Patent Application
20050064441
The present invention relates to a method of detecting or quantifying a nucleic acid molecule present in a specimen.
Techniques for detecting a specific nucleic acid molecule present in a biological specimen are of importance in the fields of fundamental research and clinical study. The techniques are particularly important in analyzing proteins, which express and function at specific organs, at a nucleic-acid level. The techniques are also important in investigating expression of proteins and how to control the expression of proteins in the nervous system and the immune system associating signal transduction system. The techniques are extremely important in genetic diagnosis since mutant genes associated with genetic diseases, genes associated with cancer, and genes associated with viruses can be identified by these techniques.
A representative technique for detecting a nucleic acid molecule is a hybridization method. In this method, a target nucleic acid is hybridized with a nucleic acid probe having a complementary sequence of the,target nucleic acid. Thereafter, the target nucleic acid can be identified using the probe. A drawback of this method resides in that it is difficult to detect a target nucleic acid if it is present in low numbers (e.g., about 1 to 1000 copies). -Furthermore, the specificity is low. Therefore, it is difficult to recognize and detect analogous sequences distinguishably.
In a device called a DNA chip, various types of nucleic-acid probes are immobilized on a base. Detection using the detection method employing the DNA chip is much easier, compared to conventional hybridization methods. However, the probes immobilized on the base differ in optimal conditions, so that a false positive hybridization reaction may take place. This is a problem with this method. Furthermore, the design of the probe to be immobilized on the base must be changed in accordance with the nucleic acid to be detected. This means that an appropriate DNA chip must be prepared every time the nucleic acid to be detected is changed. Whereas, in clinical tests, various types of nucleic acids must be detected. Under these circumstances, conventionally a wide variety of DNA chips are required.
Conventional techniques, including the aforementioned methods, are complicated since an operation consists of many steps. In addition, a long detection time and a well-trained operator are required.
Gene diagnosis is usually used as a definite diagnosis. Therefore, a quick diagnosis is required without the possibility of miss-diagnosis. However, conventional methods do not fully satisfy these requirements.
An object of the present invention is to provide a method of detecting a target nucleic acid molecule from a specimen, with a high specificity. According to the present invention, it is possible to detect and quantify only a specific nucleic acid accurately and quickly from a specimen containing a plurality of types of nucleic acid molecules.
Another object of the present invention is to provide a method of detecting a target sequence which is present few in number, thereby saving the number of DNA chips required. A further object of the present invention is to provide a method which can be easily carried out in a few steps.
The aforementioned objects can be attained by the present invention shown below.
There is provided a method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen, comprising:
Two probes are employed to detect the target nucleic acid in the method of the present invention. Therefore, in this method, a stable hybridization can be performed while preventing a non-specific binding. Furthermore, the target nucleic acid can be identified by detecting the probe (A+B). Therefore, the detection can be made with a high sequence-specificity and a high detection accuracy.
Furthermore, in the present invention, a target nucleic acid is detected by detecting the flag. The advantage of the present invention basically resides in obtaining information regarding a target nucleic acid by substituting the information with the flag, and then detecting or analyzing the flag, as is explained later. An operator can design the flag appropriately. Hence, no matter what type of nucleic acid is used as a detection target, a common DNA chip is indiscriminately used for detection without preparing DNA chips varied depending upon detection targets. It is therefore possible to save the number of DNA chips required. Furthermore, the number of probe types to be immobilized on the DNA chip can be reduced. If the design of the flag is modified, the detection efficiency of a target nucleic acid can be tremendously improved. Simultaneously, the detection step can be simplified.
For example, if the flag is designed so as to have a stable nucleic acid sequence, stable detection results can be obtained. The flag may be constituted of a plurality of units. In the case where a plurality of units are used, it is possible to encode the information of the target nucleic acid. A wide variety of information items of a target nucleic acid can be converted into codes which vary depending upon the combination of the plural units contained in the flag sequence. Furthermore, if the code is constructed with DNA sequence, the DNA code itself can be amplified (nucleotide sequence). If the DNA code uniform in length and stability is amplified by using the same primer pair, the quantification of DNA can be excellently made compared to the conventional method of amplifying a target nucleic acid itself. Therefore, a target sequence can be accurately detected and quantified, even if it is present in low numbers of copies. If it is devised that the DNA code is converted to a numerical value by using a specific algorithm, a calculation based on a DNA molecular reaction can be attained. By such a conversion, the analysis of personal genetic information such as blood types and SNPS can be simplified. In the present invention, it is possible to read out the information of a target nucleic acid from codes of the aforementioned units.
Furthermore, a further object of the present invention is to provide a method of simultaneously detecting a plurality of types of target nucleic acids from a specimen in a simple operation.
To attain the object, probes A1 to An (n is an integer of 2 or more) as well as probes B1 to Bn (n is an integer of 2 or more) are prepared with respect to the corresponding target nucleic acids N1 to Nn (n is an integer of 2 or more) and the aforementioned steps are repeated in the same manner as above. According to the present invention, individual target nucleic acids can be simply and easily detected with a high specificity. When a plurality of types of target nucleic acids are detected, the use of a flag is particularly useful.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate presently preferred embodiments of the invention, and together with the general description given above and the detailed description of the preferred embodiments given below, serve to explain the principles of the invention.
