday junction complexes C1, C2, C3, and C4 are subject to branch migration conditions, where T1 and T2 and tails T3 and T4 are different, so branch migration is dependent on their tails (that Hybridization proceeds only away from (initiating 4-way Holliday complex formation). In the absence of a mismatch between A and B, the branch migration of complexes C1, C2, C3, and C4 proceeds far away from the tail to the other end of the partial duplex. As a result, four holiday complexes C1 and C2
, C3, and C4 each decompose into double strands (FIG. 1). Alternatively, if there is a mismatch between A and B, branch migration of complexes C1, C2, C3, and C4 progressing away from the tail is stopped by the mismatch and stabilized Holliday junction complex C1, C2, C3 and C4 are formed (Fig. 2). In certain embodiments of the invention, branch migration is carried out in the presence of ions such as Mg ⁺⁺ , which ions enhance the propensity of the mismatch to interfere with spontaneous DNA transfer and thus such mismatches. It stabilizes the Holliday junction complex with. The preferred concentration range of Mg ⁺⁺ is 1-10 mM. Stabilization is due to other ions (especially
Note that divalent cations such as Mn ⁺⁺ or Ca ⁺⁺ ), or a suitable combination of ions. In a particularly preferred embodiment, branch transfer is achieved by incubation at 65 ° C. for about 20-120 minutes in a buffer containing 4 mM MgCl ₂ , 50 mM KCl, 10 mM Tris-HCl (pH 8.3). A description of suitable branch transfer conditions for the formation of stabilized Holliday junctions as a result of single base mismatches is described, for example, in Panyutin and Hsieh, (1993) J. Mol. Bi.
ol., 230: 413-24, which is incorporated herein by reference in its entirety.

6.3.4 Detection of Holiday Structures Therefore, stable 4-way Holliday junction complexes (C1, C2, C3, and
Detection of C4) can be used to indicate the presence of a difference between DNA sequences A and B. On the other hand, the absence of a stabilized 4-way Holliday complex can be used to indicate the lack of difference between DNA sequences A and B. In accordance with the present invention, stabilized Holliday junctions exhibiting a difference between nucleic acids A and B are detected by any of the methods described below. For example, a stabilized Holliday junction can be detected by a molecule capable of specifically recognizing and binding to the Holliday junction.

6.3.4.1 Detection Using Molecules Specific for 4-Way Nucleic Acid Complexes In one embodiment, molecules having specificity for the Holliday structure are used to detect the presence of a stabilized Holliday structure, thereby The presence of differences between nucleotide sequence A and polynucleotide sequence B is detected.

The presence of the Holliday structure can be detected using any molecule known to those of skill in the art to bind specifically to the Holliday structure. In a preferred embodiment, the protein is used to bind to a stabilized Holliday junction and to detect its formation. Many proteins from different organisms have been shown to bind specifically to Holliday junctions. These proteins include, but are not limited to, E. coli RuvA, RuvC, RuvB, RusA, RuvG; R.
Includes proteins / mutants obtained from uvA, RuvC, RuvB, RusA, RuvG. Moreover, such proteins include RuvA, RuvC, RuvB from various other organisms.
, RusA, RuvG homologues (including functional homologues), eg, RuvA, Ru from mammalian origin
There are homologues of vC, RuvB, RusA, and RuvG, Cce1 and spCce1 from yeast, Hjc from Pyrococcus furiosusa, and other resolvases and recombinases that can specifically bind to Holliday structures.

In a particularly convenient embodiment of the invention, thermostable proteins are used to detect the presence of Holliday structures. Such thermostable proteins include thermophiles (Thermus aquaticus, Thermus flavus).
lavus), Thermus thermophilus, and other thermophilic organisms known to those of skill in the art). Hj from Pyrococcus furiosusa
c is one good example of a suitable thermostable protein with specificity for the Holliday structure.

For the preparation and properties of many such proteins useful in the practice of the present invention, see
See, for example, the following references, all of which are incorporated herein by reference in their entirety: Davies and West, supra;
Whitby et al., Supra; Iwasaki H. et al., 1992. "Escherichia coli RuvA and RuvC proteins specifically interact with Holliday junctions and promote branch migration." Genes De
v. 6: 2214-20; Parsons CA et al., 1992. "Interaction of Escherichia coli RuvA with the synthetic Holliday junction of the RuvB protein," Proc. Natl. Acad. Sci. USA 89: 5452-56.
Traneva IR et al., 1992. "Purification and Characterization of the RuvA and RuvB Proteins of Escherichia coli" Mol. G
En. Genet. 235: 1-10; Rafferty JB et al., 1996. "Crystal Structure of the DNA Recombinant Protein RuvA and its Model for Binding to Holliday Junctions", Science 274: 415-21.
Hargreaves D. et al., 1999. "Crystallization of Escherichia coli RuvA in Complex with Synthetic Holliday Junction", Acta Crystallogr D. Biol Crystallogr 55 (Pt 1): 263.
-5; Hargreaves D. et al., 1998. "Crystal structure of Escherichia coli RuvA with bound DNA Holliday junctions at 6 A resolution", Nature Struct Biol. 5 (6): 441-6; Dund.
erdale HJ et al., 1994. "Cloning, overexpression, purification and characterization of E. coli RuvC Holliday Junction Resolvase," J. Biol. Chem. 267 (7): 5187-94.
Ariyoshi M et al., 1994. "Atomic Structure of RuvC Resolvase: Holliday Junction-Specific Endonuclease from Escherichia coli", Cell 78 (6): 1063-72; Sharples G
J et al., 1994. "Intermediate Processing in Recombination and DNA Repair: Identification of a New Endonuclease That Cleaves a Holliday Junction Specifically", EMBO 13 (24)
: 6133-42; Rice P et al., 1995. "Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration," C
ell 82 (2): 209-20; Bujacz G. et al., 1995. "High-resolution structure of the catalytic domain of avian sarcoma virus integrase," J. Mol. Biol. 253 (2): 333-46; Rice P. et al., 19
96. "Retroviral integrase and its relatives," Curr Opin Struct Biol
6 (1): 76-83; Suck D, 1997. "DNA recognition by structure-selective nucleases", Biopo
lymer 44 (4): 405-21; White MF et al., 1997. "The yeast degrading enzyme CCE1 opens the structure of a 4-way junction," J. Mol. Biol. 266 (1): 122-34; Whitby MC et al., 19
97. “S. Pombe (S. Homologous to CCE1 from S. cerevisiae)
new Holliday junction degrading enzyme from P. pombe), J. Mol. Biol. 271
(4): 509-22; Bidnenko E. et al., 1998. "Lactococcus lactis phage operon encoding an endonuclease homologous to RuvC"
, Mol Microbiol 28 (4): 823-34; Raaijmakers H et al., 1999. "X-ray structure of T4 endonuclease VII: a DNA junction resolver with a novel fold and a unique normal domain-exchanged dimer structure", EMBO 18 (6): 1447-58; Komori K et al.
1999. "Holiday Junction Resolvase from Pyrococcus furiosus: Functional Similarity to Escherichia coli RuvC Provides Evidence for a Conserved Mechanism of Homologous Recombination in Bacteria, Eukaryotes, and Archaea"", Proc. Na
tl. Acad. Sci. USA 96 (16): 8873-8; Komori K et al., 2000. "Mutation analysis of the Pyrococcus furiosus Holliday Junction Resolvase Hjc revealed functionally important residues for dimer formation, junction DNA binding and cleavage activity." J. Biol. Chem . (September 2000 issue); Sharpl
es GJ et al., 1999. "Bacterial Holiday Junction Processing: RuvABC, RecG
And considerations from the evolutionary conservation of RusA ", J Bacteriol 181 (8): 5543-50; Sharples.
GJ et al., 1993. "Escherichia coli RuvC mutant defective in cleavage of synthetic Holliday junctions," Nucleic Acids Research 21 (15): 3359-64.

