KR100442837B1 - Probe design method for the detection of neighboring SNPs or nucleotide sequence mutations - Google Patents

Abstract

In the present invention, when two or more SNP or nucleic acid sequence variants exist at adjacent positions (within a base range of 1 to 50 bp), each of the mutated bases can be accurately determined, so that any one of the two or more mutations can be detected. A probe capable of positioning a probe in a zone, including a probe in the zone to detect a variation occurring in an adjacent position of the mutation, and detecting a case in which two or more variations occur simultaneously in the adjacent position. It is about the design and design of the probe to include the combination in the above zone.

According to the method of designing the probe of the present invention, it is intuitive to know whether there is a variation in the adjacent position, whether the simultaneous variation occurs in the adjacent position, and whether the variations occur at the same time.

Description

Probe design method for the detection of neighboring SNPs or nucleotide sequence mutations}

The present invention relates to a probe design method for detecting adjacent SNP or nucleic acid sequence mutations. More specifically, the present invention precisely determines the mutated base by controlling the background noise that appears during the detection of several SNP or nucleic acid sequence mutations within adjacent sequences (between 1 and 50 bases). It is about how to design and design a DNA chip.

The human genome contains several types of mutations, including SNPs (Single Nucleotide Polymorphisms), microsatellite, insertion, and loss (see Kleyn PW and Vesell ES, Science 281: 1820-1821 (1998)). . Many of the techniques of pharmacogenetic research that detect and sequence mutations with high productivity include gel electrophoresis-based genotyping, fluorescent dye-based genotyping, and molecular labeled sequencing. (molecular beacon genotyping), mass spectrometry genotyping, DNA chip-based microarray (Shi MM, Clinical Chemistry 47 (2): 164-172 (2001)).

Currently, DNA chips have been developed as an indispensable tool in experiments, diagnosis, and treatment in genomics, medicine, and biochemistry. Currently, gene expression analysis by DNA chips is one of the most widely used tools in functional genomics. In addition, it is possible to determine the sequence of nucleic acids using DNA chips or to determine the variation of SNPs and nucleic acid sequences (see: Yershov G. et al., Proc. Natl. Acad. Sci. USA, 93: 4913-). 4918 (1996)).

DNA chips require the design of nucleic acid probes to detect mutations in disease-related genes for the purpose of diagnosing and treating specific diseases. The design of such a probe is important to detect the mutations present in the target nucleic acid specifically and with high reliability through hybridization with the target nucleic acid. When hybridizing with a probe between a mutant target oligonucleotide and a mutant normal target oligonucleotide, the detection power of the mutation can be increased, that is, the difference in each signal can be maximized. The probe needs to be designed so that it

It is possible to build an array of probes on the stationary phase that are exactly complementary to the reference sequence or that contain one or more mutants, and this process is called probe design. There are many factors to consider in the design of the probe, especially in the case of oligonucleotide probes, the determination of the length and length of the probe is the hybridization melting point (Tm), the hybridization melting point difference (ΔTm), whether the hairpin structure is formed, Cross hybridization, formation, formation, mutation location, probe length, repeat sequence, etc. are considered in consideration of factors.

US Patent No. 6,027,880 provides an immobilized probe array and method for detecting mutations in the CFTR gene. U.S. Patent No. 5,837,832 discloses a DNA chip in which 100-100,000 probes are immobilized with 9-20 bases, which have at least four probe sets, one that is exactly complementary to the reference sequence, The rest is a set of probes in which one base is substituted for another at a given position.

WO 01/06013 A1 describes a method for evaluating one or more probes that can maximize the sensitivity and specificity for hybridization of target nucleic acids.

EP 1,103,910 (EP 1,103,910 A1) discloses a method for automatically selecting oligonucleic acid probes for detecting DNA mutations.

U.S. Patent No. 6,228,593 B1 is a computer system that analyzes the sequence of nucleic acid by analyzing the fluorescence intensity by hybridization of nucleic acid probes in the biochip to calculate the probability for an unknown base.

U. S. Patent No. 5,556, 749 relates to a computer system for designing optimized oligonucleotide probes and relates to calculations and designs relating to nucleic acid probes in DNA or mRNA hybridization processes. The probe designed in this invention takes into account the specificity or covalentness of GenBank DNA, mRNA sequence and candidate probe.

The above-mentioned prior art does not mention at all the design of probes that can accurately determine the modified base when several SNPs or nucleic acid sequence mutations are present in adjacent positions.

