JP2007135540A - Method for discriminate target base of dna and allele-specific primer used thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an allele-specific primer in which when a base directly adjacent to the 3' side of a target SNP base is T and a base adjacent to the base is G, possibility of false positiveness is extremely low and clear SNP discrimination is possible. <P>SOLUTION: The method for discriminating a target base of DNA is designed such that a 3' terminal base is an SNP corresponding base, the second base from the 3' terminal is G, the third base from the 3' terminal is G or T and the base sequence from the fourth base from the 3' terminal to the 5' terminal base is complementary to a base sequence from the target SNP base to a desired base from a base adjacent to three bases to the 3' side. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for discriminating a target base possessed by DNA and an allele-specific primer used therein.

  SNP is the most frequently occurring polymorphism among gene polymorphisms, and is considered to occur at a frequency of about 0.1% in the human genome.

  In fact, the existence of over 3 million SNPs has become apparent so far, indicating that SNPs are very useful as markers for genetic testing.

  Studies on the association between SNPs and diseases so far have revealed that SNPs are closely related to diseases such as diabetes and hypertension.

  As a technique for performing SNP discrimination, a technique using a primer extension reaction (including a PCR reaction) using an allele-specific primer is known.

  The allele-specific primer refers to a primer that causes a significant difference in primer extension reaction efficiency depending on the target SNP base species.

  Therefore, for example, a target SNP base species can be easily identified by performing a PCR reaction using an allele-specific primer and analyzing the amount of the PCR product.

  Usually, an allele-specific primer is simply an oligo DNA without any particular modification.

  Since the so-called usual electrophoresis method may be used for the analysis of the PCR product amount, the SNP discrimination technique using the allele-specific primer can be said to be very advantageous in terms of cost, reaction time, and ease of operation.

  As for the analysis method, not only the above-described electrophoresis method, but also a solid phase reaction can be used to detect by QCM or SPR.

  In recent years, a method for detecting PPi (pyrophosphate), which is a byproduct of the primer extension reaction, has been developed using a luciferase reaction. Energetic efforts are being made.

  The sequence design of the allele-specific primer is a very important factor in its SNP discrimination ability.

  The most classic example is that the 3 ′ terminal base corresponds to the target SNP base (ie, is complementary to one of the expected target SNP base species) and the other sequences are relative to the target DNA sequence. It is an example of being completely complementary.

  However, in this case, the reaction time and temperature, the concentration of dNTPs used in the reaction, and if the PCR method is used, a false positive problem arises unless reaction conditions such as the number of cycles are strictly defined.

  In other words, if the SNP-corresponding base at the 3 ′ end of the primer is non-complementary to the target SNP base of the sample, the primer extension reaction from that should not occur, but in many cases it occurs. End up.

  In order to solve the above-mentioned false positive problem, several new allele-specific primers have been developed so far.

  In the allele-specific primer proposed in Patent Document 1, the 3 ′ terminal base is a SNP-compatible base, and the third base from the 3 ′ terminal is set to the base adjacent to the second base 3 ′ from the target SNP base. We are developing allele-specific primers that are intentionally non-complementary.

  In Patent Document 2, the second base from the 3 ′ end is a SNP-corresponding base, and the third base from the 3 ′ end is intentionally made to the base adjacent to the 3 ′ side from the target SNP base. Non-complementary allele-specific primers have been proposed. In this case, KOD polymerase which is 3 '→ 5' exo + polymerase is used.

  In Patent Document 3, the 3 ′ terminal base is a SNP-compatible base, and the second and third bases from the 3 ′ terminal are respectively the base adjacent to the 3 ′ side from the target SNP base and the base adjacent to the other base. An allele-specific primer that has been intentionally non-complementary has been proposed. Among these, the allele-specific primer proposed in Patent Document 3 is particularly unlikely to be false positive.

  That is, the allele-specific primer of Patent Document 3 designed as described above has an SNP-corresponding base (shown as “S ′” in the figure) located at its 3 ′ end as shown in FIG. When complementary to the base (indicated as “S” in the figure), only the second and third bases from the 3 ′ end are non-complementary to the target DNA sequence (in the figure, non-complementary A simple base pair is indicated as x), and a loop-like structure can be taken (FIG. 1A).

  As a result, the polymerase binds efficiently and performs a primer extension reaction.

  Conversely, when the SNP-corresponding base located at the 3 ′ end is non-complementary to the target SNP base, all the three bases at the 3 ′ end are non-complementary and are considered to have a branched structure (FIG. 1). (B)).

  In this case, the primer extension reaction hardly occurs.

  This is because the binding efficiency of the polymerase is considered to be extremely low.

  Accordingly, the allele-specific primer proposed in Patent Document 3 has a very low possibility of false positive as described above.

  In addition, the following documents can be listed as documents related to the present invention.

