JPWO2005001125A1 - Nucleic acid mutation and polymorphism determination method - Google Patents

Abstract

In the present invention, when a mutation or polymorphism is analyzed using a DNA microarray, each spot on the DNA microarray can be obtained even when the nucleic acid molecule used as a sample is heterozygous with respect to the sequence. It is an object of the present invention to provide a means for identifying whether the signal from is completely matched or incompletely matched between the probe and the target nucleic acid. The present invention corrects the signal due to non-specific hybridization by subtracting from the raw data the signal value obtained using a perfect match probe candidate and a sample consisting only of a perfectly matched nucleic acid. When the corrected signal value is a certain value or more, the signal is identified as a signal derived from a perfect match.

Description

  The present invention relates to a method for determining mutations and polymorphisms in mutation analysis using a microarray.

In recent years, technologies related to gene diagnosis, such as analysis of gene mutation information, prediction and diagnosis of diseases, and typing of viruses, have been greatly advanced. For example, a DNA probe complementary to each of a normal gene sequence or a gene sequence having a known mutation is prepared in advance, and sample DNA is individually hybridized to various probes, and the amount of these hybrids A method for determining the presence or absence of a mutation in a sample DNA by detecting and comparing the above has been performed as a genetic diagnosis method.
For detecting the hybrid, for example, a detectable label is applied to sample DNA, and the amount of DNA is indirectly detected by detecting the label. In general, fluorescence, a radioisotope, chemiluminescence, or the like is used for such a label.
In addition, based on the hybridization reaction using the complementarity of such DNA, hundreds to tens of thousands of DNA probes are two-dimensionally arranged in a lattice pattern, and the DNA in the sample solution is placed on this array. A system called a DNA microarray that detects the information of a plurality of genes at once by hybridizing with the above has started to spread.
In DNA microarrays, it is possible to analyze the status of multiple genes at the same time. Usually, it is often used for gene expression analysis, etc., but the qualitative nature of gene mutation and SNP (single nucleotide polymorphism) It is also beginning to be used for statistical analysis.
For example, US Pat. No. 5,202,231 describes a nucleotide sequence determination method using a DNA microarray, called the “Sequence by Hybridization (SBH) method”.
In the SBH method, possible nucleotide sequences of a certain length are arranged on a solid phase on an array, and a sequence is detected by detecting a completely complementary hybrid formed by a hybridization reaction with a sample DNA. Is to determine.
Science (1996), 274, pp. 610-614 shows that the hybridization between the probe and the target sequence shows a difference in hybridization intensity between the case of perfect match and the case of containing a single base mismatch, and further a difference in signal intensity occurs. Discloses a method for determining a sequence.
In general, when examining complementarity between an oligonucleotide and a sample DNA, not limited to SBH, it is difficult to examine the presence or absence of a hybrid with one type of probe. This is because there is no absolute signal value that can be judged to be perfectly complementary because the stability of the hybrid is affected by its sequence. Therefore, in the method described in the above-mentioned document (Science), a sequence in which a base that causes one base mismatch with the target sequence is prepared in the central portion of the probe sequence of the 15-mer oligonucleotide, and this is used to make a perfect match and 1 The presence or absence of a perfect match is determined by comparing the intensity of signals derived from base mismatches.
When performing mutation analysis using a DNA microarray, a sample nucleic acid molecule may be heterogeneous with respect to the sequence (in the case of heterozygous and in the case of heterogeneous). For example, in mammalian cells, since there are paternal and maternal sequences, there may be two types of sequence species. Further, when there is a variety of sequences as in the case of cancer cells, it is conceivable that more types of sequences are mixed. Therefore, when performing mutation analysis of a sample containing such a heterogeneous nucleic acid molecule, sufficient knowledge about mutation cannot be obtained simply by distinguishing between perfect match and incomplete match. In addition, when multiple types of perfect matches are mixed in different quantitative ratios, it is possible to determine whether a signal with relatively low signal intensity is a perfect match or an incomplete match. It cannot be judged directly from the value.

The present invention has been made in view of such a problem, and even when there are a plurality of types of perfect matches between a probe and a target sequence in a sample to be analyzed, a perfect match hybrid is obtained. It provides a means that allows for reliable identification.
That is, the nucleic acid mutation and polymorphism determination method of the present invention has the following configuration.
(I) The mutation or polymorphism determination method of the present invention includes a perfect match probe that perfectly matches a target nucleic acid, and a base that causes a mismatch of at least one base to the target nucleic acid, A step (1) of contacting a labeled sample nucleic acid with a probe set comprising at least one incomplete match probe identical to the perfect match probe immobilized on a solid phase carrier;
Measuring the signal from the hybrid formed at each spot (2);
Determining a probe in the hybrid that emits a low-intensity signal equal to or lower than a predetermined signal value (threshold A) as an incompletely matched probe candidate (3);
(4) determining a probe in a hybrid that emits a signal having the highest intensity among signals equal to or higher than another predetermined signal value (threshold B) as a first perfect match probe candidate; and A step (5) of determining that the incomplete match probe candidate is an incomplete match probe, and further determining the first complete match probe as a first complete match probe when both the first perfect match probe candidates exist;
It is characterized by including.
(Ii) In the mutation or polymorphism determination method of (i) above, if the incomplete match probe candidate cannot be determined in step (3), and / or the complete match probe candidate is determined in step (4). If the determination cannot be made, the method of steps (1) to (5) is performed again after changing the contact and / or measurement conditions in step (1) and / or (2) from the original conditions. Can be included.