The term “nucleic acid” used herein refers to all DNAs including cDNA, genomic DNA, and synthetic DNA; and all RNAs including mRNA, total RNA, hnRNA and synthetic RNA.
The term “target nucleic acid” refers to, but not limited to a nucleic acid having an arbitrary sequence. Examples of preferable “target nucleic acid” are nucleic acids of causal genes for genetic diseases, oncogenes, and virus-derived nucleic acids, which may act as a marker for disorders.
The term “binding pair” is a pair of substances capable of specifically binding to each other. For example, avidin and biotin; streptoavidin and biotin; and digoxigenin and a digoxigenin antibody. However, the binding pair is not limited to these. Any pair of substances may be used as long as they mutually bind to each other. In the case of avidin and biotin, the phrase “one of the binding pair” means either avidin or biotin. The term “the other one of the binding pair” means the remaining one. The term “binding molecule” used herein can be interchangeably used with the term “one of the binding pair” or “the other one of the binding pair”. Similarly, the term “a molecule having a high affinity with the binding molecule” or the term “a molecule specifically binding to the binding molecule” may be interchangeably used with the term “one of the binding pair” or “the other one of the binding pair”.
The term “specimen” used herein refers to, but not limited to a body fluid including blood, urine, saliva, and the like. Any specimen other than the body fluid may be included. For example, when the specimen is a solid matter, it may be converted into a liquid by an appropriate method including the addition of an enzyme, surfactant or an organic solvent.
The term “complementary sequence” used herein refers to a sequence capable of hybridizing specifically with a predetermined target nucleic acid alone under appropriate conditions.
A detection method of the present invention will be explained with reference to
In the method of this example, two types of nucleic acid probes. (hereinafter, sometimes simply referred to as “probe”) shown below, a probe Al and a probe B2, are used (
The probe A1 is constituted of a sequence F′ and a binding molecule 4 bound to the sequence F′. The sequence F′ is complementary to a nucleotide sequence F of a partial region of the target nucleic acid 3. The binding molecule 4 used herein may directly bind to the sequence F′ or indirectly bind to the sequence F′ via some portion such as an arbitrary nucleotide sequence. The portion except the nucleotide sequence F of the probe Al may be referred to as a “tag”, “tag sequence Tg” or “Tg”. The arbitrary sequence, which is responsible for the indirect binding, may be a nucleotide sequence having any number of nucleotides. More preferably, the sequence may be a non-complementary sequence to the nucleotide sequence of the target nucleic acid. Alternatively, the indirect binding may be mediated by other means such as chemical or physical means. The tag Tg is required to neither bind to nor interact with any portion of the target nucleic acid. The term “non-complementary” used herein refers to a nucleotide sequence which does not hybridize with a sequence of the target nucleic acid, in particular, a sequence within an arbitral range for use in detection.
The sequence F′ used herein has a nucleotide of 1 or more. The sequence F′ preferably has 15 or more of nucleotides to attain stable hybridization.
The binding molecule 4 used herein may be either one of the pair molecules specifically binding to each other, as previously described.
The probe B 2 consists of a sequence S′ and a flag (hereinafter, sometimes represented by “FL”) bound to the sequence S′. The sequence S′ is complementary to a nucleotide sequence S, which is a part of the target nucleic acid 3. In this example, the flag consists of a double-stranded sequence linking to the sequence S′ and the marker substance 5 bound to the double stranded sequence. Generally, it is necessary that the flag itself neither binds to nor interacts with the target nucleic acid 3. Therefore, when the flag is a nucleotide sequence, the entire length is not necessarily formed of a double-strand nucleic acid. The flag may be formed of a single-stranded sequence in part or in its entirety as long as it neither binds to nor interacts with the target nucleic acid. Furthermore, the flag has any number of nucleotides. Alternatively, the marker substance 5 may be bound to the sequence S′ by any means, as detailed in Example 3 set forth later.
When the flag is a double-stranded sequence, the sequence may be formed of any types of nucleotides and may have any number of nucleotides. However, the Tm of the sequence must be sufficiently higher than those of the sequence F and the sequence S. Particularly in the case where the flag has a double-stranded sequence, the flag sequence is not directly bound to the sequence S′. More specifically, the flag sequence hybridizes with the sequence which directly bounds to the sequence S′.
As the marker substance 5 used herein, any substance is used as long as it is generally used as a marker substance. Preferable examples of the marker substance include fluorescent substances, luminescent substances, radioisotope substances such as 32p, high absorbency substances, high reflection substances, high potential substances, magnetic substances, and pigments. Particularly, fluorescent substances such as FITC are preferable.
The sequence S′ used herein has nucleotide(s) of 1 or more, preferably 15 or more.