Indeed, when examining target samples with two or more SNPs or nucleic acid sequence mutations in adjacent positions, the distance between the mutations was not sufficient, resulting in interference with hybridization reactions due to adjacent mutations. The problem is that the base cannot be analyzed accurately.

Accordingly, the present inventors have made a thorough research to overcome the above problems, and as a result, a probe capable of detecting any one of two or more variations can be located in a predetermined region, and the variation occurring at an adjacent position of the variation can be detected. Designing a DNA chip probe that incorporates a probe into the zone and includes a combination of probes within the zone that can detect the occurrence of two or more variations simultaneously in adjacent locations. The present invention has been completed by confirming whether or not the simultaneous variation occurs at adjacent positions and whether the variations occur at the same time.

It is therefore an object of the present invention to provide a probe design method for detecting adjacent SNP or nucleic acid sequence mutations.

The probe design algorithm of the present invention provides an intuitive solution to the problem of inaccurate analysis of the target sample when two or more mutations occur adjacent to each other (1 to 50 bp bases) within the probe length. to be.

Fig. 1 is a diagram showing the normal when the 50th position of the target nucleic acid sample is A, and the variation when the G is G.

Fig. 2 shows the surface of the DNA chip on which the four types of probes, T, C, A, and G, respectively, in which the bases in the 50th position are located.

Fig. 3 shows a fluorescence signal when the solid surface of Fig. 2 is treated with a normal target sample (α column) and a homogeneous target sample (β column).

Fig. 4 shows a fluorescence signal when the solid surface of Fig. 2 is treated with a normal target sample (α column) and a heterogeneous target sample (β column).

Fig. 5 shows the case where the 50th and 51st mutations of the target nucleic acid sample are adjacent to each other, and is normal when the 50th position of the target nucleic acid sample is A; If the 5th position is C, it is normal, and if it is T, the figure shows variation.

In Fig. 6A, the 51st base is the same as G, and the 50th position of the base is T,

Four different types of probes, C, A, and G, and the surface probes represent the surface of the planted DNA chip.

Fig. 6B shows that the 50th base is the same as T, and the bases at the 51st position are each G,

Four types of probes, A, C, and T, represent the surface of the DNA chip on which they are planted.

7 shows the DNA chip surfaces of FIGS. 6A and 6B treated with normal target samples (50A, 51C) (column) and the 50th homologous target samples (50G, 51C) (β column). When the 51st homologous target samples (50A, 51T) were treated, the fluorescence signal of (γ column) was shown.

Figures 6A and 6B show DNA chip surfaces treated with normal target samples (50A, 51C) (α column) and 50th heterogeneous target samples (50A, 50G, 51C) (β column). ), And when the 51st heterogeneous target sample (50A, 51C, 51T) is treated, the fluorescence signal of (γ column) is shown.

Fig. 6 shows the DNA chip surfaces of Figs. 6A and 6B treated with normal target samples (50A, 51C) (α column) and 50th + 51th covariant target samples (50G, 51T). β, γ column).

Figure 10 shows the surface of a DNA chip in which a probe and a probe are designed according to the probe design method of the present invention in the case where two mutations are adjacent to each other.

Fig. 11 shows the DNA chip surface of Fig. 10 treated with normal target samples (50A, 51C) (column) and the 50th homologous target sample (50G, 51C) (β column), and the 51st. When the homologous target samples (50A, 51T) were treated, the fluorescence signal of (γ column) was shown.

Fig. 12 shows the DNA chip surface of Fig. 10 treated with the normal target samples (50A, 51C) (column) and the 50th + 51th covariant target samples (50G, 51T) (βcolumn). The fluorescent signal of.

Figure 13 shows the case where the 50th, 51st, and 55th mutations of the target nucleic acid sample are adjacent, so that when the 50th position of the target nucleic acid sample is A, the variation is normal; If the 5th position is C, it is normal; if it is T, it is a transition; if the 5th position is T, it is normal; if it is C, it is a figure showing the variation.

Figure 14 shows a probe designed according to the probe design method of the present invention, in which case the three variants are adjacent.

Fig. 15 shows the surface of the DNA chip on which the probe of Fig. 14 is implanted.

Fig. 16 shows the DNA chip surface of Fig. 15 treated with normal target samples (50A, 51C, 52T) (column) and the 50th homologous target sample (50G, 51C, 52T) (β column). And fluorescence signals when the 51st homologous target samples (50A, 51T, 52T) were treated (column), and when the fifth 52st homologous target samples (50A, 51C, 52C) were treated (δ column). Indicates.