(Iii) In the method for determining a mutation or polymorphism of (i) or (ii) above, after the step (5),
Identifying a probe in the hybrid that is a probe other than the first perfect match probe and emits a maximum signal with a signal intensity equal to or greater than a predetermined value (threshold C) as a second perfect match probe candidate (6) ;
Step (7) of performing the following signal correction steps (a) and (b) on the signal in the hybrid containing the second perfect match probe candidate:
Separately from the steps (1) to (6), a first correction nucleic acid consisting of only a sequence that perfectly matches the first perfect match probe is brought into contact with the same probe set as the probe set, A step (a) of measuring a signal when the probe set comes into contact with only the first correction nucleic acid; and a signal value obtained in the step (a) is subtracted from the signal value measured in the step (1). Determining a first corrected signal value for the signal derived from each hybrid (b);
When the first correction signal value in the correction step (b) is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the first correction signal value is identified as a second perfect match probe; Alternatively, when the first correction signal value in the correction step (b) has a signal intensity less than the threshold value C, the probe in the hybrid having the first correction signal value is identified as not the second perfect match probe. Step (8)
Can further be included.
(Iv) In the mutation or polymorphism determination method of (iii) above, there are a plurality of spots that emit a signal value equal to or higher than the threshold value B in the step (4), and the first perfect match probe in the step (5) Is determined,
After the step (8), with respect to the plurality of probes other than the first perfect match probe (second perfect match probe candidates), the correction step ( Step (9) for performing a) and (b)
(However, in this step,
Among the plurality of second perfect match probe candidates, for the probe contained in the hybrid having a signal value that is n-1th (n is an integer not less than 2 and not more than the type of probe),
The n-th correction nucleic acid consisting only of a sequence that perfectly matches the probe in the hybrid that emits the n-1st largest signal is the same as the probe set separately from the steps (1) to (6). Measuring the signal when the probe set is brought into contact with only the n-th correction nucleic acid;
Subtracting the measured signal value from the signal value that has been cumulatively corrected using each of the first to n-1th correction nucleic acids to obtain an nth correction signal value; and When the nth correction signal value is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the nth correction signal value is determined as the nth perfect match probe, or the nth correction signal value If the signal intensity is less than the threshold C, the probe in the hybrid having the n-th corrected signal value is identified as not a perfect match probe (however, before the determination of the n-th perfect match probe, the threshold value is When there are x hybrids (x is an integer greater than or equal to 0) having the mth correction signal value (m is an integer greater than or equal to 1 and less than n) with an intensity less than C, the nth The perfect match probe is determined to be the (nx) perfect match probe))
Can further be included.
(V) In the method for determining a mutation or polymorphism of (iii) or (iv), after the correction step (b) and before the step (8),
Converting the immobilized amount of each probe in the probe set to a relative value obtained in advance, and correcting the correction signal value based on the relative value, the immobilization amount correction proportional to the immobilized amount A step (c) for setting a signal value may be further included.
(Vi) In any mutation or polymorphism determination method of (i) to (v) above, all probes in the probe set are for the sequence of either the sense strand or the antisense strand of the target nucleic acid. It can be.
(Vii) In any mutation or polymorphism determination method of (i) to (v) above, the probe in the probe set consists of a target nucleic acid sense strand and an antisense strand. Can be.
(Viii) In the method for determining a mutation or polymorphism of (vii) above, there are first perfect match probes for the sense strand and the antisense strand, and different results are obtained for each of the sense strand and the antisense strand. In such a case, a probe that gave a different result may not be identified as a perfect match probe, regardless of the difference in the result in the sense strand and the antisense strand.
(Ix) In the mutation or polymorphism determination method of any of (i) to (vii) above, when the first perfect match probe is not present in the sense strand or the antisense strand, the first perfect match probe is present It is good also as not using the result of the chain | strand which is not used for determination.
(X) In the mutation or polymorphism determination method of any one of (i) to (ix) above, the threshold A is an absolute value or relative value of a signal, or both, and the threshold B is It can be the absolute value of the signal.
(Xi) In the mutation or polymorphism determination method of any one of (iii) to (ix) above, the thresholds A and C are absolute values or relative values of signals, or both, and the threshold B Can be the absolute value of the signal.
(Xii) In the mutation or polymorphism determination method of any one of (iii) to (xi) above, the threshold values A, B, and C may be different values for the sense strand and the antisense strand, respectively. it can.
(Xiii) In the mutation or polymorphism determination method according to any one of (x) to (xii) above, the threshold A is a relative value, and the signal intensity derived from the hybrid including the first perfect match probe And 15% or less.
(Xiv) In the mutation or polymorphism determination method of any one of (x) to (xiii) above, the threshold value A is a relative value, and the signal intensity derived from the hybrid including the first perfect match probe And 10% or less.
(Xv) In the mutation or polymorphism determination method of any of (x) to (xiv) above, the threshold C is a relative value, and the signal intensity derived from the hybrid including the first perfect match probe 20% or more.
(Xvi) In the mutation or polymorphism determination method according to any one of (i) to (xv) above, the probe set includes a set of the above probe subsets, and each probe subset includes the perfect match probe and The base sequence may be extended or shortened at both ends of the incomplete match probe.
(Xvii) The method for determining a mutation or polymorphism of any of (i) to (xv) above can be for SNP typing.
(Xviii) The SNP typing of (xvii) can be performed from the top to the two determination orders for determining a perfect match probe.
(Xix) In the mutation or polymorphism determination method of any one of (i) to (xviii) above, the target nucleic acid can be K-ras gene codon 12.

Fig. 1 This figure shows the arrangement of spots on the DNA microarray used for carrying out the mutation judging method of the present invention. Figure 2 This figure shows the probe set (antisense strand side) immobilized. The target nucleic acid (K-ras gene 12th codon amplified by PCR) is brought into contact with the existing DNA microarray, and the signal derived from the hybrid formed at each spot on the DNA microarray is measured. .