Tg attached to the probe Al and FL attached to the probe B2 are preferably designed so as to be positioned at ends at the farthest distance from each other when both probes are bound to the desired regions of the target nucleic acid 3, as shown in
The probe A1 and probe B2, when they bind to the desired regions of the target nucleic acid 3, preferably have the nucleotides complementary to adjacent nucleotides on the target nucleic acid 3, at the close ends. This is advantageous in a ligation step performed thereafter. However, this is not limited thereto.
The sequences F and S of the probes A1 and B2 may be appropriately selected so as to have the same Tm by controlling the conditions including the GC content and the combination of nucleotide bases.
The example is carried out as described below. First, the probe A1 and probe B2 are prepared in accordance with the target nucleic acid 3. They are mixed, at an appropriate ratio, with the target nucleic acid 3 (
Subsequently, the mixture is incubated under arbitrary conditions suitable for hybridization for a predetermined time period to perform hybridization (
The hybridization is performed by any general method. The preferable hybridization is performed by allowing the mixture stand still at an appropriate high temperature, for example, 95° C., effective in denaturing a nucleic acid, for 5 minutes, whereby the complimentary binding of the target nucleic acid is dissociated to convert a double strand to a single strand. In the case of the single-stranded target nucleic acid, the single-strand target nucleic acid is allowed to stand still at 70° C. for 5 minutes, whereby it can be converted into a secondary structure. Subsequently, the single-strand is allowed to stand still at an appropriate temperature effective in rebinding of a complementary nucleotide sequence, for example, 55° C. for 15 minutes, the single-stranded target nucleic acid is rebound to the complementary nucleotide sequence. By the hybridization process mentioned above, both the probes A1 and B2 bind to the same target nucleic acid 3 to make a hybrid. Therefore, it is not necessary that conditions including the type of buffer and temperature are limited to those mentioned above. Any conditions may be employed as long as the probes Al and B2 are hybridized with the target nucleic acid 3 under the conditions.
Thereafter, the probe A1 is linked with the probe B2 (
This linkage is made by use of Taq DNA ligase such as Thermus aquatius (Taq) DNA ligase manufactured by New England Biolab. However, the agent used in this linkage is not limited to this. Any agent may be used as long as it is generally used in the linkage. Furthermore, both probes may be linked with any chemical means in place of the enzymatic means.
When the sequences F and S are not present on the target nucleic acid 3 next to each other, a gap-filling reaction may be performed by using DNA polymerase I and a ligase. By this reaction, nucleotides may be introduced in the gap between both proves. Any DNA polymerase and ligase may be used as long as they are generally used. Preferably, heat-resistance DNA polymerase and ligase are preferable.
Subsequently, a probe (A+B) 7 obtained in the above is dissociated from a target nucleic acid 3 (
Subsequently, a binding molecule 4, that is, one of the binding pair, is allowed to bind to a molecule having a high affinity with the binding molecule. In this manner, a prove (A+B) 7 is recovered (
A single strand having the probe (A+B) 7 and the marker substance 5 bound thereto is separated by Bound/Free separation (hereinafter, referred to as “B/F separation”). Therefore, the accuracy in detection is improved. The B/F separation is performed in accordance with the B/F separation method generally employed.
In this method, the B/F separation for the single strand tagged with the marker substance 5 may be. preferably performed using a solid-phase carrier having a nucleotide sequence complementary to the single strand fixed on an appropriate support (
Examples of the support to be used include, but not limited to, a substrate such as a silicon plate or a glass plate, particles such as beads, a container such as a test tube or a vial, a fiber, a tube including a capillary, a filter, an affinity column, an electrode and the like. It is preferable that the material of the support and surface treatment be appropriately selected depending upon how efficiently the probe is fixed.
Since the flag has an artificially synthesized sequence, the sequence may be designed such that it has properties for improving the specificity of the hybridization of the probe (A+B).
This method is also effective when a plurality of target nucleic acids are simultaneously detected.
Individual steps and the constitutions of individual probes are the same as in Example 1 except that a plurality of detection targets are used (
To-explain more specifically, target nucleic acids N1-Nn, probes A1-An, and probes B1-Bn are mixed (
To distinguish a plurality of target nucleic acids by using the same marker substance, the flags having different sequences modified depending upon the target nucleic acids may be used. Furthermore, as shown in
Alternatively, it is possible to use different marker substances depending upon the target nucleic acids. In this case, the modification of the flag and the immobilization of the complementary sequences in different regions are not necessarily performed.
If the GC contents of Fn′ and Sn′ sequences of a probe (A1+B1 ), (A2+B2) . . . (An+Bn) [hereinafter, referred to as “(A1+B1)-(An+Bn)”] and the types of nucleotides are appropriately selected, optimal hybridization conditions (e.g., temperature and salt concentration) which differ between individual probes can be overcome. When a plurality of types of flags different in sequence are used, it is preferable to use the same (common) hybridization conditions even if the types of flags differ. In this manner, detection accuracy is improved. The detection accuracy is further improved by increasing the common Tm value of the flag sequences sufficiently higher than those of the sequences Fn′ and Sn′.