  In Patent Document 4, for the rice variety identification, the 3 ′ end corresponds to the SNP site to be identified, and the third base from the 3 ′ end is replaced with a sequence complementary to the template sequence to be annealed. PCR primers are disclosed in which the substitution is a G to A, A to C, T to G, or C to A substitution.

Furthermore, in paragraph No. 0040 of Patent Document 4, “in one aspect, the present invention provides a primer in which the first, second, third, or fourth base from the 3 ′ end is substituted. This substitution may be only one or a combination of two or more, preferably only the third base from the 3 ′ end is substituted, and this primer substitution may be any substitution. Is preferably a G to T, A to C, T to G, or C to A substitution. "
US Patent Publication No. 2003/0049628 US Patent Publication No. 2003/0148301 US Patent Publication No. 2004/0197803 JP 2004-248635 A

  The allele-specific primer proposed in Patent Document 3 has very poor primer extension reaction efficiency when the SNP-corresponding base located at the 3 'end is non-complementary to the target SNP base. As a result, the false positive problem is solved, so that accurate SNP discrimination becomes possible.

  However, when the SNP-corresponding base located at the 3 ′ end is complementary to the target SNP base, the primer extension reaction efficiency is the second and third base species from the 3 ′ end and the corresponding base on the target DNA side. There is a problem that it varies depending on the seed condition (more specifically, the base species condition of the base adjacent to the 3 ′ side of the target SNP base and the base adjacent to the other base).

  More specifically, for example, the target SNP base species in the target DNA sequence is A (adenine) or G (guanine), the base species immediately adjacent to the 3 ′ side of the SNP base is C (cytosine), If the base type next to the base is T (thymine), the 3 'terminal base of the allele-specific primer proposed in Patent Document 3 is a SNP-corresponding base, so is T (when complementary to A)? C (when complementary to G) may be used.

  On the other hand, since the second and third bases from the 3 ′ end must be non-complementary to the target DNA sequence, the second is one of A, T, and C, and the third is T, Either G or C.

  Therefore, the allele-specific primer proposed in Patent Document 3 has a total of nine combinations of the second and third base types from the 3 'end to one type of target DNA sequence.

  From the standpoint that all are non-complementary, these nine types of allele-specific primers appear to exhibit approximately the same efficiency in SNP discrimination.

  However, the present inventors have found that, among these nine types of allele-specific primers, a specific allele-specific primer has a very high efficiency in SNP discrimination compared to other allele-specific primers. The present invention was completed.

  That is, an object of the present invention is to provide a method for discriminating a target base possessed by DNA which has a low possibility of false positive and can be clearly discriminated, and an allele-specific primer used therefor.

The present invention for solving the above problems is a method for discriminating a target base possessed by DNA,
This method
(1) It has a base complementary to the base expected as the target base among the four types of bases A (adenine), T (thymine), G (guanine), and C (cytosine) at the 3 ′ end. A DNA extension step in which an allele-specific primer is bound to the DNA to cause a DNA extension reaction, and (2) the efficiency of the DNA extension step is examined, and if the efficiency is high, the target base is the same as the expected base The target base is determined to be different from the expected base if the efficiency is low,
In the DNA, the base immediately adjacent to the 3 ′ end side of the target base is T, and the base adjacent to the other base is G.
The 3 ′ terminal base of the allele-specific primer is complementary to the base expected to be the target base;
The second base from the 3 ′ end of the allele-specific primer is G;
The third base from the 3 ′ end of the allele specific primer is either G or T, and the base between the fourth base from the 3 ′ end and the 5 ′ end base of the allele specific primer A base between the base on the DNA side corresponding to the fourth base from the 3 ′ end of the allele-specific primer and the base on the DNA side corresponding to the base on the 5 ′ end of the allele-specific primer Binds complementary to the sequence.

  The DNA extension reaction is preferably a primer extension reaction. More preferably, the DNA extension reaction is a primer extension reaction from only the allele-specific primer.

  It is preferable to examine the efficiency by examining the concentration of pyrophosphate produced by the primer extension reaction. In addition, it is preferable to detect the concentration of pyrophosphate as the emission intensity.

  It is also preferred that the DNA extension reaction is a PCR reaction. More preferably, the DNA extension reaction is a PCR reaction comprising the allele-specific primer and another different primer.

  In this case, it is preferable to examine the efficiency by measuring the concentration of the amplified DNA produced by the PCR reaction by electrophoresis.

  Allele-specific primers used in the above method are also included in the spirit of the present invention.

  According to the present invention, there is provided a method for discriminating a target SNP base possessed by DNA which has a very low possibility of false positive and can be clearly discriminated by SNP.

  Hereinafter, embodiments of the present invention will be described with reference to FIGS.

(Embodiment 1)
In this Embodiment 1, the base species conditions of the allele specific primer in this invention are demonstrated using FIG.

  In the present invention, as shown in FIG. 2, as a target DNA sequence, a target base (hereinafter referred to as “target SNP base”) is present at the 5 ′ end, and the second base from the 5 ′ end is T. And when the third base from the 5 'end is G.

