JP3975663B2 - Gene polymorphism analysis method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a gene (nucleic acid) polymorphism analysis method by SSCP method using capillary electrophoresis, and in particular, gene abnormalities associated with diseases such as cancer such as gene deletion and loss of heterozygosity are detected. The present invention relates to a detection method using a type marker.
[0002]
[Prior art]
Normal human cells contain a pair of parent-derived chromosomes that differ for a particular locus, for example, when one allele is wild-type and the other allele is mutated. The case is called a heterozygote. On the other hand, when both genotypes are the same, it is called a homozygote. In the case of healthy heterozygotes, the ratio of the abundance of each allele is theoretically 1: 1. When the heterozygote is analyzed by the SSCP method, two peaks corresponding to each allele such as wild type and mutant are observed, and the signal intensity ratio of the two peaks is 1: 1.
[0003]
On the other hand, abnormalities such as gene deletion and amplification occur in most malignant cancer cells. When one allele is deleted or amplified in a heterozygote, so-called loss of heterozygosity (loss of heterozygosity, LOH) occurs. In the SSCP method, LOH is observed as a phenomenon in which the signal intensity ratio of the two peaks corresponding to each allele of the heterozygote deviates from 1: 1.
[0004]
A method of diagnosing cancer using gene polymorphism, that is, individual differences in nucleotide sequence as a marker (Japanese Patent Laid-Open No. 9-201199, hereinafter referred to as Prior Art-1). The outline of will be described below. After a specific region in the base sequence of the gene is amplified by PCR using a labeled primer, the 3 ′ end is smoothed with an enzyme having a 3 ′ → 5 ′ exonuclease activity (Klenow fragment or the like). This PCR product is analyzed by a single-strand higher-order structure polymorphism analysis (SSCP) method using a slab gel type electrophoresis apparatus, and an allele having a single nucleotide polymorphism (allyl) is separated and detected.
[0005]
A normal tissue-derived DNA fragment and a cancer tissue-derived DNA fragment are labeled with the same fluorescent dye, and are measured in separate lanes using a slab gel type electrophoresis apparatus having a plurality of lanes. The ratio of cancer cells is estimated from the signal intensity ratio of two peaks A1 and A2 derived from normal tissue and the signal intensity ratio of two peaks B1 and B2 derived from cancer tissue. In the example of LOH detection of bladder cancer using a single nucleotide polymorphism in the p53 gene as an index, when the proportion of cancer cells exceeds 10%, it is considered abnormal (LOH +, positive).
[0006]
[Problems to be solved by the invention]
In the prior art-1, a slab gel type electrophoresis apparatus is used, but automation, labor saving, and speedup are required for application to routine clinical examinations. In the present invention, a capillary electrophoresis apparatus having an automatic gel filling mechanism and an automatic sample injection mechanism is used in place of the slab gel type electrophoresis apparatus in the prior art-1 to achieve automation, labor saving, and speedup. Study was carried out. However, SSCP using a capillary electrophoresis apparatus is in the research stage, and many problems remain. Attempts were made to apply the capillary electrophoresis apparatus to Conventional Technique-1, but normal tissue and cancer tissue were labeled with the same fluorescent dye and electrophoresed on separate capillaries, or twice with a single capillary. When electrophoresis was performed separately, the problem was found that the LOH could not be correctly determined due to fluctuations in the peak signal intensity due to fluctuations in measurement conditions.
[0007]
The object of the present invention is to accurately analyze LOH even if the signal of the electrophoresis peak fluctuates in the gene polymorphism analysis method for detecting LOH by performing analysis by the SSCP method using a capillary electrophoresis apparatus. It is an object of the present invention to provide a genetic polymorphism analysis method capable of obtaining highly reliable data for diagnosis.
[0008]
[Means for Solving the Problems]
In the present invention, the SSCP method using a capillary electrophoresis apparatus is used to label target DNA fragments derived from healthy cells and test cells with different fluorescent dyes, and simultaneously perform electrophoresis on the same capillary to compare the migration patterns. By doing so, accurate determination of LOH (determination of the presence or absence of loss of heterozygosity) is possible, and highly reliable data is provided for diagnosis of cancer and the like.