In contrast, in the conventional nucleic acid detection method, in which a complementary sequence of the target nucleic acid is immobilized, optimal hybridization conditions for individual probes immobilized differ in general. However, it is impossible to set the conditions suitable for all probes.
Referring to
The probes A1-An are formed of sequences F1′ to Fn′, and tag Tg1 to Tgn (hereinafter referred to as “Tg1-Tgn”) bound to the sequences F1′ to Fn′, respectively. The sequences F1′ to Fn′ are complementary to nucleotide sequences F1 to Fn, which are parts of the target nucleic acids N1-Nn, respectively. The tag Tg1 to Tgn (hereinafter referred to as “Tg1′-Tgn′) of this example are nucleotide sequences. Each of the sequences of the tags Tg1-Tgn is required not to bind to and thus not to interact with any portion of its target nucleic acid. The tags Tg1 to Tgn used herein may be directly bound to respective sequences F1′ to Fn′ or indirectly bound to them via any intermediate portion, for example, a nucleotide sequence. When the tags are indirectly bound via an nucleotide sequence arbitrarily chosen, the arbitrary sequence may be any nucleotide sequence having any number of nucleotides. The nucleotide sequence is preferably non-complementary to the nucleotide sequence of a target nucleic acid. They may be bound by a chemical or physical means.
The sequences F1′ to Fn′ used herein have nucleotides different from each other. They may be nucleotides of one or more. To attain stable hybridization, the sequences preferably have nucleotide of 15 or more.
As shown in
As the marker substance used herein, any substance may be used as long as it is a marker substance generally used, as set forth in Example 1. More preferably, each of the sequences S1′ to Sn′ has nucleotides of 15 or more.
When probe A1(An) and probe B1(Bn) are bound to respective desired regions of the target nucleic acid, as shown in
If the GC content and the nucleotide types of sequences F1′-Fn′ and S1′-Sn′ contained in these probes are appropriately selected, it is possible to overcome the differences in optimal hybridization conditions of individual probes. When a plurality of types of tag sequences are used, it is preferable that they can be hybridized well under the same (common) hybridization conditions. Furthermore, if the common Tm value of the tag sequences is set sufficiently higher than those of the Fn′ and Sn′ sequences, the detection accuracy is improved.
This example is carried out in the following manner. First, probes A1-An and probes B1-Bn are prepared in accordance with the target nucleic acids N1-Nn, as mentioned above. Then, they are mixed with the target nucleic acids N1-Nn, in an appropriate ratio (
Then, the mixture is incubated for a predetermined time under arbitrary conditions suitable for hybridization. In this manner, hybridization is performed (not shown).
The hybridization may be performed by any general method. Preferable hybridization is performed as follows. First, a target nucleic acid is allowed to stand still at a high temperature, for example, 95° C., which is effective in denaturing the target nucleic acid. In the step, the complementary binding of the target nucleic acid is denatured, with the result that a double strand is dissociated into a single strand. In the case where the target nucleic acid is a single strand, the nucleic acid is allowed to stand still at 70° C. for 5 minutes to denature its secondary structure. Subsequently, the resultant reaction mixture is allowed to stand still for 15 minutes at an appropriate temperature, e.g., 55° C. which is effective in rebinding a complementary nucleotide sequence. As a result, the single-stranded nucleic acid is rebound to the complementary nucleotide sequence.
By the hybridization step, each set of probes A1-An and probes B1-B2 bind to the same and corresponding target nucleic acid to produce hybrids. Accordingly, type of a buffer solution to be employed and temperature condition are not limited to those mentioned above. Any conditions may be employed as long as the probes A1-An and probes B1-Bn can hybridize with respective target nucleic acids.
Subsequently, the probes A1-An are bound the corresponding probes B1-Bn (
Furthermore, in the case where the sequence F and the sequence S are not placed next to each other on the target nucleic acid, the gap between them may be filled by use of DNA polymerase I and a ligase. In this way, nucleic acid bases are introduced into the gap between both probes. Any DNA polymerase I and ligase may be used as long as they are generally used. Heat resistant DNA polymerase and ligase are preferably used.
Subsequently, the probes (A1+B1) to (An+Bn) obtained above are dissociated from the target nucleic acids (
The probes (A1+B1) to (An+Bn) are recovered by using the sequences Tg1′ to Tgn′ which hybridize with complementary sequences Tg1 to Tgn (
The complementary sequences Tg1′ to Tgn′ are preferably fixed on a support. Examples of the support to be used include, but not limited to, a substrate such as a silicon plate or a glass plate, particles such as beads, a container such as a test tube or a vial, a fiber, a tube including a capillary, a filter, an affinity column, an electrode and the like. It is preferable that the material of the support and surface treatment be appropriately selected depending upon how efficiently the probe is fixed.
In Example 3, a plurality of target nucleic acids are used. However, the method used herein is applicable to a single target nucleic acid. If the number of types of the marker substances are increased, a larger number of target nucleic acids can be identified.