FIG. 3 This figure shows the signal values in FIG. 2 as relative values.
FIG. 4 This figure shows the measured signal value as a relative value when the first correction nucleic acid is brought into contact with the probe set (antisense strand side).
FIG. 5 This figure shows the first correction signal as a relative value (on the antisense strand side).
FIG. 6 This figure shows the first correction signal as a relative value to the signal value of the second perfect match probe candidate (on the antisense strand side).
FIG. 7 This figure shows the measured signal value as a relative value when the second correction nucleic acid is brought into contact with the probe set (antisense strand side).
FIG. 8 This figure shows the second correction signal value as a relative value.
FIG. 9 This figure shows that each spot on the DNA microarray is brought into contact with the target nucleic acid (K-ras gene 12th codon amplified by PCR) on the DNA microarray on which the probe set (sense strand side) is immobilized. 2 shows a signal obtained by measuring a signal derived from the hybrid formed in FIG.
FIG. 10 This figure shows the signal values in FIG. 9 as relative values.
FIG. 11 This figure shows the measured signal value as a relative value when the first correction nucleic acid is brought into contact with the probe set (sense strand side).
FIG. 12 This figure shows the first correction signal as a relative value (sense strand side).
FIG. 13 This figure shows the first correction signal as a relative value to the signal value of the second perfect match probe candidate (sense strand side).
FIG. 14 This figure is a flowchart of the procedure in the mutation or polymorphism determination method of the present invention.

(Definition)
In the present specification, the following terms are used in the meanings defined below.
“Nucleic acid” means any of DNA, DNA containing artificial nucleotides, RNA, RNA containing artificial nucleotides, PNA, and PNA containing artificial nucleotides.
“Hybrid” means a duplex formed between any of the nucleic acids described above.
The “signal” is a signal that can be appropriately detected and measured by an appropriate means, and includes fluorescence, radioactivity, chemiluminescence, and the like.
Hereinafter, the configuration, implementation method, effects, and the like of the embodiments of the present invention will be described with reference to the drawings as appropriate.
The method for determining mutations and polymorphisms of the present invention includes a perfect match probe that perfectly matches a target nucleic acid and a base that causes a mismatch of at least one base to the target nucleic acid, and other bases are the perfect match A probe set consisting of at least one incomplete match probe that is identical to the probe is immobilized on a solid phase carrier, a labeled sample nucleic acid is brought into contact, and a hybrid of each probe and sample nucleic acid in the probe set Is used in a method for determining a sequence in a target nucleic acid by detecting the signal from the target and comparing the signal intensity. The mutation and polymorphism determination method of the present invention includes a perfect match probe that perfectly matches a target nucleic acid and a base that causes a mismatch of at least one base to the target nucleic acid, and the other bases Contacting a labeled sample nucleic acid with a probe set comprising at least one incomplete match probe identical to the match probe on a solid phase carrier (1); from the hybrid formed at each spot; Measuring a signal of (2); determining a probe in a hybrid that emits a low-intensity signal below a predetermined signal value (threshold A) as an incompletely matched probe candidate (3); another predetermined signal value ( First perfect match for probes in hybrids that emit the strongest signal among signals above threshold B) A step (4) of determining as a lobe candidate; and if both the incomplete match probe candidate and the first complete match probe candidate exist, the incomplete match probe candidate is determined as an incomplete match probe; Step (5) of determining the first perfect match probe as the first perfect match probe is performed. As described above, there is no absolute signal value that is emitted when a perfect match hybrid is formed, as described above. This is because it is necessary to judge the validity of the conditions for forming the hybrid depending on the relative relationship with the case where it is determined.
When the threshold value A in step (3) is an absolute value, it can be a value that is considered to be a non-specific signal intensity under the measurement conditions. This value should be varied depending on various conditions such as the type of target nucleic acid to be used, the preparation method, and the sequence of the probe to be used, but can be determined empirically. It is self-evident. When the threshold value A is expressed as a relative value, the magnitude is set to 15% or less, preferably 10% or less, with respect to the highest intensity signal. If it has a signal value of this level with respect to the maximum intensity, there is a high probability that it is derived from an imperfect match hybrid. On the other hand, the threshold value B in step (4) is a value that is significantly larger than the non-specific signal intensity under the measurement conditions. Like the threshold value A, the type of target nucleic acid to be used, the preparation method, and the use It should be changed according to various conditions such as the arrangement of the probe to be determined and can be determined empirically. The value is obvious to those skilled in the art. When the target nucleic acid is homogeneous with respect to the sequence, one maximum peak is usually measured, whereas when multiple sequence species are mixed in the target nucleic acid, a plurality of relatively strong signals are measured. .
If it can be determined in step (3) that there is something that is thought to be derived from an incompletely matched hybrid, and that in step (4) there is at least one peak that is significantly different from the non-specific signal intensity, From these relative relationships, it can be inferred that hybridization was performed under appropriate conditions, and therefore, it is possible to identify incomplete match probes and perfect match probes in step (5). When one maximum peak is measured, the peak, and when multiple measurements are made, the probe in the hybrid that emits the highest intensity among them can be identified as the first perfect match probe. If incomplete match probe candidates cannot be determined in step (3) and / or complete match probe candidates cannot be determined in step (4), then steps (1) and / or ( 2) Since the contact and / or measurement conditions are considered to be inappropriate, this condition is changed from the original conditions, and the method consisting of the steps (1) to (5) is performed again to complete the match. Probes and incomplete match probes are determined. If the determination can be made, it can be said that the changed condition was appropriate.
The method for determining mutations and polymorphisms of the present invention is the above-described method for determining mutations or polymorphisms.