[0009]
In the present invention, gene polymorphism analysis is performed by the SSCP method using one or more capillary electrophoresis. An analysis target DNA fragment to be analyzed and a heterozygous reference DNA fragment as a reference for comparison of the analysis target DNA fragment are labeled with different fluorescent dyes. The DNA fragment to be analyzed and the reference DNA fragment are electrophoretically separated simultaneously with the same capillary. Using the peak signal intensities of the two reference DNA fragments that appear in the obtained migration pattern, the peak signal intensities of the DNA fragments to be analyzed that appear in the migration pattern are corrected, and the heterozygosity of the DNA fragments to be analyzed is corrected. A determination is made as to whether or not there is a loss. The correction of the peak signal intensity of the DNA fragment to be analyzed is executed using the signal intensity ratio of the peaks of the two reference DNA fragments.
[0010]
A PCR product using a genomic DNA derived from a healthy cell of a subject as a template is used as a reference DNA fragment, and a PCR product using a genomic DNA derived from the same subject's test cell as a template is used as a DNA fragment to be analyzed. In addition, a PCR product using a genomic DNA derived from a healthy cell of a heterozygous healthy person as a template is used as a reference DNA fragment, and a PCR product using a genomic DNA derived from a subject's test cell as a template is used as a DNA fragment to be analyzed. used. In particular, a PCR product using, as a template, genomic DNA derived from epithelial cells recovered from blood as a DNA fragment to be analyzed is used. Collection of epithelial cells from blood is performed using a gene polymorphism analyzer that includes a column that selectively adsorbs epithelial cells based on antigen-antibody reaction. Tsp polymerase is used in the PCR reaction to obtain the above PCR product.
[0011]
When the DNA fragment to be analyzed and the reference DNA fragment include a single nucleotide polymorphic site of codon 72 in exon 4 of the p53 gene, the temperature range of the reverse strand of the DNA fragment to be analyzed and the reference DNA fragment is 44 ° C. to 48 ° C. Perform electrophoresis at
[0012]
In the present invention, the secondary structure of the target DNA fragment of interest (analysis target DNA fragment, reference DNA fragment) is obtained by calculation, and the fragmentation in electrophoresis is determined using the expansion of the difference in secondary structure due to polymorphism as an index. A suitable temperature condition that increases separation (Rs) is obtained, and electrophoresis is performed under this suitable temperature condition.
[0013]
In the present invention, the secondary structure of the target DNA fragment of interest (analysis target DNA fragment, reference DNA fragment) to be amplified by PCR is calculated, and the priming site in the PCR reaction is changed to obtain a polymorphism. A suitable primer sequence that increases the separation (Rs) of fragments in electrophoresis is obtained using the expansion of the difference in secondary structure due to the above as an index, and a target DNA fragment is prepared using this suitable primer sequence.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
[Example 1]
FIG. 1 is a flowchart showing an outline of the LOH determination procedure according to the first embodiment of the present invention. The outline of the procedure in the first embodiment will be described below with reference to FIG.
[0015]
(Step A) Genomic DNA extraction from healthy cells and PCR amplification of target DNA fragment:
In Example 1, the diagnosis target was bladder cancer, the test cells were cancer cells derived from bladder cancer that leaked into the urine of the subject, and the healthy cells used in the control experiment were white blood cells in the blood of the same subject. In step A, DNA was extracted from white blood cells in blood to obtain genomic DNA of healthy cells. In Example 1, an automatic nucleic acid extraction apparatus was used to realize a quick, simple and safe extraction operation, which can be suitably applied to routine clinical tests.
[0016]
Next, using the extracted genomic DNA as a template, PCR amplification was performed using Taq enzyme (ABI, AmpliTaq Gold) in the same manner as in the prior art-1 to obtain a target DNA fragment. In Step A, since the primer having the same base sequence as the primer set used in Conventional Technique-1 was used, the region of the target DNA fragment (part of exon 4 of tumor suppressor gene p53) and length (115 nt (nuclear) o tide, base)) is the same as the prior art-1.
[0017]
However, in the prior art-1, a primer whose 5 ′ end is labeled with the fluorescent dye Cy5 for the reverse chain (Click chain) and a primer that is not labeled for the forward chain (Watson chain) are used for PCR. In Example 1, a primer (manufactured by GENSET) labeled with a ROX at the 5 ′ end of the reverse strand and a primer (manufactured by GENSET) labeled with a FAM at the 5 ′ end of the forward strand are used for PCR. Thus, target DNA was obtained as a PCR product.