  To facilitate understanding of the present invention, FIG. 2 assumes that the target SNP base is at the 5 'end. However, generally, as shown in FIG. 1, the target SNP base only needs to exist somewhere in the target DNA sequence, and does not need to exist at the 5 'end of the target DNA sequence.

  Since the 3 ′ terminal base of the allele-specific primer is a SNP-corresponding base, it appears to be complementary to either of the two predicted target SNP base types (indicated as S1 and S2 in FIG. 2). To design. In FIG. 2, this SNP-corresponding base is S1 'and is complementary to S1.

  Furthermore, the second base from the 3 'end of the allele-specific primer is G, and the third base from the 3' end is either G or T.

  A base sequence between the 4th base from the 3 ′ end of the allele-specific primer and a base at the 5 ′ end (hereinafter referred to as “residual base sequence” or “residual base sequence of allele-specific primer”) ) Is a base sequence between the base on the target DNA side corresponding to the 4th base from the 3 ′ end of the allele specific primer and the base on the target DNA side corresponding to the 5 ′ end base of the allele specific primer ( Hereinafter, it is complementary to “residual base sequence” or “residual base sequence on the target DNA side”), and these bind to each other.

  In FIG. 2, the base sequence from the 4 ′ base to the 5 ′ end base from the 3 ′ end of the allele-specific primer is from the 4 ′ base to the 3 ′ end base in the target DNA sequence. Complementary to the base sequence.

  It is desirable that the remaining base sequence of the allele-specific primer and the remaining base sequence on the target DNA side be completely complementary. However, as long as they do not adversely affect SNP discrimination, they are completely complementary. There is no need.

  In the present invention, when the SNP-corresponding base of the allele-specific primer is complementary to the target SNP base, the relationship is as shown in FIG. 2 (a). Conversely, the SNP-corresponding base of the allele-specific primer is the target SNP base. In the case of non-complementary, the relationship is as shown in FIG.

  In FIG. 2 (and FIG. 1), the symbol “X” means that two bases sandwiching the symbol X are not bonded.

  Two bases (for example, the base S1 and the base S1 'in FIG. 1 (a) and FIG. 2 (a)) in which the code “X” is not sandwiched are bonded.

  When a DNA extension reaction (ie, typically a primer extension reaction or a PCR reaction) is performed using a polymerase having no 3 ′ → 5 ′ exonuclease activity under the sequence conditions of such allele-specific primers and the target DNA. In the case of FIG. 2 (a), the reaction proceeds very efficiently, whereas in the case of FIG. 2 (b), the reaction hardly proceeds.

  The target SNP base possessed by the DNA can be discriminated from the difference in efficiency.

  That is, the efficiency of the DNA extension step is examined. If the efficiency is high, the target SNP base is determined to be the same as the predicted base. If the efficiency is low, the target SNP base is not the predicted base. It is determined that they are different.

  As a result, the possibility of false positive is extremely low, and clear SNP discrimination becomes possible.

(Embodiment 2)
In Embodiment 2, a method for discriminating SNPs by performing a primer extension reaction using the allele-specific primer in the present invention will be described.

  As shown in FIG. 3, first, a double-stranded DNA containing a target DNA sequence is prepared so that a primer extension reaction can be performed.

  Here, the target DNA sequence conditions are the same as in Embodiment 1, in which the target SNP base is present at the 5 ′ end, the second base from the 5 ′ end is T, and the third from the 5 ′ end. The base is G.

  Accordingly, in the complementary strand of the target DNA sequence, the 3 ′ terminal base is complementary to the target SNP base, the second base from the 3 ′ end is A, and the third base from the 3 ′ end. The base is C.

  A double-stranded DNA satisfying such a sequence condition is prepared so that a primer extension reaction can be performed as described above, and an allele-specific primer satisfying the sequence condition described in the first embodiment, a DNA polymerase, dNTPs, The primer extension reaction may be performed by adding a buffer and a desired salt as necessary.

  The procedure of the primer extension reaction may be a procedure known so far, and is specifically as follows.

  That is, firstly, the double-stranded DNA is placed in a high-temperature state to denature it, and then the temperature is lowered so that the allele-specific primer and the target DNA sequence in the present invention can bind.

  Thereafter, the temperature condition may be set such that the DNA polymerase can perform an extension reaction.

  As a result, if the SNP-corresponding base located at the 3 'end of the allele-specific primer is complementary to the target SNP base, the primer extension reaction from this allele-specific primer is efficiently performed.

  Conversely, if the SNP-corresponding base located at the 3 'end of the allele-specific primer is non-complementary to the target SNP base, the primer extension reaction efficiency from this allele-specific primer is extremely poor.

  Accordingly, the efficiency of the primer extension reaction may be examined by measuring the amount of pyrophosphate released as a result of the primer extension reaction during or after the primer extension reaction.