Referring now to
The probes A1-An is formed of sequences F1′ to Fn′ and tags Tg1 to Tgn bound to the sequences F1′ to Fn′, respectively. The sequences F1′ to Fn′ are complementary to nucleotide sequences F1 to Fn, which are part of the target nucleic acids N1-Nn, respectively. Tags Tg1 to Tgn of this example are nucleotide sequences which do not bind to any portion of the corresponding target nucleic acids and do not exhibit any interactions with the target nucleic acids. Tags Tg1 to Tgn may directly bind to the respective sequences F1′ to Fn′ and indirectly bind to them via an intermediate portion such as a nucleotide sequence. In the case of the indirect binding via an sequence arbitrarily chosen, the sequence may consist of any types of nucleotides and any number of nucleotides. Preferably, the sequence is non-complementary to the nucleotide sequence of the target nucleic acid. Alternatively, they may bind to each other by a chemical or physical means.
The sequences F1′ to Fn′ used herein are different each other. They may be nucleotides of one or more. To attain a stable hybridization, the sequences F1′ to Fn′ preferably have nucleotides of 15 or more.
As shown in
As the marker substance used herein, any substance may be used as long as it is generally used as a marker substance, as described in Example 1.
The sequence S′ used herein has nucleotides of 1 or more, preferably 15 or more.
When probe A1(An) and probe B1(Bn) are bound to respective desired regions of the target nucleic acid, as shown in
If the sequences F1′-Fn′ and S1′-Sn′ included in the probes are constructed by appropriately setting the GC content and the combination of nucleic acid bases, hybridization of the probes can be appropriately performed by overcoming different-optimal hybridization conditions of individual probes. When a plurality of types of tag sequences and flag sequences are used, it is preferable that they can be hybridized under the same (common) hybridization conditions. Furthermore, if the common Tm values of the tag sequences and flag sequences is set sufficiently higher than those of the sequences Fn′ and Sn′, the detection accuracy is improved. Furthermore, if a plurality of flag sequences are used with respect to a single tag sequence, the number of the target nucleic acids distinguishable from each other can be tremendously improved.
This example can be carried out in the following manner. First, the probes A1-An and probes B1-Bn are prepared in accordance with the target nucleic acids N1-Nn, as mentioned above. Then, the probes are mixed with the target nucleic acids N1-Nn in an appropriate ratio (
Subsequently, the mixture is incubated for a predetermined time under arbitrary conditions suitable for hybridization. In this manner, hybridization is performed (not shown).
The hybridization may be performed in any general method. Preferable hybridization is performed as follows. First, a target nucleic acid is allowed-to stand still at a high temperature, for example, 95° C., which is effective in denaturing the target nucleic acid. In the step, the complementary binding of the target nucleic acid is denatured, with the result that a double strand is dissociated into a single strand. Subsequently, the single-stranded nucleic acid is allowed to stand still for 15 minutes at an appropriate temperature, e.g., 55° C. which is effective in rebinding a complementary nucleotide sequence,. As a result, the single-stranded nucleic acid is rebound to the complementary nucleotide sequence.
By the hybridization step, each set of probes A1-An and probes B1-Bn bind to the same and corresponding target nucleic acid to produce hybrids. Accordingly, the type of a buffer solution to be employed and temperature conditions are not limited to those mentioned above. Any conditions may be employed as long as the probes A1-An and probes B1-Bn can hybridize with respective target nucleic acids.
Subsequently, the probes A1-An are bound the corresponding probes B1-Bn (
Furthermore, in the case where the sequence F and the sequence S are not placed next to each other on the target nucleic acid, the gap between them may be filled by use of DNA polymerase I and a ligase. In this way, nucleic acid bases are introduced into the gap between both probes. Any DNA polymerase I and ligase may be used as long as they are generally used. Preferably, heat resistant DNA polymerase and ligase are used.
Subsequently, the probes (A1+B1) to (An+Bn) obtained above are dissociated from the target nucleic acids (not shown). The dissociation is performed by denaturation treatment such as thermal denaturation. For example, when the dissociation is performed by the thermal denaturation, the reaction is performed under physiological conditions at 85° C. or more, preferably, 90° C. or more. However, the temperature conditions are not limited thereto.
The probes (A1+B1) to (An+Bn) are recovered by using the sequences Tg1′ to Tgn′ which hybridize with complementary sequences Tg1 to Tgn (
Subsequently, respective marker substances are detected or quantified to detect and quantify the target nucleic acid (
The complementary sequences Tg1′ to Tgn′ and FL′ to FLn′ used herein are preferably fixed on a support. Examples of the support to be used include, but not limited to, a plate such as a silicon plate or a glass plate, particles such as beads, a container such as a test tube or a vial, a fiber, a tube including a capillary, filter, an affinity column, an electrode and the like. It is preferable that the material of the support and surface treatment may be appropriately selected depending upon how efficiently the probe is fixed.