[0018]
Finally, the 3 ′ end of the PCR product was blunted in the same manner as in the method described in Prior Art-1. That is, Klenow fragment (Klenow enzyme) was added and allowed to react at 37 ° C. for 30 minutes. NTP And the target 115 nt target DNA fragment was obtained.
[0019]
(Step B) Genomic DNA extraction from test cells and PCR amplification of target DNA fragment:
Basically, by the same procedure as in step A, test cells were obtained from the same subject, genomic DNA extraction was performed, and PCR amplification was performed.
[0020]
Step B is different from Step A in that the test cells are cells derived from the bladder that leaked into the urine, and the cells were collected from the urine of the subject and subjected to DNA extraction and amplification. In the extraction of genomic DNA from the collected cells, an automatic nucleic acid extraction apparatus was used as in Step A. In step B, during PCR amplification based on the genomic DNA obtained from the test cells, the 5 ′ end of the reverse strand was labeled with FAM, and the 5 ′ end of the forward strand was labeled with ROX. Target DNA fragments were obtained using primers.
[0021]
(Step C) Electrophoresis and polymorphic separation detection of amplified DNA fragments based on healthy cells:
In Example 1, a DNA sequencer (ABI) having one capillary is used as an electrophoresis apparatus, and electrophoresis is basically performed based on the SSCP conditions recommended by ABI, and PCR amplification products having a single nucleotide polymorphism are separated. Detection was performed. This device is equipped with a mechanism that automatically fills the capillary with the electrophoresis polymer and a mechanism that automatically injects the sample on the autosampler into the capillary, enabling full automatic continuous measurement and suitable for routine clinical testing. is there. This device has a function that can detect a plurality of different emission wavelengths (hereinafter referred to as multi-wavelength detection), pre-evaluates the mutual interference of fluorescence using a standard fluorescent dye, prepares a correction matrix in advance, The effect of interference is removed by matrix conversion, and a plurality of fluorescent dyes can be detected independently.
[0022]
Sample preparation conditions are as follows. Mix 1 µL of PCR reaction solution diluted 1/20 to 1/50 with water, 0.5 µL of size standard (ABI GeneScan 500 TAMRA), 12 µL of formamide, heat denature at 94 ° C for 2 minutes, and release to room temperature. Cool down. However, as described in the process D, the electrophoresis of the process C and the process D was actually performed with the same capillary at the same time. Therefore, the PCR reaction solution used was 1 μL in total, 0.5 μL derived from healthy cells in Step C and 0.5 μL derived from test cells in Step D.
[0023]
The electrophoresis conditions by the SSCP method in Example 1 are as follows. The capillary dimensions are an effective length of 30 cm, a total length of 41 cm, an inner diameter of 50 μm, and an outer diameter of 375 μm. Sample injection conditions were 15 kV and 5 seconds. The electrophoresis voltage is 15 kV. The running buffer is a 1 × TBE buffer solution containing 10% by weight of glycerol. The electrophoresis polymer was based on the electrophoresis buffer, and ABI GeneScan Polymer was added at 9% by weight.
[0024]
FIG. 2 is a diagram showing an example of an electrophoresis pattern obtained by performing electrophoresis by the SSCP method on the above sample, which is Example 1 of the present invention. The horizontal axis in FIG. 2A corresponds to time (t), and t increases from left to right. The vertical axis represents the fluorescence signal intensity at a specific emission wavelength, and the peak height is proportional to the concentration of the DNA fragment labeled with a fluorescent dye that emits fluorescence at the emission wavelength. The vertical axis in FIG. 2A indicates the signal intensity of the fluorescent wavelength of the fluorescent dye FAM.
[0025]
In the previous study, the wild-type homozygous sample for the single-stranded polymorphism of the forward strand of the target DNA fragment (part of p53exon4) has a single peak at the same position as peak 12 in FIG. In the case of the mutant sample, it was confirmed that a single peak was shown at the same position as the peak 13 in FIG.
[0026]
Accordingly, the peaks in FIG. 2 (a) are derived from the wild-type and mutant fragments of the forward strand of the target DNA fragment, respectively. On the other hand, since the forward chain labeled with FAM is a sample derived from a healthy cell described in Step A, the target DNA fragment derived from the healthy cell is related to the single nucleotide polymorphism of exon4 with a wild type and a mutant type. It is understood that it is a heterojunction type. Since two peaks 12 and 13 were observed, it was shown that two target DNA fragments having this single nucleotide polymorphism can be separated and detected by the SSCP method according to Example 1. The migration time for each peak was 23.6 minutes and 23.8 minutes, and the signal intensity was 255 and 263 (arbitrary units), respectively.