Proteins that specifically bind to Holliday structures can be detected by methods known to those of skill in the art for detection and / or visualization of proteins. In fact, one advantage of the present invention is that it allows stable Holliday junction detection without the use of labeled nucleic acids. In a preferred embodiment, at least one Holliday junction specific binding protein, ie a protein capable of selectively recognizing and binding Holliday junctions, is labeled with at least one label, the protein being stable Holliday junction. Junction (eg C1
, C2, C3, and / or C4). Binding of the protein to the Holiday junction produces a signal, which is preferably quantitatively detectable. The signal is used as an indicator of the presence and amount of stable Holliday junctions, and is an indicator of the presence and amount / ratio of mismatched DNA in DNA polymorphism analysis.

In a preferred embodiment, the present invention uses at least one labeled protein to detect Holliday junctions. The label may be endogenous to the protein, for example the native fluorescence of the protein resulting from the fluorescence of amino acids such as tryptophan, tyrosine or phenylalanine, or a protein side chain capable of reacting in a detectable manner. The label can be attached to the protein during or after translation. Suitable labels include, but are not limited to, fluorescent molecules (eg, fluorescein, rhodamine, and fluorescent proteins and peptides such as GFP and GFP variants and analogs), radioactive groups,
Solid surface, oligonucleotide, enzyme, dye, chemiluminescent group, coenzyme, enzyme substrate,
There are ligands, receptors, and small organic molecules.

In a preferred embodiment of the invention, more than one protein can be used to detect stabilized Holliday junctions. The two proteins are
It may be the same protein or two different proteins or domains or subunits of a single protein. In certain embodiments, two or more proteins are capable of binding to form a dimer or higher oligomer,
In certain embodiments, oligomerization relies on binding to Holliday junctions. In a preferred embodiment, the two proteins are labeled with different labels,
Moreover, the Holliday junction-dependent complex formation between these two proteins causes the binding of the two labels. The binding of the two labels can be used as an indicator of the presence of a stable Holliday junction (Figure 3).

For example, RuvA and RuvC are themselves Ru in the absence of a holiday junction.
It is unable to form the vC-RuvA complex. However, both RuvA and RuvC can simultaneously bind to the Holiday junction and form the RuvA-RuvC-Holiday junction complex. Both RuvA and RuvC, as well as many RuvC mutants, have been cloned, overexpressed and purified. In a preferred embodiment of the invention, RuvA and RuvC can each be fused with different fluorophores to act as donor / acceptor pairs for intermolecular fluorescence resonance energy transfer (FRET). Intermolecular FRET can be used to detect donor / acceptor pair binding and characterize intermolecular relationships. The principles involved in the application of FRET for such purposes are well known in the field of molecular biology. References describing the use of FRET to detect the binding of two molecules include, for example, Heim R. 1999, Green Fluorescent Protein Foam for Energy Transfer, Methods in Enzymology 302: 408-23; Foer.
ster T. 1948, Ann Phys 2:55; Mitra RD et al., 1996, fluorescence resonance energy transfer between blue-emitting and red-shift excited derivatives of green fluorescent protein, Gene 1
73: 13-7; and Furey WS et al., 1998, use of fluorescence energy transfer to study the conformation of DNA substrates to Klenow fragment, Biochemistry 37 (9): 297.
9-90, all of which are incorporated herein by reference. Only in the presence of Holiday Junction, the fusion protein of RuvA and RuvC
It binds to each other and produces a specific FRET signal. Detection and / or quantification of the FRET signal can be used to detect and / or quantify the presence and amount of stable Holliday junctions. In general, the binding of two proteins to a stabilized Holliday junction brings the donor and acceptor pairs into close proximity, resulting in an emission ratio (ratio of emission at maximum emission wavelength of donor to acceptor).
The change occurring in or the change in emission intensity at the maximum emission wavelength of the acceptor can be used to measure / quantify the presence of Holliday junctions.

The present invention uses RuvC mutants that lack the Holliday junction-specific endonuclease activity of the wild-type enzyme but retain the ability to specifically bind to Holliday junctions. Such mutants include D7N, E66Q, D138N
, D141N, D7N, E66D, D138E, and ruvC51, which include, for example, S
aito A et al., 1995, Identification of four acidic amino acids that make up the catalytic center of RuvC Holliday Junction Resolvase, Proc. Natl. Acad. Sci. USA 92: 7470-4;
Sharples GJ et al., 1993, Escherichia coli R defective in cleavage of synthetic Holliday junctions
uvC mutant. Nucleic Acids Research, 21 (15): 3359-64.

In a preferred embodiment of the invention, the FRET donor and acceptor pair comprises a protein. Particularly suitable proteins include green fluorescent protein (GFP) and GFP mutants with suitable excitation and emission frequencies, some of which are described in the literature, eg Heim R. (supra); Foerster T. (supra). ); Mitra RD et al. (Supra); and Furey WS et al. (Supra).

In an embodiment of the invention where detection is by a single Holliday junction-binding protein, intramolecular FRET can be used to detect the presence / amount of Holliday junction (FIG. 4). A single Holliday junction-binding protein may be doubly labeled with two fluorophores (eg, two GFP mutants that function as intramolecular donor / acceptor FRET pairs). In a preferred embodiment, the sole protein used is RuvC, more preferably one of the RuvC mutants described above. The double-labeled protein undergoes a conformational change upon binding to the Holliday junction, which causes a change in the physical relationship between the two fluorophores and thus a change in the FRET signal. Changes in FRET are detectable and can be used as an indicator of the presence and amount of stable Holliday junctions, as described above for embodiments with two Holliday junction binding proteins.

When a single binding protein is used, if the protein used is capable of forming homo-oligomers or hetero-oligomers, it also results in intracellular FRET,
It can be used to detect the presence / abundance of holiday junctions (Fig. 3
). For example, E. coli RuvA exists as a tetramer in solution but can form an octamer when bound to Holliday junctions. Octamer RuvA
Formation depends on binding to the Holliday junction. Thus, RuvA alone can be used to detect Holliday junctions by intermolecular FRET. For example, one RuvA tetramer can be labeled with one fluorophore (eg, GFP or a GFP variant) such that the two fluorophores can function as intermolecular donor / acceptor FRET pairs. The RuvA tetramer of 2 can be labeled with a second fluorophore. In such embodiments of the invention, the two differentially labeled RuvA tetramers form octamers in the presence of Holliday structures.