Fig. 17 shows the case where the DNA chip surface of Fig. 15 is treated with the normal target samples (50A, 51C, and 51T) (α column), and the 50th + 51th simultaneous variation of the target samples (50G, 51T, and 51T). (β column), when the 50th + 52th simultaneous variation-target sample (50G, 51C, 522C) was processed (γ column), when the 51st + 52th simultaneous variation-target sample (50A, 51T, 5C) was processed (δ column), and the 50th + 51st + 52nd simultaneous variation? target samples (50G, 51T, 52C) (? column) are shown.

In order to achieve the object of the present invention, the present invention is to control the noise of the background when the two or more SNP or nucleic acid sequence mutations are present in the adjacent position, preferably in the base period of 1 ~ 50bp, the detection of these mutations, etc. Therefore, the present invention provides a method for designing a DNA chip probe that can accurately determine a modified base.

That is, the probe design algorithm places a probe that can detect any one of two or more variations in a predetermined region.

② include within the zone of step ① a probe capable of detecting a variation occurring at an adjacent position of the variation; And,

(3) A combination of probes capable of detecting a case where two or more variations occur simultaneously in an adjacent position is included in the ① step.

The region means a region on the DNA chip.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

1. Common cases

A DNA chip with an oligonucleotide probe was used to determine which target nucleic acid sample was at position A or G [A / G] (see Figure 1). In general, oligonucleic acid probes complementary to the target nucleic acids used are about 6 to 50 bases in length. If the base length of the probe is too short, it is economical and the specificity of hybridization bond is increased, but it is similar to that of a universal primer. There is a greater chance that unwanted hybridization will occur. On the other hand, if the base length of the probe is too long, the economy is low, and the specificity of the hybridization bond is reduced. Therefore, it is generally known that the base length of the probe of about 9 to 21 is appropriate (see US Patent 6,027,880).

In Fig. 1,-denotes any given nucleic acid base for which no mutation occurs.

If you want to read the target sample as shown in Figure 1 on the DNA chip, a probe nucleic acid of suitable length and length is designed, synthesized and planted on the DNA chip. At this time, the determination of the interval and length of the probe may determine factors such as hybridization melting point (Tm), hybridization melting point difference (ΔTm), hairpin structure formation, cross hybridization formation, mutation position, probe length, repeat sequence degree, etc. The MeltCalc ^™ program (see Schutz E and Ahsen, Biotechniques 27: 1218-1224 (1999)) makes it easy to design probes that take these factors into account.

Suppose we design a probe with four types of 20 mer as shown in Figure 2 and plant it on a solid surface. In Figure 2,-means a base completely complementary to the target sample. Here, probe # 1 places the base that can hybridize in the absence of mutation, and probe # 2 allows hybridization in the presence of mutation if it can predict how the mutation occurs. The base is located. Probes 3 and 4 locate bases that are not present in probes 1 and 2. However, the design of the DNA chip can change the position of the probe for reasons such as consistency of other probes and arrangements. The α column is indicated to indicate the fluorescent signal of the DNA chip when the normal target sample is treated, and the β column is the fluorescent signal when the mutant target sample is treated. The black circles show where each probe is located on the stationary bed.

When the normal target sample (50A, indicating that the base at position 50 is A) was treated on the DNA chip (α column) and the homologous target sample (50G, homozygote mutation) was treated (β column). Three signals can be obtained. In Figure 3, the white circle means that the signal of the DNA chip probe is on, and the black circle means that the signal is off. Therefore, when the normal target sample (50A) was treated (α column), it was found that the first probe was completely complementary, and when the mutant-target sample (50G) was treated (β column), the second probe was turned on. In actual DNA chip experiments, however, the ideal fluorescence signal is not obtained as described above (On / Off method), but the brightest probe signal (background correction) and the next bright probe signal (background signal) are corrected. If dividing the value is higher than 1.2, the brightest signal is identified as the target nucleic acid (Hacia et al. 1998).

If the normal target sample (50A) was treated and the heterozygous target sample (having the 50A and 50G at the same time), the same signal as in Figure 4 is obtained. In Fig. 4, when the normal target sample (50A) was treated (α column), the first complementary probe was turned on. However, when the heterogeneous target sample (50AG) was treated (β column), the first and second probes were simultaneously used. You can see it turned on. This is normal for heterogeneous target samples. In this case, the probe signal values of No. 1 and No. 2 are divided into the higher value among No. 3 and No. 4 probe signals, and when it is higher than 1.2, the heterologous mutation is identified as the nucleic acid of the target sample. At this time, all background signal values should be corrected.