[0027]
On the other hand, the signal of the reverse chain labeled with ROX was also separated and detected in the same manner, but was omitted from FIG. 2 because it was outside the time axis range of FIG. The migration times of the wild type and mutant type peaks of the reverse chain were 20.3 minutes and 20.4 minutes, respectively. The separation property of the reverse chain will be described later. Since the migration time of the prior art-1 is about 5 hours, Example 1 has a feature that it is about 15 times faster than the prior art-1. This is thought to be due to the fact that the adoption of a capillary method with excellent heat dissipation characteristics enabled the adoption of an electric field as high as about 366 V / cm, which speeded up migration.
[0028]
(Step D) Electrophoresis and polymorphic separation detection of target DNA fragment based on test cell:
About the target DNA fragment obtained from the test cell by the process B, the analysis regarding the single nucleotide polymorphism of exon4 was performed by analyzing by the SSCP method in the same procedure as the process C. Since the apparatus used can detect multiple wavelengths, the samples in Step C and Step D were actually mixed and electrophoresed simultaneously using the same capillary as described above. The result of electrophoresis is shown in FIG.
[0029]
The horizontal axis of FIG. 2B and the horizontal axis of FIG. 2A are time axes having a common origin and a time scale. From comparison with known samples, two peaks 14 and 15 indicate forward of the target DNA fragment. Corresponds to wild-type and mutant fragments of the chain. The vertical axis in FIG. 2 (b) indicates the signal intensity of the fluorescent wavelength of the fluorescent dye ROX, and the forward chain labeled with ROX is a sample derived from the test cell described in step B. The target DNA fragment is heterozygous for the single nucleotide polymorphism of exon4, including wild type and mutant type.
[0030]
Despite the simultaneous electrophoresis of target DNA fragments obtained from test cells and healthy cells using the same capillary by multi-wavelength detection using interference correction by matrix conversion, they were labeled with the FAM shown in FIG. 2 (a). It is understood that the signal based on the forward chain fragment of a healthy cell and the signal based on the forward chain fragment of the test cell labeled with ROX shown in FIG. 2B can be detected independently of each other. The migration times of peaks 14 and 15 were 23.6 minutes and 23.7 minutes, respectively, and the signal intensities were 255 and 274 (arbitrary units), respectively.
[0031]
The peaks 12 and 14 were confirmed to be peaks of target DNA fragments having the same sequence, respectively, by comparison with the results of migration of wild type samples labeled with FAM and ROX, respectively. Different. This is presumably because the mobility is slightly affected by the difference in fluorescent dyes. The peaks 13 and 15 are the same except that they are mutated. The signal based on the FAM-labeled reverse chain fragment derived from the test cell was outside the time axis range of FIG. 2, but the result similar to the signal based on the reverse chain fragment in Step C is similar to the result. Obtained.
[0032]
(Step E) Determination of whether or not alleles are heterozygous types:
As explained with respect to Step C, since the DNA obtained from healthy cells obtained two peaks 12 and 13 by the SSCP method, the codon 72 of the target DNA fragment (p53exon4) contains a wild type and a mutant type. Alleles were heterozygous different from each other. Therefore, it is possible to perform LOH determination regarding this locus with respect to the test cells of this subject.
[0033]
On the other hand, if only one of the peaks 12 or 13 is observed by the SSCP method, the subject is homozygous for this locus, and information on LOH can be obtained even by examining the DNA of the test cell. Therefore, the LOH determination for this locus cannot be performed. Therefore, it is necessary to test for different loci.
[0034]
(Step F) Calculation of correction coefficient from signal intensity of fragment derived from allele of healthy cell:
In the following description, variables X1 (S1, P1, C1) and X2 (S2, P2, C2) are used as X = S, P, C, but 1, 2 following X are wild type and mutation, respectively. Indicates that it corresponds to a type. As described with respect to Step C, the signal intensities S1 and S2 of the target DNA fragments corresponding to the wild-type and mutant types of the forward chain of healthy cells were 255 and 263, respectively. The correction coefficient F is defined by (Equation 1), and the value of the correction coefficient F is 263/255 = 1.031.