In an embodiment of the invention, the solid surface may act as a label (eg the surface of an optical biosensor, eg Biacore or Iasys). Optical biosensors are described, for example, in Canziani G et al., 1999, "Exploratory Biomolecular Recognition Using Optical Biosensors," Methods 19 (2): 253-69. Usually, molecules capable of binding to Holliday junctions, preferably including the proteins mentioned above, are immobilized on the surface of the biosensor. In this embodiment, the binding of Holliday junctions to the immobilized protein can produce a detectable light signal. This signal is recorded by the biosensor as an indicator of the presence and quantity of stable Holliday junctions. In this embodiment, a single label (ie, biosensor surface) is sufficient for the detection of Holliday junctions. One advantage of this embodiment of the invention is that neither the polynucleotide forming the Holliday junction nor the Holliday junction binding molecule need be labeled.

In another embodiment of the invention, LOCI (luminescent oxygen channeling immunoassay)
Can be used to detect the binding of Holliday junction binding proteins to Holliday junctions. LOCI, for example Ullman EF et al., 1
994, Luminescent Oxygen Channeling Immunoassay: Measurement of Particle Binding Dynamics by Chemiluminescence, Proc. Natl. Acad. Sci. USA 91: 5426-30.

In yet another embodiment, detecting the binding of the Holliday junction binding protein to the Holliday junction is performed by an ELISA assay. ELISA assays are well known in the art, eg, US Pat. No. 6,013,439 and Sambrook et al.
(Above).

In yet another embodiment of the present invention, mass spectroscopy is used to detect stabilized Holliday junctions. One advantage of embodiments of the present invention is that neither the polynucleotide forming the Holliday junction nor the Holliday junction binding molecule need be labeled. Formation of a 4-way Holliday junction from two double-stranded DNAs, or formation of a Holliday junction / protein complex upon binding of a Holliday junction-specific binding protein to a Holliday junction, results in the production of molecules with higher mass. It Detection of the presence / ratio of molecules with different masses by mass spectrometry can be used to detect / quantify Holliday junctions, ie to indicate the presence or absence of a difference between two DNA fragments. The use of mass spectrometry to characterize macromolecular complexes is described, for example, by Kelleher NL, 2000, "From primary structure to function: Biological considerations from large molecule mass spectra", Chem Biol. 7 (2). : R37-45.

In yet another embodiment of the invention, the SIGNATUREBIO INC. Phenotypic dynamic profiling system can be used to detect protein-protein interactions induced by Holliday junctions. One advantage of this embodiment of the invention is that neither the polynucleotide forming the Holliday junction nor the Holliday junction binding molecule need be labeled.

6.3.4.2 Detection of Stabilized Holliday Structures by Gel Electrophoresis In one aspect of the invention, the mixture resulting from subjecting the 4-way complex to branch migration conditions is separated by gel electrophoresis. Surprisingly, the stabilized Holliday structure can be detected by gel electrophoresis under suitable conditions. The presence of a stabilized Holliday structure identified by gel electrophoresis indicates the presence of differences between the target and reference sequences.

The gel electrophoresis conditions are that the stabilized Holliday structure is double-stranded, partially double-stranded and 1-stranded.
It should be chosen so that it can be separated from other complexes in the mixture, such as single-stranded polynucleotides. The actual conditions for gel electrophoresis depend on the size of the polynucleotide sequence and the partial duplex and what the sequence is.
Such conditions will be apparent to those of ordinary skill in the art. For example, if the target sequence and the reference sequence
When it is 35-300 bp long and the partial duplex is about 50-300 bp long, the stabilized Holliday structure separates at 160 V in 4% -6% TBE-PAGE gels in 30 minutes. be able to.

6.3.4.3 Detection of Stabilized Holliday Structures by Isolation In another embodiment of the invention, stabilized Holliday junctions are isolated from other molecules in a mixture, such as double-stranded and single-stranded polynucleotides. By separating, the stabilized Holliday junction is detected. Stabilized Holliday junctions were separated from double-stranded DNA by gel electrophoresis, capillary electrophoresis, chromatography (including affinity chromatography using columns coated with Holliday structure-specific binding proteins detailed above). , Isolated.

There are many ways to separate a 4-way Holliday structure from double-stranded DNA.
To determine an individual's genotype for multiple SNP positions, a set of PCR primers (F1, F2, R2, and R2) may be designed for each SNP position. A DNA fragment (partial double strand) obtained by PCR using the genomic DNA of the individual can be regarded as the target DNA. A DNA fragment of known genotype (partially double-stranded) obtained using the same set of primers is considered as reference DNA. Therefore, there will be two reference DNAs (partially double-stranded) for each SNP. Target DN for each SNP position
A (partial double-stranded) is mixed and compared with the corresponding two types of reference DNA (partial double-stranded) one by one by causing Holliday junction formation / branch migration. After PCR / branch transfer, the resulting mixture for multiple (million units) SNP positions are pooled together. The pooled DNA is then subjected to conditions that allow separation / isolation of Holliday structures (eg gel / capillary electrophoresis, chromatography). Pools of Holliday structures formed between corresponding reference DNAs for multiple SNP target DNAs are isolated / purified by various means.

Holiday junctions can be separated from double-stranded DNA by gel electrophoresis or capillary electrophoresis. The band containing the Holiday Junction DNA can then be identified and isolated. DNA from the band containing the holiday junction can then be eluted / purified from the gel band.

Alternatively, and by way of example and not by way of limitation, pools composed of Holliday junctions can be isolated by chromatography. In a preferred embodiment, solid surfaces coated / conjugated with Holliday structure-specific binding proteins (eg, columns, filters, plastics ...) Can be used to isolate / purify Holliday junctions. The presence of a specific SNP DNA fragment in the purified pool composed of Holiday Junction
It shows that the target DNA of the SNP is different from the reference DNA being compared. Alternatively, in other words, the diploid genomic DNA used to generate the target DNA for that particular SNP position has at least one copy of the SNP type that differs from the type of reference DNA used. Thus, any method that allows the true separation of DNA fragments containing isolated / purified pools of Holliday junctions can be used for SNP scoring. Many proteins from various organisms have been shown to bind specifically to Holliday junctions and have been described in detail above.

By way of example and not limitation, DNA hybridization can be used for the true separation of DNA fragments containing isolated / purified pools of Holliday junctions. In a preferred embodiment, the isolated Holliday structure-containing DNA is labeled and used as a probe to hybridize with DNA fragments / oligos immobilized on SNP chips / microarrays for SNP scoring. be able to. A positive hybridization signal for a particular SNP on the chip / microarray indicates that the diploid genomic DNA used to generate the target DNA for that particular SNP position is a different SNP type than the type of reference DNA used. Means to have at least one copy. All possible types for each SNP position were used as reference DNA (one at a time) and compared to the corresponding target DNA (forming a Holliday structure / branching), then High-specificity / accuracy of a large number (1 million units) of SN at the same time by performing Holliday structure isolation / purification and identification by hybridization using chip / microarray
For the P position, the diploid genomic DNA sample can be genotyped.