Now, a further preferable example of the present invention will be explained. If an appropriately designed flag is employed in the following method, a plurality of target nucleic acids can be efficiently detected with a high accuracy.
The following two types of nucleic acid probes, a probe A and a probe B, can be used in this method (
The probe A is formed of a nucleotide sequence F′ and a binding molecule bound to the sequence F′. The sequence F′ is complementary to a nucleotide sequence F which is a partial region of the target nucleic acid.
The binding molecule used herein is either one of two substances having specifically a high affinity with each other. For example, biotin, avidin, or streptoavidin. Furthermore, the binding molecule may bind directly to the sequence F′ or may be indirectly bind to the sequence F′ via an arbitrary sequence. The arbitrary sequence, which is responsible for the indirect binding, may be any nucleotide sequence having any number of nucleotides. More preferably, the sequence is a non-complementary sequence to the nucleotide sequence of the target nucleic acid.
The probe B is formed of a nucleotide sequence S′ and a flag. The nucleotide sequence S′ is complementary to a nucleotide sequence S which is a partial region of the target nucleic acid. The flag is a double strand. The double strand has an arbitrary sequence consisting of a plurality of units. The flag must not bind to the target nucleic acid and not exhibit any interaction with the target nucleic acid.
The sequences F′ and S′ used herein have a nucleotide of 1 or more, preferably 15 or more.
When a plurality of target nucleic acids are simultaneously detected, the flag FL of a plurality of types of units may be used. As an example, the flag FL consists of 4 units, SD, D0, D1, ED will now be explained. This flag is formed as follows. First, 22 types of units are designed. Then, two units are selected from them as primers. One is defined as the SD unit. The other one is defined as the ED unit. Subsequently, the D0 and D1 units are selected by using the remaining 20 units. If the flag is designed by varying the combination of the two units depending upon a type of nucleic acid, 100 types of nucleic sequences can be detect (
The 22 types of units are preferably designed by orthonormal nucleotide sequences. The Tm of the orthonormal nucleotide sequences fall within almost the same range, so that they do not make a stable hybrid with the non-complementary sequences. They do not form a stable secondary structure which prevents the formation of a hybrid with the corresponding complementary sequences. Therefore, the occurrence of mis-hybridization will be reduced at the time of a final detection operation, with the result that the hybrid-formation speed can be increased. It is therefore possible to improve the detection accuracy and to reduce the detection time. Furthermore, if the number and types of units are increased, 10000 types of nucleic acid sequences can be detected.
Referring to
Detection is performed as follows. First, the probe A and probe B are mixed with a target nucleic acid (
Subsequently, the probe A, probe B, and target nucleic acid(s) are incubated for a predetermined time under the conditions suitable for hybridization (
Both the probes A and B hybridize with the same target nucleic acid by the hybridization thus performed (
Subsequently, the probes A and B, both being hybridized with the same nucleic acid, are ligated to each other (
The Tm value of flag FL is preferably set at a temperature higher than those of the sequences F′ and S′. If the Tm value is set in this manner, it is possible to prevent denaturation of the flag, which lowers the detection sensitivity, during the heating and cooling operations at the time of hybridization, ligation, and denaturation involved in this detection method.
Subsequently, the information on the flag FL obtained is subjected to the B/F separation. More specifically, the binding molecule attached to the probe (A+B) is captured on a solid-phase carrier via a binding molecule to be paired up with the binding molecule of the probe (A+B) (
Examples of the solid phase carrier are a plate, beads, a container, a fiber, a tube, a filter, an affinity column, an electrode and the like. Preferably, beads are used.
The flag FL of the probe (A+B) bound to the binding molecule is denatured into a single strand (
After completion of the PCR reaction, a PCR product, that is, a double-stranded sequence, is recovered by permitting the binding molecule to bind to a solid-phase carrier immobilized (
Subsequently, the single-stranded flag sequence FL attached on the immobilized carrier is analyzed as follows. The immobilized carrier having the single-stranded flag sequence FL bound thereto, is equally divided into ten-portions (in this case, D1 unit consists of D1-1 to D1-10). To individual ten portions, one of D1-1′ to D1-10′ labeled with a marker molecule and all D0′ sequences (D0-1′ to D0-10′) are added to hybridize them with the flag sequence FL.
Subsequently, two nucleic acid molecules thus hybridized are ligated to each other. The ligation conditions and the definitions for the marker substance are the same as previously mentioned. Thereafter, the molecule ligated is denatured and recovered in a liquid phase.
The nucleic acid molecules labeled with the marker molecule are analyzed by hybridizing them with a DNA chip or a DNA capillary on which the nucleic acid molecules of D0-1 to D0-10 are previously immobilized. Particularly, the DNA capillary is advantageous since the nucleic -acid molecules which have been divided into D0-1 to D0-10 portions, can be simultaneously analyzed. The analysis can be easily made by the DNA capillary.
As an example, the case of the flag FL which is designed by using 10 types of sequences, D0-1 to D0-10, and 10 types of sequences, D1-0 to D1-10 will now be explained. Since the sequence D0-1 is immobilized at position 1 of
In this example, 10 types of units are used. However, the units are not limited to 10 types. More than 10 types or less than 10 types may be used.