After step (5),
Identifying a probe in the hybrid that is a probe other than the first perfect match probe and emits a maximum signal with a signal intensity equal to or greater than a predetermined value (threshold C) as a second perfect match probe candidate (6) ;
Step (7) of performing the following signal correction steps (a) and (b) on the signal in the hybrid containing the second perfect match probe candidate:
Separately from the steps (1) to (6), a first correction nucleic acid consisting of only a sequence that perfectly matches the first perfect match probe is brought into contact with the same probe set as the probe set, A step (a) of measuring a signal when the probe set comes into contact with only the first correction nucleic acid; and a signal value obtained in the step (a) is subtracted from the signal value measured in the step (1). Determining a first corrected signal value for the signal derived from each hybrid (b);
When the first correction signal value in the correction step (b) is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the first correction signal value is identified as a second perfect match probe; Alternatively, when the first correction signal value in the correction step (b) has a signal intensity less than the threshold value C, the probe in the hybrid having the first correction signal value is identified as not the second perfect match probe. Step (8)
Can further be included.
The threshold value C can be an absolute value or a relative value with respect to the signal value with the highest intensity. However, using the relative value with respect to the signal with the highest intensity, in the signal correction step described below, It becomes easier to perform the correction, and the setting of the threshold becomes less complicated. As a relative value to be converted here, it is preferable that the relative value when the maximum intensity is 100 is expressed as a percentage.
In step (6), a hybrid in a hybrid other than the hybrid including the first perfect match probe and emitting a signal having a signal intensity equal to or higher than a predetermined value (threshold C) is identified as a perfect match probe candidate. To be done. Here, the threshold value C, like the threshold values A and B, should be varied depending on various conditions such as the type of target nucleic acid to be used, the preparation method, and the sequence of the probe to be used, and can be determined empirically. The value is obvious to those skilled in the art. As a specific value, when the threshold value C is expressed as a relative value, it can be set to a magnitude of 20% or more with respect to the signal intensity derived from the hybrid including the first perfect match probe.
In step (7), the following signal correction steps (a) and (b) are performed on the signal from the hybrid including the second perfect match probe candidate.
Here, the correction step is
Separately from the steps (1) to (6), a first correction nucleic acid consisting of only a sequence that perfectly matches the first perfect match probe is brought into contact with the same probe set as the probe set, Performing a step (a) of measuring a signal when the probe set is in contact with only the first correction nucleic acid; and the signal value obtained in the step (a) is the signal value measured in the step (1). The step (b) of obtaining the first corrected signal value for the signal derived from each hybrid is performed by subtracting from the above.
In the above correction step (a), the hybridization with the first correction nucleic acid consisting only of the sequence that perfectly matches the first perfect match probe may cause a plurality of complete matches in the target nucleic acid. If there is a signal, the signal intensity is relatively strong, but it is unknown whether it is due to an incomplete match or another perfect match that is contained in a small percentage of the target nucleic acid sample. There is to clarify it. Even in the case of hybridization with the first correction nucleic acid consisting only of a sequence that perfectly matches the first perfect match probe, the complete match probe performs non-specific hybridization with the correction nucleic acid, A certain signal may be generated. Therefore, the signal value derived from this non-specific hybridization is obtained in the correction step (a), and in the next step (b), the signal value derived from each hybrid is subtracted from the already obtained signal value. Determine the signal value. The corrected signal value obtained by going through such a step is obtained by subtracting the signal emitted by the hybrid generated by nonspecific hybridization of each probe, and was caused only by specific hybridization. Signal value.
After performing the correction step, when the first correction signal value in the correction step (b) is a signal intensity equal to or higher than the threshold value C, a probe in the hybrid having the first correction signal value is When the signal is identified as a perfect match probe or the correction signal value in the correction step (b) has a signal intensity less than the threshold value C, the probe in the hybrid having the first correction signal value is designated as the second perfect match probe. The step (8) of identifying that there is no is performed. In the case of the first perfect match probe, it was included in the hybrid emitting the strongest signal and could be relatively identified by the presence of at least one incomplete match. However, in the case of these intermediate levels of signal intensity, i.e., signals that are weaker than the signal from the hybrid containing the first perfect match probe but are considered to be greater than the non-specific signal value, It is necessary to identify whether it is due to a perfect match contained in a small proportion in the nucleic acid or a non-specific signal at a slightly higher level than usual. This discrimination is very important when measurement is performed under conditions where non-specific hybridization occurs at a relatively high frequency. In the present invention, since a correction value obtained by subtracting a signal resulting from non-specific hybridization occurring between the target nucleic acid and each perfect match probe is used, the correction value is a relative intensity equal to or higher than the threshold value C. It is possible to determine that a hybrid that emits the signal includes a perfect match probe that is not derived from non-specific hybridization and is not background.
As described above, the present invention is not limited to the basic method for identifying only the first perfect match, the method for re-measurement after changing the conditions when the measurement conditions are not suitable, and the other than the first perfect match. The second perfect match is also identified. The present invention further includes a plurality of perfect matches after the first perfect match (complete matches existing at a lower abundance ratio than the first perfect match) such as “second perfect match, third perfect match,...”. Even in the case where there are a plurality of them, it is possible to provide a method of sequentially identifying all of them.
In this aspect, the present invention provides a case where there are a plurality of spots that emit a signal value equal to or higher than the threshold value B in the step (4), and the first perfect match probe is determined in the step (5), that is, the first perfect match. This applies when there are multiple perfect matches after the match. This is applied when the first perfect match has already been identified (that is, it has been proved that the contact conditions were appropriate), and the second perfect match and the subsequent are identified.