For example, a method of genotyping multiple polymorphic positions is illustrated in FIG. Figure 5
In the target DNA, the four forward primers (F / R1, F / R2, F / R3, and F / R
4) and one reverse primer. 4 forward primers F / R
1, F / R2, F / R3, and F / R4 are polynucleotide sequences F and 4 complementary to the target DNA.
And one of the two tail sequences T1, T2, T3, and T4. The reverse primer comprises a polynucleotide sequence complementary to the target DNA and any universal tail UT. PCR is performed as described above to generate the target partial duplex. The forward and reverse primers are chosen such that the resulting partial duplex contains the polymorphism of interest in the target DNA. A reference DNA corresponding to the target DNA and containing a sequence known as a polymorphism is amplified with the same primers to produce a reference partial duplex. The target duplex and the reference duplex can form a 4-way structure and are mixed under conditions where branch migration occurs. The stabilized Holliday structure can be isolated using any of the methods described above. The isolation of the Holliday structure indicates a difference between the target and reference sequences.

Notably, multiple target DNAs can be assayed simultaneously. Partial duplexes from each unique target DNA are prepared with a unique set of PCR primers. For example, when using two target DNAs, the partial duplex corresponding to the first target DNA is prepared using the primers corresponding to tails T1, T2, T3 and T4. Partial duplexes corresponding to the second target DNA can be prepared with primers corresponding to tails T5, T6, T7 and T8. Tail T1, T2, T3 and T4 are T5
, T6, T7 and T8 are not supported. Reference partial duplexes for each reference DNA are prepared with primers corresponding to the primers used for the corresponding target DNA. All target partial duplexes correspond to the corresponding reference partial 2 in the same mixture.
The stabilized Holliday structure can be recovered in a single step since it can contact the strands. The recovered polynucleotide can optionally be amplified by PCR, eg, using any universal tail UT. The recovered polynucleotide can then be identified by techniques known to those of skill in the art. For example, the recovered polynucleotide may have tails T1, T2, T3 and T4 or tails T5, T6,
It can be identified by hybridizing with oligonucleotides specific for T7 and T8. Hybridization can be performed, for example, on a gene chip,
This can be done using an array of oligonucleotides or labeled beads coated with oligonucleotides. The presence of a detectable hybridization signal indicates that the particular target DNA is different from its corresponding reference DNA. For example, hybridization of the recovered polynucleotide to the oligonucleotides corresponding to tails T5, T6, T7 and T8 indicates that the second target DNA is different from its corresponding reference DNA.

Importantly, the same detection device (eg, gene chip, array of oligonucleotides, or labeled beads coated with oligonucleotides) can be used to perform genotyping of any polymorphism. . The detector is specific for the tail corresponding to the PCR primers used in the above method but not for the target DNA.

6.4 Method for Detecting Differences Between Two Nucleic Acids by Detecting Stabilized Holliday Structures on Solid Substrates In another aspect, the invention provides a method for separating 4-way complexes on solid substrates. And provide reagents. One partially double-stranded polynucleotide corresponding to the polynucleotide sequence is immobilized on a solid support and contacted with another polynucleotide under conditions where a 4-way complex can be formed and branch migration can occur. This array is
For example, it may be a target sequence that forms a 4-way complex with the nucleic acid corresponding to the reference sequence,
The reverse is also acceptable. If the target and reference sequences are identical, branch migration proceeds to completion and one duplex is released from the solid surface. If there is a difference between the target sequence and the reference sequence, branch migration is stopped and the Holliday junction structure is stabilized on the solid substrate. The detection of a fixed Holiday junction structure is
The difference between the target sequence and the reference sequence is shown.

The solid substrate can be any solid substrate known to those of skill in the art. Solid substrates include any material known to those of skill in the art upon which the polynucleotide can be immobilized. Suitable materials include, for example, metals, polymers, glass, polysaccharides, nitrocellulose and the like. The solid substrate may also take any form including beads, discs, slabs, strips, or any other form capable of carrying a polynucleotide. The polynucleotide is any means known to those of skill in the art for immobilizing molecules,
It is bound to a solid substrate. The polynucleotide is non-covalently or covalently attached to the solid substrate, eg, via a linker or directly. In a preferred embodiment, the polynucleotide is immobilized on nitrocellulose by UV crosslinking.

To determine whether there are differences / mutations between the two DNA fragments A and B, the two
Each of the two DNA fragments (A or B) is first labeled with one forward primer (F)
And tail / tag # 1 or tail / tag # 2 by PCR amplification using either of the two reverse primers (R1 and R2) (FIGS. 6A and 6B) (FIGS. 6A and 6B). The resulting four DNA duplexes are A1 (A + tail / tag # 1); A2 (A + tail / tag # 2); B
1 (B + tail / tag # 1); B1 (B + tail / tag # 2).

One of the duplexes, eg A1, is immobilized on a solid substrate. A mixture of double-stranded A2 / B1 / B2 was added to immobilized double-stranded A1 and then subjected to branch transfer conditions, resulting in partial double-stranded A ′, A ″, B ′, B ″. Form a transient holiday structure (Figs. 6A and 6B). A
When there is a mismatch / difference between B and B, the transient holiday structure becomes a stabilized holiday structure / junction (Fig. 6B). When there is no mismatch / difference between A and B, the transient Holliday structure / junction breaks down into double strands (Fig. 6A).

Alternatively, one of the duplexes, eg A1, is immobilized on a solid substrate. Next, denature A1 and hybridize with A2 to immobilize partially double-stranded A'and A '' immobilized on a solid substrate.
(Figs. 6A and 6B). The mixture of B1 and B2 (preferably a ratio of 1: 1) is then subjected to boiling / denaturing conditions and then to reannealing conditions to allow partial duplexes B'and B'in the mixture. 'Form a mold (Figures 6A and 6B). B1 containing B'and B '' partial duplexes
The / B2 mixture is then added to the A'and A '' partially double-stranded immobilized solid substrate. The resulting mixture is then subjected to branch transfer conditions resulting in partial duplex A ', A'', B
', B' form a transient holiday structure (Figures 6A and 6B). If there is no difference between A and B, the transient Holliday structure / junction will rapidly disassemble into a double strand (Figure 6A).
).

Unbound material is then optionally washed away from the solid substrate using PCR / branch transfer buffer or any buffer that does not disrupt the Holliday structure.
Only when there is a mismatch / difference between A and B a stable 4-way holiday structure is formed on the glass surface, which is not washed out (Fig. 6B).

Next, a reagent is added to the solid substrate, which can not only specifically bind to the Holliday structure but can also be detected directly or indirectly. The reagent is a GFP (or other fluorescent) labeled Holliday junction specific binding protein (eg RuvC, Ru
vA, RusA and their homologues) and other similar reagents detailed herein, but not limited thereto. Then, optionally, all reagents not specifically bound to the surface are washed away. The specifically bound reagent is then detected and / or quantified by any method suitable for that reagent. Such a method has been described in detail above. The specifically bound reagent shows the presence of a stabilized Holliday structure on the solid substrate and thus the difference between A and B.

6.5 Methods for Identifying SNPs Genetic variations involving SNPs each contain at least two possible versions at variable positions within the DNA sequence. To determine which version of a target DNA sample (diploid or haploid) has at a particular variable position, the reference DNA sequence shows each possible variation at that variable position with each reference. Compare with DNA sequence. DNA mutations are known to exist in forms other than SNP. The present invention
Not only can SNPs be detected, but polymorphisms including multiple bases, multi-base-paired deletions, insertions and missense mutations can also be detected. But SN
Genotyping with P will be used to explain (but not limit) the application of the invention for genotyping various genetic variations in diploid individuals (eg, humans).