  As a result, the possibility of false positive is extremely low, and clear SNP discrimination becomes possible. The means for examining the efficiency of the primer extension reaction is not limited to the method for measuring the amount of pyrophosphate.

  For example, the primer extension reaction is performed in a state where the 5 ′ end side of the allele-specific primer in the present invention is fixed on a crystal oscillator, the change in frequency is analyzed, and the primer extension reaction is monitored. The elongation reaction efficiency may be examined.

  Alternatively, in the state where the 5 ′ end side of the allele-specific primer in the present invention is fixed to the gold surface, the primer extension reaction is performed, and the surface plasmon resonance (SPR) phenomenon is analyzed to monitor the primer extension reaction. Primer extension reaction efficiency may be examined.

  In any case, it is sufficient that the primer extension reaction efficiency can be examined.

  Here, the specific procedure for performing the primer extension reaction is not limited to the above procedure.

  That is, in the above procedure, all necessary reagents are mixed in the reaction solution in advance and the primer extension reaction is performed. For example, first, a buffer in which double-stranded DNA containing the target DNA sequence and allele-specific primer are dissolved is used. Prepare the solution (if necessary, add the desired salt), perform the denaturation step at high temperature and bind to the target DNA sequence with the allele-specific primer, and then add the DNA polymerase and dNTPs in order. It may be done. In any case, the step may be a step capable of performing a primer extension reaction.

  Also, here, when the DNA containing the target DNA sequence is originally single-stranded, unlike the double-stranded case described above, there is no need for a step under high-temperature conditions in the sense of denaturing the double-stranded DNA.

  However, in order to control the binding reaction between the single-stranded DNA and the allele-specific primer, it is necessary to first adjust the dissociated state of both of them. More preferably, it is before the binding step between the primer and the target DNA sequence.

  Here, it is more preferable that the DNA polymerase has weak or no 3 '→ 5' exonuclease activity. That way the SNP discrimination is more accurate.

  Furthermore, the DNA polymerase may be exposed to high temperatures depending on the procedure of the primer extension reaction.

  In that case, it is necessary to use a heat-resistant material such as that used in PCR, for example, but this is not particularly necessary when the primer extension reaction is carried out by a procedure that is not exposed to such a high temperature.

  The method for measuring the amount of pyrophosphate is described in Nucleic Acids Research, 2001, Vol. 29, no. There are methods proposed in 19e93, WO 03 / 0786655A1, and Japanese Patent Application Laid-Open No. 2004-141158.

  However, it is not limited to these, and it is sufficient that the amount of pyrophosphate can be measured.

(Embodiment 3)
In the third embodiment, a method for discriminating SNP by performing PCR using the allele-specific primer in the present invention will be described.

  As shown in FIG. 4, first, a double-stranded DNA containing a target DNA sequence is prepared so that a PCR reaction can be performed. Here, the target DNA sequence conditions are the same as in Embodiment 1, in which the target SNP base is present at the 5 ′ end, the second base from the 5 ′ end is T, and the third from the 5 ′ end. The base is G.

  Accordingly, in the complementary strand of the target DNA sequence, the 3 ′ terminal base is complementary to the target SNP base, the second base from the 3 ′ end is A, and the third base from the 3 ′ end. The base is C.

  A double-stranded DNA satisfying such a sequence condition is prepared so that a PCR reaction can be performed as described above, and an allele-specific primer that satisfies the sequence condition described in the first embodiment, and the allele-specific primer and forward A PCR reaction may be performed by adding a second primer in a primer / reverse primer relationship, a DNA polymerase for PCR, dNTPs, Mg ions, a buffer, and, if necessary, a desired salt.

  As a result, if the SNP-corresponding base located at the 3 ′ end of the allele-specific primer is complementary to the target SNP base, the template DNA in the region sandwiched between the allele-specific primer and the second primer is efficiently amplified. Is done.

  Conversely, if the SNP-corresponding base located at the 3 'end of the allele-specific primer is non-complementary to the target SNP base, the template DNA in the region sandwiched between this allele-specific primer and the second primer is almost amplified. Not.

  Therefore, if the amount of amplification of this template DNA is examined by a method such as electrophoresis after the PCR reaction, the possibility of false positive is extremely low and clear SNP discrimination becomes possible.

  Alternatively, the amount of pyrophosphate released as a result of the PCR reaction may be measured during or after the PCR reaction.

  Even in this case, the possibility of false positive is extremely low, and clear SNP discrimination is possible.

  More preferably, the DNA polymerase for PCR has weak or no 3 '→ 5' exonuclease activity.

  The method for measuring the amplification amount of the template DNA is not particularly limited, and may be other than the above-described electrophoresis method.

  The method for measuring the amount of pyrophosphate is described in Nucleic Acids Research, 2001, Vol. 29, no. There are methods proposed in 19 e93, WO 03/0786655 A1, and Japanese Patent Application Laid-Open No. 2004-141158.