2. Problems when several SNPs or nucleic acid sequence variations exist within adjacent sequences (between 1 and 50 bases)

If two or more mutations in the target sample occur, if the positions between the mutations are not within the length of a probe (1 to 50 bp), they can be interpreted as independent mutations. Or if a nucleic acid mutation occurs, a problem occurs.

If the 50th mutation and the 51st mutation are adjacent (8 bp apart in Figure 5), then the 50th mutation is assumed to be A → G and the 51st mutation is C → T.

If the 50th and 51st mutations are adjacent to each other, the design of the probe will inevitably have only two probes overlapping. That is, the probe for reading the 50th variation (A → G) can be designed as shown in Fig. 6A. In Fig. 6A, the α column represents the fluorescence signal of the DNA chip when treated with the normal target sample, the fluorescence signal when the 50th variant target sample is processed, and the γ column is the fluorescence when the 51th variant target sample is processed. Marked to indicate signal. The black circles show where each probe is located on the stationary bed.

In the same way, a probe for reading the 51st variation (C → T) can be designed as in Fig. 6B.

Treatment of normal target samples (50A, 51C) on DNA chips such as FIGS. 6A and 6B (α column) and 50th homologous target samples (50G, 51C) (β column), and 51 When the first homologous target sample (50A, 51T) is treated (column), a signal as shown in Fig. 7 can be obtained.

As shown in Fig. 7, when the normal target samples (50A, 51C) were processed, it was found that the first probe of the α column was turned on, so that it was in good agreement with the prediction. When processing the 50th homologous target sample (50G, 51C), one can generally expect probe 2 of 50β and probe 1 of 51β to be turned on. Because the shift in position 50 occurred, position 51 is normal. However, in the 50 th β probe, the second probe was turned on to make a good prediction. In the 51 th β probe, no probe was turned on and buried with the background signal. In such cases, the experimenter or interpreter will be very confused whether this phenomenon is the result of a false test or is it a disturbance of the hybridization reaction due to adjacent mutations. This prevented the 1 st probes (50T, 51G) of 51β from turning on because they were not independent of each other because there was not enough distance between the 50th and 51st variations of the variant target sample. Likewise, when the 51st homologous target samples (50A, 51T) were processed, the 2nd probe turned on for the 51st γ probe, which was well predicted, but the 50th γ probe did not turn on any probe, so it was like a background signal. It is buried.

Thus, if there is an adjacent variation, an algorithm for the array of probes may not be determined, which may lead to considerable confusion.

However, in the case where the above-described variation is a heterogeneous variation rather than a homogeneous variation, the interpretation of the experimental result is the same as in the case where there is an independent variation, as described later. When the normal target samples (50A, 51C) were treated on the DNA chips such as FIGS. 6A and 6B (α column) and the 50th heterologous target samples (50A, 50G, 51C) (β column), When the 51st heterogeneous target sample (50A, 51C, 51T) is treated (column), the same signal can be obtained.

As shown in the figure, when the normal target samples (50A, 51C) were processed, it was found that the probes of the α column were all turned on, which was in good agreement with the prediction. When the 50th heterogeneous target sample (50A, 50G, 51C) is treated, probes 1 and 2 of 50β and probe 1 of 51β are turned on. Similarly, when the 51st heterogeneous target sample (50A, 51C, 51T) is processed, probes 1 of 50γ and probes 1 and 2 of 51γ are turned on. This result shows that when the 50th and 51st mutations are heterogeneous, they generate signals without interfering with each other.

It is quite possible that the DNA of a patient with a disease caused by some genetic predisposition may have more than one base mutation. If two or more base mutations are adjacent to each other, it can be inferred from the diagram that one probe is not turned on by using the above-described one to four probes due to mutual interference. In case of normal target samples (50A, 51C), all probes of α column were turned on, which was in good agreement with the prediction, and when the 50th and 51st covariate target samples (50G, 51T) were (β, γ columns), one The probe did not turn on either. This is theoretically the 50th and 51st variations, so it may be natural to turn them off, but as mentioned above, if no probe is turned on, it will be buried with a background signal, causing confusion to the experimenter or interpreter. Given.

In Fig. 2, the β column and the gamma column are samples of the same base in terms of the target samples (50G and 51T) in which both the 50th and 51st mutations occur at the same time. However, the β and γ columns mean DNA samples drawn from different patients (sources).