Target DNA is amplified from the genomic DNA of interest to genotype a genomic DNA sample (X / X) at a particular SNP location. Two more reference DNAs (A or B
) Is amplified from either of two reference genomic DNA samples (A / A or B / B) or a cloned DNA fragment containing two reference DNA sequences (A or B). All three of these DNA samples are amplified by PCR with two common sets of unlabeled primers under standard PCR conditions. Genomic DNA PCR conditions are similar to STS PCR conditions (the primer concentration of PCR from genomic DNA is about 500 nM to 1000 nM for the forward primer and about 250 μM to 500 μM for each of the two reverse primers). Yes, lower primer concentrations (about 250 nM) can be tolerated in PCR using cloned DNA fragments. Each primer set contains a total of three primers: one forward primer (LF or RF) and two reverse primers (LR1, LR2 or RR1, RR2). The LF hybridizes to the target sequence to the left of the SNP position of interest. As far as the feasibility of PCR amplification is concerned, the LF is designed to be at a minimum distance from the SNP position in question. LF is preferably SNP
<10 bp away from position. LR1 and LR2 share the same 3'terminal portion (LR) that hybridizes to the target sequence to the right of the SNP position of interest. As far as the feasibility of PCR amplification is concerned, LR1 / LR2 are designed to be at a minimum distance from the SNP position in question.
The 3'end of LR1 / LR2 preferably corresponds to the base pair next to the SNP position in question. RF is
Hybridizes to the target sequence to the left of the SNP position of interest. As far as the feasibility of PCR amplification is concerned, the RF is designed to be at a minimum distance from the SNP location in question. RF
The 3'end of preferably corresponds to the base pair next to the SNP position in question. LR1 and LR2 are
It shares the same 3'end portion (RR) that hybridizes to the target sequence to the right of the SNP position of interest. As far as the feasibility of PCR amplification is concerned, RR1 / RR2 are designed to be at a minimum distance from the SNP position in question. The 3'end of RR1 / RR2 is generally 5 to 500 bp,
And preferably will be less than 10 bp.

By using two primer sets (LF / LR1 / LR2 and RF / RR1 / RR2), two target DNA amplicons, L (X / X) and amplified from the target genomic DNA sample, were obtained. There is R (X / X). Amplicon L (X / X) is a primer set LF / LR1 / L
Obtained using R2. The amplicon R (X / X) is obtained using the primer set RF / RR1 / RR2. 4 reference DNAs from 2 reference DNA samples (A / A and B / B) by using 2 primer sets (LF / LR1 / LR2 and RF / RR1 / RR2)
Amplicon, L (A / A), R (A / A), L (B / B), R (B / B) are produced. Amplicon L (X / X)
Are mixed separately with amplicon L (A / A) and amplicon L (B / B) in a ratio of 1: 1 to give a mixture L (X / X) / (A / A) and L (X / X) / (B / B) is formed. Amplicon R (X / X) is
Separately mixed with amplicon R (A / A) and amplicon R (B / B) in a ratio of 1: 1 to give a mixture R (X / X) / (A / A) and R (X / X) / (B / B) is formed. The resulting four mixtures L (X / X)
/ (A / A), L (X / X) / (B / B), R (X / X) / (A / A) and R (X / X) / (B / B) are then modified / Reannealed and subjected to branch transfer conditions (2 min at 95 ° C in PCR buffer containing Mg ⁺⁺ then 30 min at 65 ° C). The presence and amount of stable Holliday junctions are then detected using the Holliday junction specific binding proteins described above. The detected signals of the four mixtures were recorded and all possible genotypes (Fig. 7
And genotype the target genomic DNA sample at the SNP location of interest by comparing with the barcodes in Table 1). The present invention will not only allow genotyping of the target genomic DNA sample at the SNP location of interest, but will also determine the presence and location of other mutations near the SNP of interest.

7. Example 1 Detection of Differences Between Two Sequences by Gel Electrophoresis This example shows that stabilized Holliday structures can be formed from polynucleotides having different sequences. The stabilized Holliday structure can be detected by gel electrophoresis according to the method of the present invention. Five regions of human genomic DNA containing known single nucleotide polymorphisms (SNPs) are tailed (tai
led) PCR amplified using a reverse primer, allowing the formation of Holliday junctions. The sequence of these regions, the position and identity of each SNP in these, and the sequence of the primer are shown in the National Center for Biotechnology Information (NCBI).
Can be found in the SNP database of. NCBI assay ID of SNP used
Were: 4215, 4217, 4213, 4141 and 4212.

Genomic DNA samples from the M08PDR panel, the smallest subset of the Human Polymorphism Discovery Resource panel containing eight individual DNA samples, were purchased from the Coriell Cell Repository (Camden, NJ). did. Two genomic DNA samples (one homozygous and one heterozygous) were amplified for each SNP. The genotype of these samples is Lishanski, 2000,
Clinical Chemistry 46 (9), 1464-1470. The primer sequences for amplifying genomic DNA were as follows.

[0104] Where F is the forward PCR primer, R is the reverse PCR primer, and t1
And t2 are common “tail” sequences (underlined) for all five amplicons. Forward and reverse primer sequences are published in the NCBI SNP database.

PCR amplification was performed using a PTC-200 DNA engine thermocycler (MJ Research Inc., Waltham, MA). Thirty-five cycles of PCR were performed with denaturation at 94 ° C for 10 seconds, reannealing at 62 ° C for 15 seconds, and extension at 72 ° C for 45 seconds. Prior to cycling, a 10 minute incubation at 95 ° C was performed to activate AmpliTaq Gold® DNA polymerase (Applied Biosystems, Foster City, CA), followed by 2 minutes of denaturation at 95 ° C and 65% cycling. Incubation (reannealing and branch transfer) was performed for 30 minutes at ° C. The reaction mixture (100 μl) was diluted with 10 ml of commercially available AmpliTaq buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% gelatin).
0 ng genomic DNA, 2.5 U AmpliTaq Gold® DNA Polymerase, 200 μM each d
NTPs and 250 nM of each primer were included.

5 μl of the PCR product subjected to branch migration was mixed with 1 μl of 6 × TBE loading buffer (Invitrogen Corp., San Diego, Calif.) And a 4-12% gradient TBE precast polyacrylamide gel (Invitrogen Corp. , San Diego, CA). Xcell SureLock (R) Mini-Cell Electrophoresis Device (Invitrogen
Corp., San Diego, CA) was run at 165V for 30 minutes in 1 × TBE buffer. The gel was stained with SYBR Gold fluorescent dye (Molecular Probes, Eugene, OR) and the band was DR-45M Dark Reader (registered trademark) transilluminator (Clare
It was visualized by Chemical Research, Denver, CO).

FIG. 8A shows a photograph of a typical gel. 1x 4-12% gradient precast gel
Staining with SYBR Gold was done by running 1 in TBE buffer at 65V for 30 minutes. NCBI Assay I
D, the length of each polymorphism and amplicon is shown in the lower part of the gel. Lanes 3, 6 and 13 are size markers, 100 bp DNA ladder (Invitrogen Corp.,
San Diego, CA). Arrows indicate undisassembled Holliday junction bands.