  However, it is not limited to these, and it is sufficient that the amount of pyrophosphate can be measured.

(Embodiment 4)
In the fourth embodiment, a SNP discrimination kit including an allele-specific primer in the present invention will be described.

  Users perform the primer extension reaction and pyrophosphate measurement reaction using the SNP discrimination kit described here, and the target SNP base species in their samples are very unlikely to be false positives and accurately. Discrimination becomes possible.

  The SNP discrimination kit according to the fourth embodiment includes an allele-specific primer reagent tube containing the allele-specific primer according to the present invention shown in the first embodiment for a target DNA sequence, and a DNA polymerase reagent tube containing a DNA polymerase. And a buffer reagent tube containing a buffer to which a desired salt is added if necessary, a dNTPs reagent tube containing dNTPs, and a pyrophosphate reagent tube containing a reagent for measuring pyrophosphate.

  Therefore, users first prepare a sample of single-stranded DNA or double-stranded DNA containing their target DNA sequence so that a primer extension reaction can be performed, and then allele contained in the allele-specific primer reagent tube. Using the specific primer and the DNA polymerase contained in the DNA polymerase reagent tube, the buffer contained in the buffer reagent tube, and the dNTPs contained in the dNTPs reagent tube, a primer extension reaction was performed according to the method described in the second embodiment. Just do it.

  Pyrophosphate released into the reaction solution during the primer extension reaction can be measured using a pyrophosphate measurement reagent contained in the pyrophosphate reagent tube.

  Therefore, the target SNP base species is very unlikely to be false positive based on the measurement result, and can be accurately discriminated.

  The reagent for measuring pyrophosphate is not particularly limited as long as it can measure pyrophosphate, but it is not limited to Nucleic Acids Research, 2001, Vol. 29, no. 19 If the luminescence reaction by luciferase is used as shown in e93, ATP sulfurylase for converting pyrophosphate into ATP, luciferin that reacts with ATP to perform a luminescence reaction (wavelength 520 nm), and the reaction Contains catalytic luciferase.

  These may be mixed in a single tube, or may be included in separate tubes.

  In any case, users can use pyrophosphoric acid released in the process of the primer extension reaction using these reagents for measuring pyrophosphoric acid, Nucleic Acids Research, 2001, Vol. 29, no. 19 Pyrophosphate can be optically measured according to the method shown in e93.

  Furthermore, in this case, dATP remaining unreacted in the primer extension reaction reacts with luciferin. More preferably, a tube containing an enzyme for decomposing these unreacted dATP is also included as part of the kit.

  This enzyme is preferably added to the primer extension reaction solution before the reaction with the above luciferase or before the conversion reaction from pyrophosphate to ATP, and the dATP decomposition reaction is performed.

  With regard to the pyrophosphate measurement reagent, if the pyrophosphate electrochemical measurement method shown in WO 03 / 077865A1 is used, pyrophosphatase for hydrolyzing pyrophosphate into phosphate and reaction with this phosphate GAPDH (glyceraldehyde 3-phosphate dehydrogenase) that catalyzes the reaction of GAP (glyceraldehyde 3-phosphate) with NAD, and phosphate-GAP-NAD, and the reaction of phosphate-GAP-NAD The resulting electron mediator (oxidized form) reacts with NADH and diaphorase that catalyzes the reaction between NADH and electron mediator.

  These may be mixed in a single tube, or may be included in separate tubes. In any case, the user can use pyrophosphoric acid released in the process of the primer extension reaction electrochemically in accordance with the method shown in WO 03/0786655 A1, using pyrophosphoric acid as a reagent. Acid can be measured.

For the pyrophosphate measurement reagent, if the electrochemical measurement method of pyrophosphate disclosed in Japanese Patent Application Laid-Open No. 2004-141158 is used, the pyrophosphate is hydrolyzed to phosphoric acid and H ⁺ transports H ^{+. +} -PPase - film containing ^{(H +} pyrophosphatase) and comprises a dye optical characteristics vary depending on the ^{H +} concentration.

  These may be mixed in a single tube, or may be included in separate tubes. In any case, the users can use the pyrophosphoric acid released in the process of the primer extension reaction optically according to the method disclosed in Japanese Patent Application Laid-Open No. 2004-141158. Pyrophosphate can be measured.

  There are many known methods for measuring pyrophosphate other than those listed here. Therefore, of course, the pyrophosphate measurement reagent is not limited to those listed here, but may be configured to include all or a part of the reagents necessary for pyrophosphate measurement.

  Instead of the pyrophosphate measuring reagent, a fluorescent indicator that binds to double-stranded DNA, such as Cyber Green I, may be included in the kit of the fourth embodiment.

  That is, when the primer extension reaction is performed in the presence of a fluorescent indicator that binds to double-stranded DNA typified by Cyber Green I, it is possible to analyze the primer extension reaction process in real time by monitoring the fluorescence. SNP discrimination can be performed from the analysis result.