As described above, when examining a target sample in which mutations occur in adjacent nucleic acid bases, the probe at the position where the mutation occurs is turned on, but in the case of a probe adjacent to the mutation, no probe is turned on, so the position is normal. It is not accurate whether the mutation has occurred, whether it is an error due to a false test, or if it is a disturbance of the hybridization reaction due to adjacent mutations. This prevented all adjacent probes from turning on because they were not independent of each other because there was not enough distance between variant target samples. In addition, when mutations occur in adjacent nucleic acid bases at the same time, the signal is not turned on for all adjacent probes, which makes it difficult to determine the mutation.

3. How to solve the problem when there are several SNP or nucleic acid sequence mutations in adjacent sequences (1-50 bases)

In the present invention, the present invention proposes a method of designing a probe that solves the above two problems. First, the probe corresponding to the modified base of the adjacent probes is included in its position to significantly increase the confidence of the adjacent mutation. Second, because neighboring mutations can occur at the same time, place a probe underneath to test its combination.

(1) Probe design method in the case of two adjacent variations (refer to Figure 10)

As shown in Fig. 10, it can be seen that the 5th probe of the 50th and 51st transition positions, as indicated by the arrow, was planted by bringing the 2nd probe of each opponent, and the 6th probes were the 50th and 51st It is for examination when the mutation position occurs at the same time.

When the normal target samples (50A, 51C) were treated on the DNA chip designed as shown in Figure 10 (α column) and the 50th homologous target samples (50G, 51C) were treated (β column), and the 51st homology When the mutant target samples (50A, 51T) are treated (column), the same signal as in Fig. 11 can be obtained. By arranging the probes as above, it is clearly known whether they are normal target samples or each adjacent variation samples.

As shown in Fig. 11, when the normal target samples (50A, 51C) were processed, it can be seen that since the first probe of the α column was turned on, the prediction was well performed. When the 50th mutant target sample (50G, 51C) was processed, the 2nd probe of the 50th β probe turned on to predict well, and the 5th probe of the 51st β probe turned on to the 51st position of this target sample. Is a normal sample without variation, but it is intuitive to see that probe 5 was turned on instead of probe 1 because of the variation in the adjacent 50th position. In the case of the simple probe array described above, no probe of the 51st β probe is turned on and buried with a signal in the background, which is largely distinguished from the result of a false test, an adjacent mutation, or confusion.

Similarly, when the 51st homologous target samples (50A, 51T) were processed, the 2nd probe turned on for the 51st γ probe and predicted well, and the 50th γ probe turned on 5 so that the 50 mutation was normal. We can see at a glance that the sample is turned on, but instead of probe 1, because of the variation in the adjacent 51st position.

Therefore, when examining a target sample with adjacent mutations, by arranging the probes as above, at least one probe is turned on at one transition position, which gives an intuitive confidence in the variation that is distinguished from the background noise. will be.

In addition, the target sample (50G, 51T) in which the 50 and 51 mutations occurred simultaneously in the design of the probe was very confusing in interpreting whether the probe resulted from a false test because no probe was turned on in the simple probe array as before. Now, each probe is now turned on six times, so it's intuitive to see that it's a simultaneous coincidence (cf. Fig. 12), and one probe is turned on at least one of the transitions to distinguish it from background noise at once.

As a result of the analysis of the mutated bases as described above, it is intuitive to know whether mutations occur at adjacent positions at the same time. In this example, only two mutations occur at the same time, but two or more mutations may be adjacent to each other. The case makes it intuitive to know which combinations of positions occur at the same time.

This probe design algorithm can solve the problem of not being able to accurately analyze the mutated base analysis of the target sample when two or more mutations occur adjacent to each other (1 to 50 bp base) within the probe length. .

(2) Probe design method with three adjacent variations (refer to Fig.13 to Fig.16)

It will be described how the three variations are adjacent using the probe design algorithm.

The 50th variation is assumed to be A → G, the 51st variation is C → T, and the 52nd variation is T → C (see Fig. 13). If the 50th, 51st and 52nd variations are adjacent, the probe Inevitably, the three probes will inevitably overlap. That is, a probe for detecting the 50th, 51st, and 52th mutations can be designed as shown in Fig. 14.

In Fig. 14, arrows indicated that the 5th probe of the 51st and 52nd mutant positions is taken from the 2nd probe of the 50th transition position, and the 52nd 6th and 50th 5th probes are the 51st transition position. Probe 2 was taken and planted, and the 50th and 51st probes were taken and planted with the 2nd probe at the 52nd transition position.