For each SNP in Figure 8A, two lanes are shown: one homozygous (left),
The other is a heterozygote (right). The length of the amplicon varies from 122 bp to 245 bp. When there is a single base sequence difference between the two alleles, a relatively weak and slowly migrating band (indicated by an arrow) is only present in the heterozygous sample for each SNP. The slower band corresponds to the longer amplicon. Serial dilution experiments (not shown) showed that the amount of DNA in these bands was approximately equal to the expected amount of undegraded Holliday junction (1/16 of total material). The slowly migrating band disappears upon digestion with the wild-type E. coli RuvC protein, a Holliday junction-specific endonuclease (not shown). Based on this evidence, the slowly migrating band indicates a clearly undegraded Holliday junction and thus the presence of two alternative alleles in the genomic DNA.

Therefore, performing rapid electrophoresis of unlabeled PCR products is sufficient for SNP genotyping, as illustrated in the experiment of FIG. 8B. In this experiment, all eight genomic DNA samples from the M08PDR panel were amplified using SNP4215 specific primer sets and subjected to branch migration. The 6% TBE gel was run in IX TBE buffer at 165V for 30 minutes and stained with SYBR Green. SYBR Green (Molecular Pro
bes, Eugene, OR), only 5 samples (1, 3, 5, 6, and 8) pre-identified as heterozygous for A / G SNP (Lishanski, 2000, supra) were stained for branch migration. An undegraded Holliday junction band due to inhibition is shown (indicated by arrow).

[0110] 8. Example 2 RuvA and mutant RuvC proteins, the present embodiment that specifically bind to stabilized Holiday Ja Nkushon is stabilized Holliday junction structure, a protein according to the method of the present invention Is specifically bound by and can be detected. The particularly stabilized Holiday junction structure is specifically bound to RuvA and RuvC,
was detected.

In E. coli, the RuvA and RuvC proteins are part of a multi-subunit RuvABC complex that promotes homologous recombination by degrading Holliday junctions. Shinagawa and Iwasaki, 1996 Trends In Biological Sciences 21,
107-111; Eggleston and West, 2000, J. Biol. Chem. 275, 26467-26476. in
Their binding to synthetic Holliday junctions in vitro has been extensively studied. Davies and West, 1998, Current Biology 8, 725-727; Whitby et al., 1996.
, J. Mol. Biol. 264, 878-890. The 22 kDa RuvA protein exists as a homotetramer in solution and specifically binds to synthetic Holliday junctions either as a tetramer or as an octamer, depending on protein concentration. The 19 kDa RuvC protein binds to the Holliday junction as a dimer and cleaves them,
Holliday junction specific endonucleases or resolves that initiate their degradation. Several mutants of the RuvC protein have been isolated, which retain specific binding activity but lack endonuclease activity. Saito et al., 1995, Biochemistry 92, 7470-7474.

Recombinant RuvA and RuvC (D7N mutant) proteins expressed and purified from E. coli were kindly provided by Dr. Hideo Shinagawa (Osaka University, Japan). PCR amplification and branch transfer of samples selected from the M08PDR panel were performed as described in Example 1. In addition to SNPs 4215, 4212, 4213, and 4141 of Example 1, two more
One SNP was included in the experiment: SNP 3989 and 4216. The following primer set was used.

[0113]

Protein binding was detected by band shift experiments as follows. 5 μl of the PCR product subjected to branch transfer was mixed with 1 μl of 0.25 μM RuvA protein or 1 μl of 0.5 μM mutant RuvC protein (diluted with 1 × AmpliTaq buffer, see Example 1) at room temperature for 5-10 Incubated for minutes. 1 μl of 6
B. Sample loading buffer was added and the sample was loaded onto a precast polyacrylamide gel, which was run and stained as in Example 1.

FIG. 9A shows an experiment comparing RuvA and RuvC binding to samples that are homozygous for SNP4215 (left) or heterozygous (right). Sample (A = RuvA; C =
Mutant RuvC) in IX TBE on a 4-12% gradient polyacrylamide gel.
Flowed in buffer at 165V for 30 minutes and stained the gel with SYBR Green. A fairly thin holiday junction band is shown with white dots. No binding was observed in homozygotes (no shifted band), while mobility of Holliday junction bands in heterozygous samples was shifted by protein binding.

FIG. 9B shows the binding of RuvA under conditions ensuring its specificity for Holliday junctions for four PCR products corresponding to four different SNPs. The experimental conditions were the same as in Figure 9A above. Only heterozygous samples were used in this binding experiment. For all SNPs, the Holliday junction band underwent a change in electrophoretic mobility upon RuvA binding (-not adding RuvA, and +
Indicates that RuvA was added).

FIG. 9C provides yet another example of binding of RuvA, mutant RuvC, and a mixture of these two proteins to Holliday junctions for three PCR products corresponding to three different heterozygous SNPs. To do. The experimental conditions were the same as in Figure 9A above except that the gel was run for 75 minutes and stained with SYBR Gold. A ＝ RuvA ； C
= Mutant RuvC; A + C = RuvA + RuvC (PCR product added to premixed protein).

The experiments of this example show that under certain conditions (relatively low protein concentrations) RuvA and mutant RuvC are specific for the undegraded Holliday junction formed by PCR amplified DNA subjected to branch migration. Indicates that they are bound together. Thus, these binding reactions can be used to distinguish (ie, SNP genotyping) a sample with two alleles of a given polymorphism from a sample with one allele.

9. Example 3 Specific binding of stabilized Holliday structure by RuvA and RuvC This example shows that the stabilized Holliday structure is as described in the method of the present invention.
It shows that it can be specifically bound by a complex of two proteins. Simultaneous specific binding of Holliday structures by two proteins (RuvA and mutant RuvC) probing the Holliday structures with antiserum to the two proteins (provided by Dr. Hideo Shinagawa (Osaka University, Japan)) Indicated by

Protein binding was performed as described in Example 2. A heterozygous sample for SNP4216 was used. When both RuvA and mutant RuvC were included in the ligation reaction, they were premixed before adding the PCR product. Gel electrophoresis conditions were the same as in Figure 9C. After staining with SYBR Gold, the gel was incubated briefly with IX Tris-Glycine running buffer (25 mM Tris base, 192 mM Glycine, pH 8.3, Invitrogen Corp., San Diego, CA) containing 0.1% SDS.
Electrophoretic transfer of proteins from this gel onto a nitrocellulose membrane (0.2 μm pore size) was performed using the Xcell II® Blot Module (Invitrogen Corp., San
Diego, CA) at 20V for 2 hours. The transfer buffer is
12 mM Tris base-96 mM glycine (pH 8.3), 20% methanol. Membrane, 50m
Rinse briefly with 1 × TBST buffer (Roche Molecular Biochemicals, Mannheim, Germany) containing M Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween20. The membrane was then loaded with 1% blocking reagent (Roche Molecular Biochemica) in 1 × TBST.
(Is, Mannheim, Germany). This step and all following incubation and washing steps were performed at 4 ° C. with constant shaking. After the blocking step, the membranes were incubated for 1 hour in 10 ml of 1: 1000 diluted rabbit anti-RuvC antibody in 1% blocking solution. The membrane was washed by changing 20 ml of 1 × TBST 4 times and each washing for 5-10 minutes. Membranes were then incubated for 30 minutes in 10 ml of a 1: 1000 dilution of ImmunoPure® goat anti-rabbit IgG (alkaline phosphatase conjugate) in 1% blocking solution. Again, 20 ml of 1 × TBST was exchanged 4 times and each wash was done for 5-10 minutes to wash the membrane.
44 μl of 75 mg / ml nitroblue tetrazolium chloride (NBT) and 33 μl of 5-bromo
-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP), 10 ml of 0.1M
A substrate solution for alkaline phosphatase was prepared in addition to Tris-HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl ₂ . Both NBT and BCIP, Life Technologies,
Obtained from Rockville, MD. Color development occurred within 5-10 minutes.