  A fluorescent indicator that binds to double-stranded DNA typified by Cyber Green I may be contained alone in a single tube, or may be contained in a buffer reagent tube or the like. What is necessary is just to set it as the kit structure which can mix an indicator with a primer extension reaction liquid.

  Similarly, a TaqMan probe may be included in the kit of Embodiment 4 instead of the pyrophosphate measurement reagent.

  In other words, if the TaqMan probe is designed to bind to the desired region in the sequence where the primer extension reaction proceeds, the primer extension reaction process can be analyzed in real time by monitoring the fluorescence dependent on this TaqMan probe. Therefore, SNP discrimination can be performed from the analysis result.

  The TaqMan probe designed as such may be included in a single tube alone, or may be included in a buffer reagent tube or the like, and the TaqMan probe can be mixed with the primer extension reaction solution. A kit configuration may be used.

  Here, it is more preferable that the DNA polymerase has weak or no 3 '→ 5' exonuclease activity. That way the SNP discrimination is more accurate. Furthermore, the DNA polymerase is more preferably heat-resistant.

(Embodiment 5)
In the fifth embodiment, a SNP discrimination kit including an allele-specific primer in the present invention will be described.

  By using the SNP discrimination kit described here, the possibility that the target SNP base type in each sample is false positive is extremely low and the PCR reaction for accurately discriminating can be performed. .

  The SNP discriminating kit in the fifth embodiment is the second allele-specific primer in the present invention shown in the first embodiment with respect to the target DNA sequence and the relationship between the allele-specific primer and the forward primer / reverse primer. Primer reagent tubes containing primers, PCR DNA polymerase reagent tubes containing PCR DNA polymerase, buffer reagent tubes containing a buffer to which Mg ions and a desired salt are added as needed, and dNTPs reagents containing dNTPs It consists of a tube.

  Accordingly, the users first prepare a double-stranded DNA sample containing their target DNA sequence so that a PCR extension reaction can be performed, and then the allele-specific primer and the second primer contained in the primer reagent tube. PCR according to the method described in Embodiment 3 using the PCR DNA polymerase contained in the PCR DNA polymerase reagent tube, the buffer contained in the buffer reagent tube, and the dNTPs contained in the dNTPs reagent tube A reaction may be performed.

  More preferably, the kit according to the fifth embodiment includes a reagent for examining the PCR reaction efficiency. Examples of reagents for examining PCR reaction efficiency include reagents for measuring pyrophosphate, fluorescent indicators that bind to double-stranded DNA, such as Cyber Green I, and TaqMan probes, as in the third embodiment.

  The reagent for measuring pyrophosphate can measure pyrophosphate released into the reaction solution during the PCR reaction. Therefore, the target SNP base species is very unlikely to be false positive based on the measurement result, and can be accurately discriminated. About the kind of reagent for a pyrophosphate measurement, it is the same as Embodiment 4, and should just be the structure containing all or one part of a reagent required for a pyrophosphate measurement.

  For the fluorescent indicator and TaqMan probe that bind to double-stranded DNA, such as Cyber Green I, the PCR reaction process can be monitored in real time by performing the PCR reaction in the presence of these, and the target SNP base is determined based on the results. Species are very unlikely to be false positives and can be accurately identified.

  Therefore, these may be contained alone in one tube, or may be contained in a buffer reagent tube or the like, and a kit configuration capable of mixing these reagents into a PCR reaction solution may be used. .

  As described above, the allele specific primer, the SNP discrimination method using the allele specific primer, and the SNP discrimination kit including the allele specific primer in the present invention have been described through the first to fifth embodiments.

  These are not particularly limited to SNPs, and the same effect can be obtained for discriminating single-base differences due to mutations, or for discriminating single-base differences when artificially mutating a specific base. Needless to say.

  More specific examples of the present invention will be described below.

Example 1
In Example 1, the target base species in λDNA was determined by PCR reaction using the allele-specific primer in the present invention.

  First, mutant λDNA-6997-GC was prepared by artificially replacing the 6997th T / A base pair in λDNA (Takara Bio Inc.) with a G / C base pair.

And the discrimination of the substitution of these base pairs,
5′-GTCTGCCGCACCTATGGCTGGTGT-3 ′ (λ-TGT : SEQ ID NO: 1 )
5′-GTCTGCCGCACCTATGGCTGGGGT-3 ′ (λ-GGT : SEQ ID NO: 2 )
It was decided to carry out by PCR reaction using two kinds of primers consisting of as forward primers.

  The two types of primers are called λ-TGT and λ-GGT in this order.

Here, the 3 ′ terminal base T of the above-mentioned two kinds of primers is a double-stranded DNA sequence 5′-GTCTGCGCACCCTGGGCTGCAT-3 ′ / 5′-ATGCAGCCCATAGTGGCGCAGAC-3 ′ (SEQ ID NO: 3) consisting of the 6976th to 6997th wild type λDNA. ) , 5′-ATGCAGCCATAGGTGCGCAGAC-3 ′ (the antisense strand of SEQ ID NO: 3) is complementary to the 5 ′ terminal base A.