Probes 7, 8, 9, and 10 are for examination when mutations occur simultaneously at the 50th, 51st, and 52nd transition positions. Probe 7 is the (50, 51) th and 8th probes ( The 50th, 52th and 9th probes are used to check for mutations at the (51, 52) th and 10th probes at the (50, 51, 52) th position. The order of the probes is not important here, and it is important to design the probes including the number of cases where all two or more variations of the three transition positions can be examined.

In Figure 15 and Figure 16, the α column represents the fluorescent signal of the DNA chip when the normal target sample is processed, and the β, γ, and δ columns represent the fluorescence signal when the 50th, 51st, and 52th variant target samples are processed, respectively. Marked to indicate.

When the normal target samples (50A, 51C, 52T) were treated on the newly designed DNA chip as described above (α column) and when the 50th homologous target samples (50G, 51C, 52T) were treated ( β column), when treated with 51st homologous target samples (50A, 51T, 52T) (γ column), and when treated with 52nd homologous target samples (50A, 51C, 52C) (δ column) You can get a signal like By arranging the probes according to the new algorithm as above, it is clear whether the sample is a normal target sample or each adjacent side sample.

As shown in Fig. 16, when the normal target samples (50A, 51C, 52T) were processed, it can be seen that since the first probe of the α column was turned on, the prediction was well performed (α column). When the 50th mutant target sample (50G, 51C, 52T) was processed, the 2nd probe of the 50th β probe was turned on to predict well, and the 5th probe of the (51, 52) β probe was turned on to make this target. It is intuitive to see that the (51, 52) position of the sample is a normal sample without variation, but probe 5 is turned on instead of probe 1 because of the variation of the adjacent 50th position. Thus, the simple probe array can be distinguished from the case of confusion in interpreting whether the probe of the (51, 52) beta probe is not turned on and buried with a signal in the background, which is the result of the false test.

Similarly, when treating the 51st homologous target sample (50A, 51T, 52T) (γ column) and the 52nd homologous target sample (50A, 51C, 52C) (δ column), It can be seen that probe # 2 of the position is turned on, and other probes are turned on probe # 5 or # 6 indicating an adjacent mutation.

Thus, when examining a target sample with adjacent mutations, by arranging the probes as above, at least one probe is turned on at one transition position, thereby providing intuitive confidence in the variation that is distinct from background noise. Will be.

In the design of the above probe, the target sample where variations 50, 51, and 52 occurred simultaneously by multiple combinations (where the combination of three variants is (50 + 51), (50 + 52), (51+ 52), and (50 + 51 + 52), irrelevant in order), it was confusing that no probes were turned on in the simple probe array as before, resulting in a false test or simultaneous variation by combination. Since each of the seven to ten probes is turned on, it is intuitive to know that this is a simultaneous coincidence (Ref .: ７ ７). can do.

Based on the analysis of the mutated bases as described above, it is intuitive to know whether simultaneous mutations occur in adjacent positions, and whether or not mutations occur simultaneously in combinations of positions.

As a result, the probe design algorithm described above intuitively solves the problem that when two or more mutations occur adjacent to each other (1 to 50 bp bases) within the length of the probe, the mutated base analysis of the target sample cannot be accurately performed. That's how it can be.

In the above examples, we showed how to improve the analysis of mutated bases using the above probe design algorithm for the case where two and three variants are adjacent. Even if four or more mutations occur within any probe length (1 to 50 bp bases), the probe design method described above can solve both problems when detecting adjacent SNP or nucleic acid sequence variations. do.

As described above, even if two or more SNP or nucleic acid sequence variants exist in adjacent sequences (1-50 base intervals), the detection method of the adjacent SNP or nucleic acid sequence may be detected using the probe design method of the present invention. It solves all the problems, and greatly improves the confidence of whether there is a variation in the adjacent position, and also whether or not the variations occur at the same position and whether the variations occur at the same time. Intuitively.

Claims (2)

(a) placing a probe in a constant region capable of detecting any one nucleotide variation out of two or more nucleotide variations; (b) including a probe within the zone of step (a) that can detect nucleotide variations occurring at adjacent positions of the nucleotide variations; And (c) incorporating a combination of probes capable of simultaneously detecting two or more nucleotide variations at adjacent positions into the region of step (a), wherein the two or more monobasic polymorphisms at adjacent positions (SNPs) Or a probe for detecting nucleotide sequence variation. The method of claim 1, wherein the adjacent position is designed to be characterized by being within a base length of 1 to 50 bp.