Next, in this aspect, after the step (8), the plurality of probes having a high signal intensity for probes other than the first complete match probe (second complete match probe candidates) Step (9) for performing the correction steps (a) and (b) is performed in order. In this step (9), the probes after the second perfect match probe (second perfect match probe candidates) are contacted with an appropriate nucleic acid for correction in order from the one having the strongest signal intensity to obtain a correction signal. , Second perfect match, third perfect match,... More specifically, for the identification of the second perfect match probe, the second correction signal value is obtained using the second correction nucleic acid, and for the identification of the third perfect match probe, the third correction nucleic acid is used. This means that the third correction signal value is obtained.
However, in this embodiment, the candidate for the second perfect match probe is not a second perfect match probe because noise due to non-specific binding or the like is particularly large. Since what was supposed to be the third perfect match probe may eventually be identified as the second perfect match probe, the name of the perfect match probe may need to be corrected. For example, in the above case, it is necessary to appropriately adjust the number of the “3” part of the initial third perfect match to obtain the second perfect match. To do this, do the following:
First, among the plurality of second perfect match probe candidates, for a probe included in a hybrid having a signal value that is n−1th (n is an integer not less than 2 and not more than the type of probe), The n-th correction nucleic acid consisting only of a sequence that perfectly matches the probe in the hybrid that emits the (n-1) -th largest signal, separately from the steps (1) to (6), is the same probe as the probe set The signal is measured when the probe set is brought into contact with the set and the probe set is in contact with only the n-th correction nucleic acid. Here, a plurality of second perfect match probe candidates are determined in the order of strong signal by the correction method in the case of the first perfect match probe in the same manner as in the case of the first perfect match probe. In addition, “n is an integer not less than 2 and not more than the type of probe” means that the second perfect match probe having the first strongest signal is 2-1 = 1, and the first largest signal is It is to do. For the nucleic acid for correction, since the first nucleic acid for correction is already used, n is set to 2 or more in order to distinguish it from the n-th (n is 2 or more) correction nucleic acid. Regarding the upper limit of n, the maximum number of second perfect match probe candidates is up to the number equal to the type of probe. Here, “type of probe” is used to determine one mutation or polymorphic part. This shows the number of types of probes included in the set of sense strand probes or the number of types of probes included in the set of antisense strand probes.
Next, the measured signal value is subtracted from the signal value that has been cumulatively corrected using each of the first correction nucleic acid to the n-1 correction nucleic acid to obtain the nth correction signal value. . Here, the correction signal value is obtained using the signal measured with the nucleic acid for correction. For example, in the case where the third perfect match is to be identified (ie, when n = 3), the identification of the first and second perfect match probes has already been made before the identification of the third perfect match probe. Considering that each identification step has been subjected to a correction step using the first and second correction nucleic acids, and the signal value should be corrected cumulatively by that amount. Will be.
Next, when the n-th corrected signal value is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the n-th corrected signal value is determined as the n-th perfect match probe, or When the n-corrected signal value has a signal intensity less than the threshold C, the probe in the hybrid having the n-th corrected signal value is identified as not a perfect match probe. Here, it is determined whether or not the perfect match probe candidate can be identified as the perfect match probe by comparing the threshold value with the correction signal value. Through this process, the nth perfect match probe candidate is finally recognized as the nth perfect match probe, and even one of the perfect match probe candidates is identified as not a perfect match probe. In this case, it is necessary to appropriately correct the “nth” n. For the correction, before the determination of the nth perfect match probe, x spots (m is an integer less than or equal to 1 and less than n) having an mth correction signal value with an intensity less than the threshold C (x is 0). If there is an integer above, the nth perfect match probe is determined to be the (nx) perfect match probe. This is because, for example, in the step of identifying the second perfect match probe (when n = 3) performed before the determination of the third perfect match probe, the second perfect match probe candidate is identified as not being a perfect match. If so, x = 1 (ie, if there was one spot with a second corrected signal value with an intensity less than threshold C), so the third perfect match probe to be determined here is This means that it should be finally determined as the second perfect match probe (since 3-1 = 2). In addition, if the second and third perfect match probe candidates are both identified as not perfect matches before the fourth perfect match probe is determined, x = 2. The 4 perfect match probe means that it should be finally determined as the second perfect match probe (since 4-2 = 2).
In the mutation and polymorphism determination method of the present invention, each probe in the probe set previously determined after the correction step (b) and before the step (8) is further fixed. The method may further include a step (c) of converting the phase amount into a relative value and setting the correction signal value based on the relative value as an immobilized amount correction signal value proportional to the solid phase amount. This makes it possible to perform more accurate mutation determination by correcting the difference when the amount of the solid phase to the solid phase carrier (for example, DNA microarray) of each probe is large.
Here, the amount of the probe immobilized on the carrier can be measured by quantifying a signal emitted from a fluorescent dye or the like previously introduced into the probe.
In the mutation and polymorphism determination method of the present invention, all the probes in the probe set may be directed to either the sense strand or the antisense strand of the target nucleic acid, or in the probe set. The probe may consist of one for the sense strand of the target nucleic acid and one for the antisense strand. In terms of the cost of preparing probes, etc., there are advantages to using either the sense strand or the antisense strand, but the use of both the sense strand and the antisense strand ensures the determination of mutation and polymorphism. It is considered preferable for sex. In this case, when the first perfect match probe for each of the sense strand and the antisense strand exists and a different result is obtained for each of the sense strand and the antisense strand, the probe that gave the different result is sensed. Regardless of the difference in results in the strand and antisense strand, it is possible not to identify a perfect match probe. Furthermore, when the first perfect match probe is not present in the sense strand or the antisense strand, it is possible not to use the result of the strand in which the first perfect match probe is not present in the determination. Thus, the reliability of the measurement result can be increased by using only the result with high certainty.