The bands thus shown containing the RuvC protein (lanes 2 and 3 in FIG. 10,
Panel II) was marked with a pencil. The membrane is then reblocked and the rabbit anti
Reprobed with a 1: 2000 dilution of RuvA antibody.

Bands containing the RuvA protein are found in lanes 1 and 3 (FIG. 10, panel).
III). The positions of the bands shown in lane 3 for the anti-RuvC antibody and then for the anti-RuvA antibody are exactly the same. Its mobility is slightly faster than that of the RuvA (lane 1) and RuvC (lane 2) complexes, presumably due to the increased compactness of the RuvAC complex.

In each panel of FIG. 10, lane 1 is heterozygous PCR product + RuvA, lane 2 is heterozygous PCR product + RuvC (mutant), and lane 3 is heterozygous PCR product + (RuvA + RuvC). ) Is shown. Panel I, SYBR Gold stained gel, Panel II, membranes probed with anti-RuvC antibody, and Panel III, anti-Ruv.
Membrane re-probed with C antibody. For staining with anti-RuvC antibody, see Panel I.
Also seen on Lane 2 of II.

10. Example 4 Detection of Stabilized Holliday Junctions via LOCI This example was stabilized by detecting the intermolecular interaction of two molecules with specificity for the Holliday structure. The method of detecting the holiday structure will be described.

This example is based on the homogeneous chemiluminescence detection method LOCI (Ullman et al., 1994, Proc. Natl. Acad.
Sci. USA 91, 5426-5430; Packard BioScience, Meriden, CT). This takes advantage of the fact that chemiluminescence occurs when two particles (donor and acceptor) are in close proximity due to biological interactions. PCR products are prepared from the reference and target sequences described in Examples 1-3. The PCR products are contacted under conditions that allow the four-chain complex to form and branch migration to occur.

After branch transfer, the stabilized Holliday junction structure, if present, is contacted with biotinylated RuvA (b-RuvA) and the unlabeled mutant RuvC. The complex is then contacted with an antiserum specific for RuvC (eg mouse or rabbit). The resulting complex is contacted with streptavidin-coated donor beads and acceptor beads appropriately coated with anti-mouse or anti-rabbit IgG.

The mixture is then irradiated with laser light at 680 nm, which produces singlet oxygen in the photosensitizer in the donor beads. Singlet oxygen induces chemiluminescence in the compound in the acceptor beads. When a stabilized holiday structure is formed,
A signal is observed due to the difference between the reference and target polynucleotides. If there is no holiday junction (no inhibition of branch migration)
, No signal is observed.

11. Example 5 Detection of Stabilized Holliday Structures Using RuvA This example shows that two molecules of the same protein can be individually labeled with different labels,
It shows that it can be used for the detection of the difference between the target polynucleotide and the reference polynucleotide.

The RuvA protein exists as a tetramer in solution and two such tetramers bind to Holliday junctions to form octamers. 1 RuvA
The tetramer is labeled with biotin in vitro (b-RuvA) and the other tetramer is labeled with fluorescein (f-RuvA). Labeled RuvA molecules can be used to detect stabilized Holliday structures.

PCR products are prepared and subjected to branch transfer conditions as described in Examples 1-4. If present, the stabilized Holliday structure is contacted with b-RuvA and f-RuvA. The resulting mixture is then contacted with streptavidin-coated donor beads and anti-fluorescein antibody-coated acceptor beads.

Interactions between donor beads and acceptor beads are detected as described in Example 4. The presence of the donor-acceptor signal indicates the difference between the target and reference sequences.

12. Example 6 Detection of Stabilized Holliday Structures via FRET This example demonstrates an efficient and sensitive detection of differences between two polynucleotides based on fluorescence resonance energy transfer (FRET). The method will be described.

Published Crystal Structure (Rafferty) of RuvA Complex with Synthetic Holliday Junction
Et al., 1996, Science 274, 415-421; Hargreaves et al., 1998, Nature Struct. Biol.
6, 441-446; Ariyoshi et al., 2000, Proc. Natl. Acad. Sci. USA 97, 8257-8262.
Roe et al., 1998, Mol. Cell 2, 361-372) show that the distance between two RuvA tetramers can be in the FRET range of 10-100 Å.

To generate a FRET signal in the presence of a stabilized Holliday structure, one tetrameric RuvA is labeled with a donor member of the FRET pair (eg, fluorescein) and a second tetrameric RuvA is labeled. Corresponding acceptor member (eg tetramethylrhodamine or QSY7 dye (Molecular Probes, Eugene, Oregon))
Label with.

PCR products are prepared and subjected to branch transfer conditions as described in Examples 1-4. If present, the stabilized Holliday structure is contacted with both labeled RuvA tetramers. After binding, the donor fluorophore is excited at the appropriate wavelength using a spectrofluorometer and FRET is detected by the appearance of acceptor sensitized fluorescence.

No FRET is observed in the absence of Holiday junctions (no inhibition of branch migration in the PCR product). The presence of the FRET signal indicates a stabilized Holliday structure and a difference between the two polynucleotide sequences.

Alternatively, in a similar manner, other pairs of molecules that interact specifically with Holliday are
Label with FRET pair. For example, RuvA and the mutant RuvC are combined into a FRET pair 2
Label with three different fluorescent dyes and detect their assembly on Holliday junctions.

In another example, the RuvA molecule was modified with two different GFP mutants (Heim, 1999, Methods i).
n Enzymology 302, 408-423; green fluorescent protein)
Introduce donor and acceptor fluorophores into RuvA. Vectors for cloning and expressing such fusions are available from Aurora Bioscience (San Diego, CA). This approach does not require chemical modification of the protein. Or Ru
The vA and mutant RuvC are fused to two different GFP mutants to detect their assembly on Holliday junctions.

13. Example 7 Detection of Stabilized Holliday Structures via BRET This example provides another sensitive and efficient method for detecting differences between polynucleotide sequences. The method of this example is based on the bioluminescence resonance energy transfer (BR
ET; Packard BioScience, Meriden CT). The BRET method detects energy transfer from the bioluminescent donor luciferase to the acceptor GFP.

PCR products are prepared and subjected to branch transfer conditions as described in Examples 1-6. The stabilized Holliday structure, if present, was fused to luciferase.
Contact with both RuvA and RuvA fused to GFP. RuvA and luciferase and G
FP fusions are prepared with commercially available cloning and expression vectors, respectively.