[0035]
[Expression 1]
F = S2 / S1 (Expression 1)
(Step G): From the signal intensity of the fragment derived from the allele of the test cell and the correction coefficient, the ratio of the abundance of the allele in the test cell is determined:
As described in Step D, the signal intensities P1 and P2 of the target DNA fragments corresponding to the wild type and the mutant type of the forward strand of the test cell were 255 and 274, respectively. The ratio T of the abundance of alleles in the test cell is defined by (Equation 2), and T = 274/255 / 1.031 = 1.042.
[0036]
[Expression 2]
T = P2 / P1 / F (Expression 2)
(Process H) Determination of deviation (displacement) from 1 of T:
As determined in the process G, T = 1.042, and the deviation of T from 1 is + 4.2%. A criterion for judging that the deviation from T of ± 10% or more was LOH (abnormal) was set, and in this case, the test cell was judged to have no LOH (abnormal). In this case, the deviation of T from 1 was less than 10%. However, when T ≦ 0.9 or when T ≧ 1.1, the deviation of T from 1 is ± 10% or more. It is determined that there is LOH (abnormality). This result can be provided to a doctor as data for diagnosing bladder cancer.
[0037]
In addition, since the test cell used in Example 1 was that of a healthy person, the above determination of no abnormality was a correct result. In Example 1, although each target DNA fragment was labeled with a different fluorescent dye, electrophoresis was performed simultaneously in the same capillary in Step C and Step D, so the intensity ratio of the fragments derived from healthy cells was used as a reference. It is considered that the correction of the intensity ratio of the fragments derived from the test cells functioned effectively. It should be noted that the difference in behavior between the two due to the difference in fluorescent dyes was virtually irrelevant. This is a fact that has conventionally been difficult for those skilled in the art to predict, and is one of the points of inventive step of the present invention.
[0038]
In Example 1, as a capillary electrophoresis apparatus for performing analysis by the SSCP method, for example, Ar + laser and He-Ne laser are used as a light source, and a diffraction grating and a CCD camera are used as a detector. Can also be used. The scope of application of the present invention is not limited to these apparatuses, and a wide range of general capillary electrophoresis apparatuses capable of detecting fluorescence with multiple wavelengths can be applied. In Example 1, for the sake of simplicity, the case of performing fluorescence detection from two types of fluorescent dyes is illustrated, and a method of using only one type of target DNA fragment derived from a test cell is illustrated. If each target DNA fragment is labeled with n different fluorescent dyes using an apparatus capable of detecting fluorescence from n kinds of fluorescent dyes, naturally, up to (n-1) target cell-derived target DNAs. Fragments can be measured simultaneously.
[0039]
The scope of application of the present invention is not limited to the p53exon4 region of the genomic DNA sample extracted from the cells collected from the blood and urine and the diagnosis of bladder cancer for the sample, the PCR region, and the application shown in Example 1 . The present invention can be applied to samples obtained from cells derived from other living organisms, and single nucleotide polymorphisms, microsatellite polymorphisms, short tandem repeat polymorphisms, etc. in other gene regions can be used as markers. . It can also be applied to genetic diagnosis of other diseases where LOH is an index. The present invention can be similarly applied to analysis tests using other capillary electrophoresis apparatuses when the signal intensity ratio needs to be evaluated with high accuracy.
[0040]
The unique effect of Example 1 is that it is possible to unify all the electrophoresis conditions except for the difference in fluorescent dyes by simultaneously electrophoresing a fragment derived from a healthy cell and a fragment derived from a test cell through the same capillary. . Even if there are fluctuations in electrophoresis conditions that affect the signal intensity ratio of allele-derived fragments that are the criteria for determining LOH, the effects of fluctuation factors can be offset and high-precision measurement can be achieved. is there.
[0041]
[Example 2]
Example 2 is the same as Example 1, but the enzymes used for PCR amplification and the steps after PCR are different. In Step A and Step B of Example 1, Taq polymerase was used as a PCR enzyme. In this case d at the 3 'end NTP A PCR product to which one (N = A, T, G, C) is added is generated, and a ghost peak caused by the PCR product has a problem of hindering quantitative analysis by the SSCP method. In order to avoid this defect, the PCR product was treated with Klenow enzyme to smooth the 3 ′ end, and dNTPs at the protruding end of the 3 ′ end were removed.