Similarly, the second base G from the 3 ′ end of the above-mentioned two kinds of primers is non-complementary to G / T and the second base T from the 5 ′ end of 5′-ATGCAGCCATAGGTGCGCAGAC-3 ′ (SEQ ID NO: 3). It is a relationship.

Although the third base species from the 3 ′ end of the two kinds of primers are different (T in the case of λ-TGT, G in the case of λ-GGT), 5′-ATGCAGCCATAGTGGCGCAGAC-3 ′ (SEQ ID NO: 3 ) In the non-complementary relationship with the third base G from the 5 ′ end.

The sequences from the 4 ′ base to the 5 ′ end of the above-mentioned two kinds of primers are similarly 3 from the 4 ′ base from the 5 ′ end of 5′-ATGCAGCCATAGGTGCGCAGAC-3 ′ (SEQ ID NO: 3). 'Completely complementary to the sequence up to the terminal base.

On the other hand, 3 of the two kinds of primers 'tribasic termini, mutant λDNA-6997-GC duplex DNA sequence consisting of 6976 to 6997 th 5'-GTCTGCGCACCTATGGCTGCA G -3' / 5'- C TGCAGCCATAGGTGCGCAGAC- 3 ′ (SEQ ID NO: 4) 5′- C TGCAGCCATAGGTGCGCAGAC-3 ′ (antisense strand of SEQ ID NO: 4) is non-complementary to the 3 bases at the 5 ′ end (underlined portion is the site of mutagenesis) .

As a reverse primer, DNA consisting of 5′-GAATCACGGTATCCGGCTGCCGCTGA-3 ′ (SEQ ID NO: 5) was used.

The reverse primer consists of 7,406 to 7,430 th λDNA double-stranded DNA sequence 5'-TCAGCGCAGCCGGATACCGTGATTC-3 '(SEQ ID NO: 6) / 5'-GAATCACGGTATCCGGCTGCGCTGA-3 ' of, 5'-TCAGCGCAGCCGGATACCGTGATTC-3 '(SEQ No. 6 sense strand) and is completely complementary.

  If the reaction proceeds well by the PCR reaction with λ-TGT and λ-GGT, a 455 bp DNA amplification product should be obtained.

  The experiment is as follows.

First,
2 μL of enzyme mixture of Light Cycler-FastStart DNA Master SYBER Green I kit (Roche Diagnostics)
10 μg / mL of the above wild-type λDNA or mutant λDNA-6997-GC,
1 μM of either λ-TGT or λ-GGT 1 μM of the above reverse primer 1.6 mM MgCl ₂
20 μL of a reaction solution containing was prepared.
Using this reaction solution, LightCycler, a thermal cycler manufactured by Roche Diagnostics, denaturation step: 94 ° C., 10 seconds, annealing step: 58 ° C., 10 seconds, extension step: 72 ° C., 10 seconds, number of cycles: PCR reaction was performed as 20 cycles.

  Each PCR reaction result was analyzed using Bioanalyzer 2100, which is a system for DNA electrophoresis manufactured by Agilent Technologies.

  The analysis results are shown in FIG.

  These results show the concentration (nM) of the target DNA fragment after each PCR reaction.

  From this result, the target DNA fragment was not detected in the case of mutant λDNA-6997-GC when either of the above λ-TGT and λ-GGT was used as a forward primer.

  On the other hand, in all cases of wild-type λDNA, the target DNA fragment was 32 nM or more.

  That is, it can be said that the single base difference in this example could be clearly discriminated by performing the PCR reaction using the above λ-TGT and λ-GGT as a forward primer.

(Comparative Example 1)
In this comparative example 1, as a comparative experiment of the above example 1, an attempt was made to discriminate the difference in one base between the wild type λDNA and the mutant type λDNA-6997-GC of example 1 using the following five types of primers. It was.

That is, 5′-GTCTGCGCACCTATGGCTGAGT-3 ′ (λ-AGT : SEQ ID NO: 7 ),
5′-GTCTGCCGCACCTATGGCTGACT-3 ′ (λ-ACT : SEQ ID NO: 8 ),
5′-GTCTGCCGCACCTATGGCTGTTT-3 ′ (λ-TTT : SEQ ID NO: 9 ),
5′-GTCTGCGCACCTATGGCTGTCT-3 ′ (λ-TCT : SEQ ID NO: 10 ),
5′-GTCTGCGCACCTATGGCTGGTT-3 ′ (λ-GTT : SEQ ID NO: 11 )
The same experiment as in Example 1 was performed using five types of primers having the sequence of

  These five types of primers are called λ-AGT, λ-ACT, λ-TTT, λ-TCT, and λ-GTT in this order.