After conjugation, the donor fluorophore was excited at the appropriate wavelength using a spectrofluorometer,
BRET is detected by the appearance of sensitized fluorescence of the acceptor. No excitation light source is required for detection of the BRET signal. The presence of the BRET signal indicates a difference between the polynucleotide sequences.

Alternatively, RuvA and mutant RuvC were added to luciferase and GFP, respectively.
Can be fused to (or vice versa) and their assembly on the Holliday junction is detected by BRET.

The above discussion contains specific theories for the mechanisms involved in the present invention. These theories are in no way intended and should not be construed as limiting the invention.

The present invention should not be limited in scope by the particular embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are considered to be within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated herein by reference in their entireties.

[Brief description of drawings]

FIG. 1 provides a schematic representation of an embodiment of the invention for detecting differences between two related nucleic acid sequences using PCR, where the lack of differences between the two related sequences indicates complete branch migration. And cause the decomposition of the Holiday Junction Complex.

FIG. 2 provides a schematic representation of an embodiment of the present invention for detecting differences between two related nucleic acid sequences using PCR, wherein the presence of the differences between the two related sequences prevents branch migration. Then
Causes formation of a stable Holliday junction complex.

FIG. 3 provides a schematic showing one method of using Holliday junction specific binding proteins to detect differences between two related nucleic acid sequences, where a mutation in one of the two related sequences is Present, the branch migration is stopped and a stable Holliday junction complex is formed, which can be detected using labeled Holliday junction specific binding protein.

FIG. 4 provides a schematic diagram showing another method of using Holliday junction specific binding proteins to detect differences between two related nucleic acid sequences, where a mutation in one of the two related sequences is Present, the branch migration is stopped and a stable Holliday junction complex is formed, which can be detected using labeled Holliday junction specific binding protein.

[Figure 5]   FIG. 5 provides a schematic representation of the genotyping method for target DNA.

FIG. 6A shows that by degrading a 4-way complex immobilized on a solid substrate,
A schematic diagram of a method of comparing two nucleic acids is provided. FIG. 6B provides a schematic representation of a method for detecting the difference between two nucleic acids by detecting a stabilized Holliday structure immobilized on a solid substrate.

FIG. 7 is a schematic representation of an embodiment of the invention for genotyping diploid genomic DNA samples at specific SNP positions using PCR.

FIG. 8A provides the results of a representative gel electrophoresis experiment in which stable Holliday structures are detected. FIG. 8B provides the results of a representative gel electrophoresis experiment in which differences in nucleotides within several pairs are detected.

FIG. 9A illustrates the mobility shift of stable Holliday structures upon protein binding. FIG. 9B illustrates the specific binding of stable Holliday structures by RuvA. Figure 9C
Illustrates the specific binding of stable Holliday structures by RuvA, mutant RuvC, and a mixture of RuvA and RuvC.

FIG. 10 illustrates specific interactions of stable Holliday structures with both RuvA and RuvC.

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 60 / 234,229 (32) Priority date September 21, 2000 (September 21, 2000) (33) Priority claiming countries United States (US) (31) Priority claim number 60 / 234,363 (32) Priority date September 22, 2000 (September 22, 2000) (33) Priority claiming countries United States (US) (31) Priority claim number 60 / 242,770 (32) Priority date October 23, 2000 (October 23, 2000) (33) Priority claiming countries United States (US) (31) Priority claim number 60 / 242,840 (32) Priority date October 23, 2000 (October 23, 2000) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, I S, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Yang, Wendy             United States California             94043, Mountain View, Rock Door             Venue 1901, Apartment             # 108 (72) Inventor Lishansky, Ara             United States California             95126, Saint Joe's, Meridian             Way 840, # 110 F-term (reference) 4B024 AA11 CA04 GA25 HA12                 4B063 QA12 QA17 QA20 QQ42 QR08                       QR14 QR42 QR56 QR66 QS25                       QS34 QX02

Claims (18)

[Claims] 1. A method for detecting the presence of a difference between two related polynucleotide sequences, comprising: a. Forming a 4-way complex containing both of the polynucleotide sequences in double-stranded form; b. The 4-way complex is subjected to conditions under which branch migration occurs, where if there is a difference between two related nucleic acid sequences, the branch migration in the 4-way complex is stopped and the 4-way complex is stable. If there is no difference between the two related nucleic acid sequences, a complete strand exchange occurs and the 4-way complex is broken down into two double-stranded nucleic acids, branch migration in the 4-way complex. In this way a stabilized 4-way complex is formed; c. Selecting the stabilized complex or its degraded double-stranded product after branch transfer, the 4-way complex. Specific binding of first reagent that recognizes automatically Subject to conditions and binding of the reagent to the 4-way complex produces a detectable signal; d. Produced by specific binding of the first reagent to the 4-way complex. A method as described above, wherein a signal is detected, the signal is associated with the presence of the difference between the nucleic acid sequences, and no detection of the signal is associated with no difference between the nucleic acid sequences. 2. The method of claim 1, wherein the difference is a mutation. 3. The method of claim 1, wherein the nucleic acid sequence is DNA. 4. The method of claim 1, wherein the 4-way complex comprises Holliday. 5. The method of claim 1, wherein the first reagent is a chemical that binds to the Holliday junction and produces a specific detectable signal when bound to the Holliday junction. 6. The method of claim 5, wherein the first reagent is a dye. 7. The method of claim 1, wherein the first reagent is a Holliday junction binding protein. 8. The method of claim 7, wherein the holiday junction binding protein is a recombinase or a resolvase. 9. The method of claim 7, wherein the Holiday Junction Binding Protein is thermostable. 10. The holiday junction binding protein is RuvA, RuvC,
8. The method of claim 7, selected from the group consisting of RuvB, RusA, RuvG, Cce1, and spCce1. 11. The method of claim 5, wherein the generation of the detectable signal comprises a conformational change in the Holliday junction binding protein. 12. The method of claim 1, wherein producing a detectable signal comprises Holliday junction-induced binding of a Holliday junction binding protein and a nucleic acid forming a Holliday junction complex. 13. The method of claim 1, wherein producing the detectable signal comprises specific Holliday junction binding-induced fluorescence of the first reagent. 14. The method of claim 1, wherein at least one of the related polynucleotide sequences is not detectably labeled. 15. The method of claim 14, wherein none of the relevant polynucleotide sequences are detectably labeled. 16. The method of claim 1, wherein the stabilizing complex is subjected to conditions that allow specific binding of a second reagent that selectively recognizes the 4-way complex, wherein: Simultaneous binding of the first and second reagents to the 4-way complex produces a detectable signal; and b. Specificity of the first and second reagents to the 4-way complex. Binding is detected as an indication of the presence of a 4-way complex, the signal of which is associated with the presence of differences in the nucleic acid sequences and the undetectable signal being associated with the absence of differences between the nucleic acid sequences, The above method. 17. The method of claim 9, wherein the first reagent and the second reagent are Holliday junction binding proteins. 18. The signal is directed to the Holliday junction, the first signal.
10. The method of claim 9, which is produced by intimate binding of a reagent of claim 1 to a second reagent.