[0042]
In Example 2, by using Platinum Genotype Tsp polymerase (Life Technologies / GibcoBRL) as a PCR enzyme, the addition of dNTP to the 3 ′ end was suppressed and the Klenow enzyme treatment was omitted. According to Example 2, the Klenow enzyme reaction, which requires time, labor and cost, was omitted. Further, by omitting one enzyme reaction step, error factors such as poor reproducibility due to inactivation of the enzyme were eliminated. Therefore, Example 2 has a specific effect that a rapid, simple, inexpensive and highly reliable clinical test can be performed. Various PCR enzymes that are considered to be difficult to add dNTP to the 3 ′ end have been reported. As a result of comparison of various enzymes, the above Tsp was most suitable.
[0043]
FIG. 3 is Example 2 of the present invention. For products obtained by PCR amplification of the exon4 region of the p53 gene using various polymerases, the fragment length was measured using a capillary electrophoresis apparatus packed with a denaturing gel POP4 (ABI). It is an example of the result of having analyzed. 3A shows AmpliTaq Gold (ABI), FIG. 3B shows Platinum Genotype Tsp, FIG. 3C shows Pyrobest (Takara Shuzo), and FIG. 3D shows Pfx (Life Technologies). / GibcoBRL) is an example of an electrophoresis pattern of PCR products obtained using each as a polymerase.
[0044]
In the migration patterns of FIGS. 3 (a) and 3 (c), fragments 17 and 18 having a larger number of bases are generated as by-products in addition to the desired 115nt fragment 16 as a PCR product. In the electrophoresis pattern of FIG. 3D, a fragment 19 having a shorter base number than the 115nt fragment 16 is generated as a PCR product. As shown in FIG. 3 (b), the PCR product migration pattern using Tsp polymerase contains almost no by-products with a length other than the target 115nt fragment 16. Therefore, by using Tsp as a polymerase in the PCR reaction, a DNA fragment having a smooth 3 ′ end can be obtained, and the Klenow enzyme reaction can be omitted.
[0045]
Example 3
FIG. 4 is a flowchart showing an outline of the LOH determination procedure according to the third embodiment of the present invention. The procedure of the third embodiment is similar to the procedure of the second embodiment, but the calculation method of the correction coefficient F is different.
[0046]
(Step I): In Example 2, the correction coefficient F was determined using a target DNA fragment obtained by PCR amplification of genomic DNA obtained from healthy cells of a subject as a standard sample. In Example 3, target DNA fragments obtained by PCR amplification of genomic DNA obtained from cells of other individuals or other organisms, not necessarily from the subject's own healthy cells, or target DNA obtained by chemical synthesis Fragments were used alone or mixed.
[0047]
When used alone, the sample must be obtained from a heterozygous healthy cell, but this is not the case when used in a mixed manner, but obtained from a homozygous or LOH cell. Or it may be a chemically synthesized product. Of course, when the target DNA fragment is obtained from homozygous or LOH cells or by chemical synthesis, not only the wild type but also the mutant type sample is prepared and mixed to obtain a composition similar to the heterozygous type. (In this case, the composition ratio is not necessarily strictly 1: 1 for the following reason). In any case, in Step I, a target DNA fragment having a heterozygous-like composition corresponding to Step A in Example 1 and Example 2 is prepared as a standard sample stock.
[0048]
(Step J): The concentrations C1 and C2 of fragments (wild type and mutant in Example 3) derived from alleles of a target DNA fragment (standard sample stock) having a heterozygous similar composition are quantified. Various methods can be used to quantify the concentration. In Example 3, primary samples of wild type and mutant types (concentration test completed) were prepared, and the standard sample stock and each primary standard sample were used for analysis by the SSCP method using an electrophoresis apparatus. The concentrations C1 and C2 of each fragment derived from the allele of the standard sample stock were quantified by comparing the height of the migration peak corresponding to the fragment derived from each of the type and the mutant type.
[0049]
As another method for determining the concentrations C1 and C2, prepare a heterozygous primary standard sample that has been confirmed to be free of LOH (concentration tested, in this case, the concentration of wild type and mutant type is the same), The concentrations C1 and C2 are determined by comparing the SSCP pattern with that of the standard sample stock. Alternatively, target DNA fragments are prepared for each of the wild type and the mutant type, and the concentration of each fragment is determined by measuring the UV intensity in advance and measuring the fluorescence intensity by the interaction with an intercalator such as PicoGreen (manufactured by Molecular Probes). After the determination, the concentrations C1 and C2 are determined by mixing them at a predetermined ratio. Alternatively, by collecting a large number of healthy heterozygous individuals, confirming that they do not have LOH, and mixing the target DNA fragments obtained from them in approximately equal amounts, the ratio is approximately 1: 1. There is also a method in which a standard sample stock that can be regarded as having a close concentration ratio is prepared, the total concentration C of the target DNA fragments is obtained, and the concentrations C1 and C2 are determined by (Equation 3).