The “DNA capillary” used herein is a device for detecting a target nucleic acid. Since a complementary sequence to the target nucleic acid is bound inside the device, the target nucleic acid can be detected by binding the target nucleic acid to the complementary sequence. As shown in
In the method of this example, since an orthonormal nucleotide sequence is used as each unit of the flag sequence FL, hybridization can be uniformly performed under the same conditions, e.g., a reaction temperature. Therefore, the detection can be made with a high accuracy while preventing mis-hybridization.
Furthermore, since numerous analyses can be performed simultaneously under the same conditions, the time required for the detection can be reduced.
According to the method of the present invention, complicated genomic information expressed by a nucleotide sequence of DNA can be converted into numerical values. Moreover, if calculation is made by using a DNA molecular reaction, analysis of various types of information and complicated genetic information mutually associated can be easily made. In addition, after a nucleic acid is encoded to a nucleic acid code, the nucleic acid code can be amplified easily. Therefore, even if the target sequences are present in low copy numbers, the target sequences can be accurately and quantitatively detected. Furthermore, it is possible to compress numerous data by encoding the sequence. Therefore, a number of detections can be made by using a fewer number of devices, such as a DNA chip or a capillary array.
The term “encode reaction” used herein is referred to converting a nucleotide sequence into codes based on orthonormal nucleotide sequences. The “encode reaction” includes the steps of
The term “decode reaction” used herein is referred to reading the code converted in the above and thereby reproducing original information. The step of
In Example 5, a single type of target nucleic acid is detected. If a plurality of types of flag sequences are designed, a plurality of types of target nucleic acids can be simultaneously detected by the same steps.
An application example of Example 5 is shown in
The resultant sequences D11′-D1n′ obtained are detected by using a chip or a capillary array to which the sequences D01-D1n are immobilized (
To demonstrate that the method of the present invention is useful in practice, the following experiment was performed. The sequence used herein and how to bind the sequence are shown in
(1) Target Sequence and Modified Sequence
A sequence r-32-f1 was used as a target sequence (
(2) Probe and Detection Sequence
A nucleotide b-16A was used as a probe A (
(3) Experimental Method
The target sequence was tried to be detected by using the aforementioned probes. First, a probe B solution containing 500 pmol of probe B in a TE buffer solution of pH 8.0 was mixed with a solution for a detection sequence in an equal amount. The resultant mixture was incubated at 95° C. for one minute and decreased in temperature to 25° C. for 7 minutes. Consequently, a double-strand probe B was obtained.
The double-stranded probe B obtained, the target sequence, and the probe A were mixed. The ligation reaction was performed using a Taq DNA ligase (manufactured by New England Biolab) of 20 U in 100 μL of a ligantion buffer solution. The ligation reaction was performed by gradually reducing temperature from 70° C. to 5 over 5 minutes to anneal the probes A, B and the target sequence. A ligation reaction was performed by adding TaqDNA ligase at 55° C. for 15 minutes. Thereafter, the temperature of the reaction is reduced to room temperature. After the ligation reaction, the specimen was added to magnetic beads on which avidin is immobilized. The resultant mixture was subjected to pre-cold wash at 30° C. to remove the target sequence unreacted. Further, cold wash was performed at the same temperature as above to remove the target sequence. After the supernatant was removed, 100 μL of TE buffer solution was added. The resultant solution was subjected to hot wash at 70° C. to recover the detection sequence.
(4) Experimental Results
The experimental results will be described below.
The specificity of the method of this example was investigated and the results are shown in
The following experiment was performed to demonstrate, in particular, the specificity and quantitative properties of the encode reaction and the decode reaction of the method of the present invention. The sequence used herein is shown in
7-1 Specificity of Encode Reaction
(1) Experimental Method
First, a probe BIATP (S′IGTP+SD+D0IGTP+D1IGTP+ED, 0.3 nM) for IGTP, a probe BLRG-47 (S′LRG-47+SD+D0LRG-47 +D1IGTP+ED, 0.3 nM) for LRG-47, a probe AIGTP (0.3 nM) for IGTP, and an IGTP gene (0.3 nM-0.003 nM) serving as a target gene, were mixed in a ligation buffer solution, which is supplied together with Taq DNA ligase (TaqDNA ligase, manufactured by New England Biolab). The temperature of the solution mixture obtained was reduced from 70° C. at a rate of 3° C./minute. When the temperature reached 55° C., a ligase (20 units) was added to make a total volume of 30 ML. Thereafter, a ligation reaction was performed for 15 minutes, 10 mg/mL of dynabeads M-280, streptoavidin (1 μL) were added and mixed for 15 minutes. After the supernatant was removed, 20 ML of Tris EDTA buffer solution (hereinafter, simply referred to as “TE”) was added. The resultant mixture was washed at 60° C. to clean probes A and B unreacted and the target nucleic acid sequence (cold wash or room-temperature wash). The washing was performed twice. After the supernatant was removed, 20 μL of TE was added. The flag sequence encoded was recovered at 95° C. Similarly, the encode reaction using LRG-47 was evaluated by ligating probes A and B responsive to the target gene LRG-47 gene. In the encode reaction, the probe ligated was subjected to PCR using the SD sequence and ED sequence as primers. Measurement was performed by capillary electrophoresis for single-strand.