More specifically, the probe set is composed of a set of two or more probe subsets, and each probe subset extends or shortens the base sequence at both ends of the complete match probe and the incomplete match probe. If an overlapping probe is used, a more accurate mutation or polymorphism can be determined.
In the method for determining a mutation or polymorphism of the present invention, SNP typing or oncogene mutation determination, for example, codon 12 of the K-ras gene can be used as a target nucleic acid. Specific examples using codon 12 of the K-ras gene are described in the examples below.

In the following, the mutation determination method of the present invention will be described using more specific sequences.
FIG. 1 is a diagram showing the arrangement of spots on a DNA microarray used for carrying out the mutation judging method of the present invention. As a probe to be immobilized on the microarray, probes for the sense strand and the antisense strand of the 12th codon sequence of the K-ras oncogene were used. In the figure, spot 1 has the perfect match probe (corresponding to the sense strand) shown in SEQ ID NO: 1, and in spots 2 to 7, the incomplete match probe shown in SEQ ID NO: 2 to 7 (on the sense strand) Arranged). The incomplete match probe has CGT, TGT, AGT, GCT, GAT, and GTT as sequence portions that were GGT in the complete match probe, respectively. In spots 8 to 14, a perfect match probe and an incomplete match probe (having the sequences of SEQ ID NOs: 8 to 14, respectively) for the antisense strand were similarly arranged. In the following, each of the incomplete match probes (SEQ ID NOs: 2 to 7) in both the sense strand and the antisense strand is referred to as a CGT probe, a TGT probe, an AGT probe, a GCT probe, a GAT probe, and a GTT probe. is there. These probe groups were designed and synthesized based on sequences available from databases such as GenBank.
The immobilization of each probe on a DNA microarray was performed by a method known in the art. Specifically, spotting was performed by a micro-dispensing system using a piezo element.
In preparing the labeled nucleic acid, a nucleic acid extracted from cancer cells collected from a human body was used as a template. Using this extracted nucleic acid and K-ras oncogene primer (Takara Shuzo Co., Ltd., Cat # 7112; those shown in SEQ ID NOs: 15 and 16) in PCR, a labeled target nucleic acid labeled 5 'with FITC is prepared. did. Samples amplified by PCR were confirmed by 8% NuSieve (FMC) agarose electrophoresis. About 50 μg of the amplified target nucleic acid was brought into contact with the above-described DNA microarray on which each probe was immobilized.
Hybridization between the probe and the target nucleic acid was performed under the following conditions.
Hybridization solution: 3xSSPE (sodium phosphate buffer, technical data: DNA microarray and the latest PCR method, see Cell Engineering separate volume Genome Science Series 1 Shujunsha), 10% (V / V) ExpressHyb (Clontech) target nucleic acid Suspended in a 10% solution;
Analyzer: An experimental system in which a cooled CCD camera is connected to Olympus BX-51TFR (a means for enabling sample solution driving and temperature control is mounted around the reaction filter, and an image of the fluorescent spot is further displayed. Designed to be automatically recorded);
Operation: 50 μl of the reaction solution prepared with the hybridization solution is added to the reaction part of the dedicated chamber where the microarray with a diameter of 6 mm is installed, the hybridization reaction is performed at 50 ° C., the fluorescent image is taken, and the image is taken to the hard disk Saved in.
The resulting image was analyzed using analysis software programmed for mutation analysis and the image signal was digitized. The digitized signal was further analyzed as follows.
As the threshold A that defines the low intensity signal, a value of the absolute intensity of the signal 15 was set. CGT probes, TGT probes, and AGT probes showing signals below threshold A were used as non-specific probes for target nucleic acids, that is, incomplete match probe candidates (see FIG. 2).
On the other hand, as the threshold value B, an absolute signal intensity of 100 was set. The GGT probe contained in the spot emitting the highest intensity signal was determined as the first perfect match probe candidate (see FIG. 2).
Since both the probe candidate equal to or lower than the threshold A and the probe candidate equal to or higher than the threshold B exist, the incomplete match probe candidate is determined as an incomplete match probe, and the first complete match probe candidate is further determined as the first complete match probe ( Antisense strand side).
Next, as the threshold C, 20% was set as the signal value with respect to the signal intensity of the first perfect match probe. The relative value of the signal value of each probe when the signal value of the first perfect match probe (antisense strand side) is 100% is shown in FIG. A GAT probe that is a probe in which a probe other than the first perfect match probe (antisense strand side) is immobilized and is immobilized on a spot that emits a signal intensity equal to or higher than the threshold C is used as a second complete probe. It was determined as a match probe candidate (see FIG. 3).
Next, separately from the determination of the first perfect match probe (antisense strand), the first correction nucleic acid consisting of only the sequence that perfectly matches the first perfect match probe (antisense strand) is used as the probe. The same probe set as the set was contacted on each spot of the DNA microarray, and the signal when the probe set was in contact with only the first correction nucleic acid was measured. For the first correction nucleic acid, a labeled control nucleic acid labeled with FITC at the 5 ′ end is prepared by performing PCR using a DNA whose nucleotide sequence is known to have GGT in advance by a DNA sequencer as a template. This was used. The measured values of the hybridization with the first correction nucleic acid are shown in FIG. In this figure, the signal value of the GGT probe is taken as 100%, and the relative value is shown. The value obtained by subtracting this relative value from the relative value in FIG. 3 is shown in FIG.
In FIG. 5, since the signal value (relative value) of the GAT probe is equal to or greater than the threshold value C, the GAT probe was determined to be the second perfect match probe (antisense strand side).