These are the same as λ-TGT and λ-GGT used in Example 1, and the 3 ′ terminal base (T) is complementary to the 5 ′ terminal (A) of 5′-ATGCAGCCATAGGTGCCAGCAC-3 ′ (SEQ ID NO: 3). It is.

The second and third bases from the 3 ′ end are non-complementary to the second and third bases from the 5 ′ end of 5′-ATGCAGCCATAGGTGCGCAGAC-3 ′ (SEQ ID NO: 3) .

Its remaining sequence is completely complementary to the remaining sequence of 5′-ATGCAGCCATAGTGGCGCAGAC-3 ′ (SEQ ID NO: 3) .

  The analysis results are shown in FIG.

  These results show the concentration (nM) of the target DNA fragment after each PCR reaction as in FIG.

  From this, first, in any of λ-AGT, λ-ACT, λ-TTT, λ-TCT, and λ-GTT, in the case of wild-type λDNA, a target DNA fragment of 7 nM or more and 13 nM or less was detected slightly. On the other hand, the mutant λDNA-6997-GC is not detected at all.

  The concentration of the target DNA fragment in the case of the combination of these five kinds of primers and wild type λDNA (note the size of the vertical axis in FIGS. 5 and 6) is the combination of λ-TGT or λ-GGT and wild type λDNA. This is clearly smaller than the above case, and it can be seen that the discrimination ability of λ-TGT and λ-GGT is superior.

  As described above, as understood from Example 1 and Comparative Example 1, when the base immediately adjacent to the 3 ′ end of the target SNP base is T and the base next to the other base is G, allele specific If the second base from the 3 ′ end of the sex primer is G and the third base is either T or G, very high discrimination ability can be obtained.

  According to the present invention, there is provided a method for discriminating a target SNP base possessed by DNA having a very low possibility of false positive and capable of distinct SNP discrimination.

Diagram showing the relationship between template DNA and allele-specific primers in the prior art The figure which showed typically the relationship between the template DNA and allele specific primer in this invention The flowchart figure of the SNP discrimination | determination method of Embodiment 2. The flowchart figure of the SNP discrimination | determination method of Embodiment 3. The graph showing the experimental results of Example 1 The graph showing the experimental results of Comparative Example 1

Explanation of symbols

1: Allele-specific primer in the prior art 2: Template DNA in the prior art
3: Allele-specific primer in the present invention 4: Template DNA in the present invention

Claims (9)

A method for discriminating a target base possessed by DNA, comprising:
The method
(1) DNA having an allele-specific primer having a base complementary to the base expected at the 3 ′ end among the four types of bases A, T, G, and C attached to the DNA (2) examining the efficiency of the DNA extension step; if the efficiency is high, the target base is determined to be the same as the expected base; The target base has a discrimination step in which it is discriminated that it is different from the predicted base;
In the DNA, the base immediately adjacent to the 3 ′ end side of the target base is T, and the base adjacent to the other base is G.
The 3 ′ terminal base of the allele-specific primer is complementary to the base expected to be the target base;
The second base from the 3 ′ end of the allele-specific primer is G;
The third base from the 3 ′ end of the allele specific primer is either G or T, and the base between the fourth base from the 3 ′ end and the 5 ′ end base of the allele specific primer A base between the base on the DNA side corresponding to the fourth base from the 3 ′ end of the allele-specific primer and the base on the DNA side corresponding to the base on the 5 ′ end of the allele-specific primer A method of binding complementary to a sequence.   The method of claim 1, wherein the DNA extension reaction is a primer extension reaction.   The method according to claim 1, wherein the DNA extension reaction is a primer extension reaction from only the allele-specific primer.   The method according to claim 2, wherein the efficiency is examined by examining the concentration of pyrophosphate produced by the primer extension reaction.   The method of Claim 4 which detects the density | concentration of the said pyrophosphate as luminescence intensity.   The method of claim 1, wherein the DNA extension reaction is a PCR reaction.   The method according to claim 1, wherein the DNA extension reaction is a PCR reaction comprising the allele-specific primer and another different primer.   The method according to claim 6, wherein the efficiency is examined by measuring the concentration of amplified DNA produced by the PCR reaction by electrophoresis. An allele-specific primer for discriminating a target base possessed by DNA,
In the DNA, the base immediately adjacent to the 3 ′ end side of the target base is T, and the base adjacent to the other base is G.
The 3 ′ terminal base of the allele-specific primer is complementary to a base expected as the target base among four types of bases A, T, G, and C;
The second base from the 3 ′ end of the allele-specific primer is G;
The third base from the 3 ′ end of the allele specific primer is either G or T, and the base between the fourth base from the 3 ′ end and the 5 ′ end base of the allele specific primer An allele-specific primer, wherein the sequence binds complementarily to a base sequence on the 3 ′ end side from a base adjacent to 3 bases on the 3 ′ end side from the target base.

Images