(2) Experimental Results.
7-2. Quantitative Properties of the PCR Amplification After the Encode Reaction
(1) Experimental Method
The uniformity and quantification property of the PCR performed after the encode reaction were confirmed by using the following method. The experiment was performed with respect to the flag sequences for detecting IGTP and LRG-47. The flag sequences for detecting IGTP and LRG-47 include both SD sequence and ED sequence. PCR reactions of both the flag sequences for IGTP and LRG-47 were performed by use of the SD sequence and ED sequence. The amounts of strands of both sequences added to the PCR reaction fall within 100 pM to 10 fM. The cycle of the PCR reaction consisting of 94° C./30 seconds, 65° C./30 seconds, 65° C./60 seconds, and 72° C./30 seconds, was repeated 25 times.
(2) Experimental Results
7-3. Specificity and Quantitative Properties of the Decode Reaction
(1) Experimental Method
The sequences used herein are the same as mentioned above. More specifically, an SD+D0IGTP+D1IGTp+ED sequence added with biotin (hereinafter, also referred to as “b-code 1”) and SD+D0LRG-47+D1LRG-47+ED (hereinafter, also referred to as “b-code 2”) were subjected to a decode reaction. 0.2 μL of the b-code 1 and b-code 2 (concentration: 100 μM) were mixed with a TE buffer (49.8 μL) containing 1M NaCl and 40 μL of dynabeads M-280 streptoavidin (10 mg/mL). The resultant solution was stirred for 15 minutes, washed, at room temperature, and then, the supernatant was removed. To the obtained beads, a TE buffer solution of 50 μL was added, and the mixture was washed at room temperature. After the supernatant was removed, a further 50 μL of TE buffer solution was added to the beads obtained. The resultant beads were washed at 80° C. and the supernatant was removed. Thereafter, the resultant beads was washed at room temperature with a ligation buffer and the supernatant was removed. Subsequently, a reverse strand relative to D1 and reverse strands relative to D0IGTP and DoLRG-47 were added to the resultant beads to a final concentration of 400 nm, individually. The temperature of the resultant mixtures was raised to 95° C. to convert the reverse strand into a single-strand reverse strand. The temperature was further reduced at a rate of 7° C. per minute. Subsequently, Taq ligase of 20 units were added at 60° C. to the resultant mixture, the ligation reaction was performed for 15 minutes. After the supernatant was removed, 50 μL of the TE buffer was added to the reaction solution. The resultant mixture was washed at room temperature. Thereafter, the supernatant was removed, 50 μL of the TE buffer was again added. The temperature of the reaction solution was increased to 80° C. and the supernatant was recovered. In this manner, the probes ligated was recovered. The streptoavidin beads (40 μL) were added to the supernatant obtained and further a TE buffer solution (12.5 μL) containing 1M NaCl was added, and stirred for 15 minutes. Thereafter, the supernatant was recovered at 80° C., an aliquot (20 μL) of the supernatant was added to each of two tubes. The probe (b-1) having the sequence corresponding to DOIGTP having biotin added thereto and the probe (b-2) having the sequence corresponding to DOLRG-47 having biotin added thereto were added to each of two tubes in an amount of 100 μmol. To the resultant solution, further 16 μL of TE buffer solution containing 2M NaCl was added. The resultant mixture was increased to a temperature to 95° C. Thereafter, the temperature of the mixture was reduced to 25° C. at a rate of 10° C. per minute. In this manner, hybridization was performed. To this solution, 40 μL of the streptoavidin was added and the solution mixture was stirred for 15 minutes. Thereafter, the supernatant was removed and 40 μL of TE buffer solution was added to the resultant mixture. The mixture was washed at 40° C. Subsequently, the supernatant-was removed and the TE buffer solution (40 μL) was further added, and the temperature of the resultant mixture was increased to 70° C. The supernatant was recovered, and each supernatant was subjected to capillary electrophoresis for a single strand.
(2) The Experimental Results
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11-283148 | Oct 1999 | JP | national |
| 11-283437 | Oct 1999 | JP | national |
This is a Continuation Application of PCT Application No. PCT/JP00/06919, filed Oct. 4, 2000, which was not published under PCT Article 21(2) in English. This application is based upon and claims the benefit of priority from the prior Japanese Patent Applications No. 11-283148, filed Oct. 4, 1999; and No. 11-283437, filed Oct. 4, 1999, the entire contents of both of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09872881 | Jun 2001 | US |
| Child | 10712715 | Nov 2003 | US |
| Parent | PCT/JP00/06919 | Oct 2000 | US |
| Child | 09872881 | Jun 2001 | US |