Since the GCT probe in the spot emitting the strongest signal after the GAT probe has a signal intensity equal to or higher than the threshold C when the signal of the GAT probe is 100% (see FIG. 6), The same correction was performed, and it was confirmed whether or not the GCT probe was a perfect match probe.
Separately from the determination of the first perfect match probe (antisense strand side), the second nucleic acid for correction consisting of only the sequence that perfectly matches the second perfect match probe (antisense strand side) is the same as the probe set. The probe set was contacted on each spot of the DNA microarray, and the signal when the probe set was in contact with only the second correction nucleic acid was measured. For the second correction nucleic acid, a labeled control nucleic acid labeled with FITC at the 5 ′ end is prepared by performing PCR using a DNA whose nucleotide sequence is known in advance by a DNA sequencer as a template. This was used. The measured values of hybridization with the second correction nucleic acid are shown in FIG. In this figure, the signal value of the GAT probe is taken as 100%, and the relative value is shown. FIG. 8 shows a value obtained by subtracting this relative value from the relative value in FIG.
In FIG. 8, since the signal relative value of the GCT probe after subtraction is equal to or greater than the threshold value C, the GCT probe was determined to be the third perfect match probe (antisense strand side).
The following steps were performed in the same manner as described above except that a probe for the sense strand was used instead of the probe for the antisense strand.
Similarly to the above, the value of the absolute intensity of the signal 15 was set as the threshold value A that defines the low intensity signal. CGT probes, TGT probes, and AGT probes showing signals below the threshold A were used as non-specific probes for the target nucleic acid, that is, incomplete match probe candidates (see FIG. 9).
On the other hand, for the threshold B, unlike the case of antisense, an absolute signal intensity of 90 was set. The GGT probe contained in the spot emitting the highest intensity signal was determined as the first perfect match probe candidate (see FIG. 9).
Since both the probe candidate equal to or lower than the threshold A and the probe candidate equal to or higher than the threshold B exist, it is determined as an incomplete match probe candidate and an incomplete match probe, and the first complete match probe candidate is further determined as the first complete match probe ( Sense strand side).
Next, as the threshold C, 20% was set as the signal value with respect to the signal intensity of the first perfect match probe (sense strand side) in the same manner as described above. FIG. 10 shows the relative value of the signal value of each probe when the signal value of the first perfect match probe (sense strand side) is 100%. A GAT probe that is a probe in which a probe other than the first perfect match probe (on the sense strand side) is immobilized and a probe that emits a signal intensity equal to or higher than the threshold C is matched to the second perfect match. It was determined as a probe candidate (see FIG. 10).
Next, separately from the identification of the first perfect match probe, the first correction nucleic acid consisting only of the sequence that perfectly matches the first perfect match probe (sense strand side) is put into the same probe set as the probe set. On the other hand, it was made to contact on each spot of a DNA microarray, and the signal when the probe set contacted only the first correction nucleic acid was measured. For the first correction nucleic acid, a labeled control nucleic acid labeled with FITC at the 5 ′ end is prepared by performing PCR using a DNA whose nucleotide sequence is known to have GGT in advance by a DNA sequencer as a template. This was used. The measured values of the hybridization with the first correction nucleic acid are shown in FIG. In this figure, the signal value of the GGT probe is taken as 100%, and the relative value is shown. FIG. 12 shows a value obtained by subtracting this relative value from the relative value in FIG.
In FIG. 12, since the signal value (relative value) of the GAT probe is equal to or greater than the threshold value C, the GAT probe was determined to be the second perfect match probe (sense strand side).
The GCT probe in the spot that emitted the next strongest signal after the GAT probe does not have a signal intensity equal to or higher than the threshold C when the signal of the GAT probe is 100% (see FIG. 12). It was determined that it was not a probe. Therefore, the GCT probe has different results depending on whether a probe corresponding to the antisense strand is used or a probe corresponding to the sense strand. If this is provided, it is determined that this is not a perfect match probe.
From the above results, it was found that only the GGT probe and the GAT probe were perfect matches, and the target nucleic acids corresponding to these sequences were contained in the sample.
In the above description, the DNA microarray in which the probe is immobilized on the substrate and the type of the probe is specified by the position has been described as an example. However, the hybridization reaction can be detected in a state where a plurality of types of probes can be identified. The present invention can be applied to any configuration. Specifically, there is no particular limitation as long as the probe is solid-phased on the substrate, and the probe is solid-phased using glass, silicon wafer, various porous substrates, gel, microtiter plate or beads as a base material. Can be used.
INDUSTRIAL APPLICABILITY In the present invention, when analyzing nucleic acid mutations and polymorphisms, even if a nucleic acid molecule as a sample is heterogeneous with respect to its sequence, the measured signal is a probe and Is more accurate with respect to the nucleic acid sequence in the sample compared to conventional methods for analyzing mutations and polymorphisms. Allows you to get information.

[Sequence Listing]

Claims (19)

A perfect match probe that perfectly matches the target nucleic acid and at least one incomplete match probe that includes a base that causes a mismatch of at least one base to the target nucleic acid, and the other bases are identical to the perfect match probe A labeled sample nucleic acid is contacted with a solid phase carrier on which a probe set comprising: (1);
Measuring the signal from the hybrid formed at each spot (2);
Determining a probe in the hybrid that emits a low-intensity signal equal to or lower than a predetermined signal value (threshold A) as an incompletely matched probe candidate (3);
(4) determining a probe in a hybrid that emits a signal having the highest intensity among signals equal to or higher than another predetermined signal value (threshold B) as a first perfect match probe candidate; and A step (5) of determining that the incomplete match probe candidate is an incomplete match probe, and further determining the first complete match probe as a first complete match probe when both the first perfect match probe candidates exist;
A method for determining mutations or polymorphisms. If incomplete match probe candidate cannot be determined in step (3) and / or complete match probe candidate cannot be determined in step (4), contact in step (1) and / or (2) The method for determining mutation or polymorphism according to claim 1, further comprising performing the method comprising the steps (1) to (5) after changing the measurement conditions from the initial conditions. After step (5),
Identifying a probe in the hybrid that is a probe other than the first perfect match probe and emits a maximum signal with a signal intensity equal to or greater than a predetermined value (threshold C) as a second perfect match probe candidate (6) ;
Step (7) of performing the following signal correction steps (a) and (b) on the signal in the hybrid containing the second perfect match probe candidate:
Separately from the steps (1) to (6), a first correction nucleic acid consisting of only a sequence that perfectly matches the first perfect match probe is brought into contact with the same probe set as the probe set, A step (a) of measuring a signal when the probe set comes into contact with only the first correction nucleic acid; and a signal value obtained in the step (a) is subtracted from the signal value measured in the step (1). Determining a first corrected signal value for the signal derived from each hybrid (b);
When the first correction signal value in the correction step (b) is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the first correction signal value is identified as a second perfect match probe; Alternatively, when the first correction signal value in the correction step (b) has a signal intensity less than the threshold value C, the probe in the hybrid having the first correction signal value is identified as not the second perfect match probe. Step (8)
The method for determining a mutation or polymorphism according to claim 1 or 2, further comprising: When there are a plurality of spots that emit a signal value equal to or higher than the threshold value B in the step (4), and the first perfect match probe is determined in the step (5),
After the step (8), with respect to the plurality of probes other than the first perfect match probe (second perfect match probe candidates), the correction step ( Step (9) for performing a) and (b)
(However, in this step,
Among the plurality of second perfect match probe candidates, for the probe contained in the hybrid having a signal value that is n-1th (n is an integer not less than 2 and not more than the type of probe),
The n-th correction nucleic acid consisting only of a sequence that perfectly matches the probe in the hybrid that emits the n-1st largest signal is the same as the probe set separately from the steps (1) to (6). Measuring the signal when the probe set is brought into contact with only the n-th correction nucleic acid;
Subtracting the measured signal value from the signal value that has been cumulatively corrected using each of the first to n-1th correction nucleic acids to obtain an nth correction signal value; and When the nth correction signal value is a signal intensity equal to or higher than the threshold value C, the probe in the hybrid having the nth correction signal value is determined as the nth perfect match probe, or the nth correction signal value If the signal intensity is less than the threshold C, the probe in the hybrid having the n-th corrected signal value is identified as not a perfect match probe (however, before the determination of the n-th perfect match probe, the threshold value is When there are x hybrids (x is an integer greater than or equal to 0) having the mth correction signal value (m is an integer greater than or equal to 1 and less than n) with an intensity less than C, the nth The perfect match probe is determined to be the (nx) perfect match probe))
The method for determining a mutation or polymorphism according to claim 3 further comprising: After the correction step (b) and before the step (8),
Converting the immobilized amount of each probe in the probe set to a relative value obtained in advance, and correcting the correction signal value based on the relative value, the immobilization amount correction proportional to the immobilized amount Step of signal value (c)
The mutation or polymorphism determination method according to claim 3 or 4, further comprising: The method for determining mutation or polymorphism according to any one of claims 1 to 5, wherein all probes in the probe set are for the sequence of either the sense strand or the antisense strand of the target nucleic acid. The method for determining a mutation or polymorphism according to any one of claims 1 to 5, wherein the probe in the probe set comprises a target nucleic acid sense strand and an antisense strand. When the first perfect match probe of each of the sense strand and the antisense strand is present and a different result is obtained in each of the sense strand and the antisense strand, the probe that gave the different result is designated as the sense strand and the antisense strand. 8. The method for judging mutation or polymorphism according to claim 7, wherein the method is not identified as a perfect match probe regardless of the difference in the result in the sense strand. 9. The method according to claim 1, wherein when the first perfect match probe is not present in the sense strand or the antisense strand, the result of the strand in which the first perfect match probe is not present is not used for determination. The method for determining a mutation or polymorphism described in 1. 10. The threshold value A is an absolute value or relative value of a signal, or both, and the threshold value B is an absolute value of a signal, according to any one of claims 1 to 9, Mutation or polymorphism determination method. 10. The thresholds A and C are absolute values or relative values of signals, or both, and the threshold values B are absolute values of signals. A method for determining the mutation or polymorphism described. The method for judging mutation or polymorphism according to any one of claims 3 to 11, wherein the threshold values A, B, and C are different values for each of the sense strand and the antisense strand. The threshold value A is a relative value and has a magnitude of 15% or less with respect to the signal intensity derived from the hybrid including the first perfect match probe. A method for determining the mutation or polymorphism described. The threshold value A is a relative value and has a magnitude of 10% or less with respect to a signal intensity derived from a hybrid including the first perfect match probe. A method for determining the mutation or polymorphism described. The threshold value C is a relative value and has a magnitude of 20% or more with respect to the signal intensity derived from the hybrid including the first perfect match probe. A method for determining the mutation or polymorphism described. The probe set is composed of a set of two or more probe subsets, and each probe subset is composed of overlapping probes that extend or shorten base sequences at both ends of the complete match probe and the incomplete match probe. The method for determining a mutation or polymorphism according to any one of claims 1 to 15. The polymorphism determination method according to any one of claims 1 to 15 for SNP typing. 18. The polymorphism determination method for SNP typing according to claim 17, wherein the determination order of the SNP typing is determined to be a perfect match probe from the top two. The mutation or polymorphism determination method according to any one of claims 1 to 18, wherein the target nucleic acid is a K-ras gene codon 12.

Images