[0050]
[Equation 3]
C1 = C2 = C / 2 (Equation 3)
Regardless of the origin of the standard sample stock in step I, the concentrations C1 and C2 of the fragments derived from alleles of the standard sample stock are determined in step J. Step I and step J provide a heterozygous (similar) standard sample stock containing fragments of known concentrations derived from alleles.
[0051]
(Step K): Electrophoresis of a standard sample stock is performed and separation detection is performed based on the base sequence polymorphism. The process K is the same as the process C in Example 1 and Example 2, and as a result, the signal intensities S1 and S2 of each fragment derived from the allele were obtained. The difference is that the sample used is a standard sample stock and that it is known to be definitely heterozygous or heterozygous, and the concentration of each fragment derived from that allele. Are C1 and C2, respectively.
[0052]
(Step F ′): A correction coefficient F is obtained from the signal intensities S1 and S2 of the fragments 1 and 2 and the concentrations C1 and C2. DNA fragments 1 and 2 correspond to the wild type and the mutant type, respectively. Step F ′ is similar to Step F in Example 1 and Example 2. The difference is that the fragments 1 and 2 are not derived from the healthy cells of the subject, but are derived from the above-mentioned standard sample stock. The concentrations of the fragments 1 and 2 are not necessarily equal, and the concentrations are given by C1 and C2. As shown in (Equation 4), this is also a point to be obtained in consideration of the concentration. Of course, in the case of the complete heterojunction type with C1 = C2, the correction coefficient F obtained by the process F ′ in the third embodiment matches the correction coefficient F obtained by the process F in the first and second embodiments.
[0053]
[Expression 4]
F = (S2 / C2) / (S1 / C1) (Equation 4)
(Step A, Step C, Step E): Step A, Step C, Step E in Example 3 are almost the same as Step A, Step C, Step E in Example 1 and Example 2. The difference is that in Example 3, the signal intensity in the SSCP analysis of the fragment obtained from healthy cells in Step C is not used in the calculation of the correction coefficient F, but only whether it is heterozygous in Step E or not. It is used only for judgment.
[0054]
(Process B, Process D, Process G): Process B, Process D, and Process G in Example 3 are substantially the same as Process A, Process C, and Process E in Example 1 and Example 2. The difference is that in Example 3 in Step D, electrophoresis of DNA fragments from the test cells is performed together with the standard sample stock in Step K. Of course, as in Example 1 and Example 2, electrophoresis may be performed together with the DNA fragment from the healthy cells in Step C, but this is not always necessary (as described above, the data on healthy cells are heterozygous. It is used only to confirm that it is present and is not reflected in the correction factor).
[0055]
When electrophoresis is performed together with DNA fragments from the healthy cells in step C, the DNA fragments from the healthy cells in step C are distinguished from the standard sample stock in step K and DNA fragments from the test cells in step D. Therefore, in PCR amplification of DNA fragments from healthy cells in step A, labeling is performed with a third fluorescent dye different from the fluorescent dye used in steps I and B.
[0056]
(Process G, Process H): Process G and Process H in Example 3 are the same as Example 1 and Example 2. The only difference is that the value obtained by measuring the standard sample stock as described in step F ′ is used as the correction coefficient F when determining the ratio T of the abundance of alleles in the test cell in step G. It is. In Example 3, when obtaining the correction coefficient F, the migration pattern of the standard sample stock is used instead of the migration pattern of the DNA fragment derived from the healthy cells of the subject. Hereinafter, a characteristic effect of the third embodiment will be described.
[0057]
First, even if the ratio T of the abundance of alleles in the target region in the subject's cells assumed to be healthy cells deviates (divides) from 1: 1, There is an effect that LOH can be calculated with high accuracy. As a case where such a case occurs, for example, when a leukocyte in blood is used as a healthy cell, the subject has leukemia.
[0058]
Second, steps I and J are not performed by the user, but are preferably performed by the device manufacturer or reagent manufacturer, and the user can purchase and use standard sample stocks of known concentrations from the device manufacturer or reagent manufacturer. In this case, there is an effect that the reliability of the analysis result can be improved. Furthermore, when it is confirmed that the subject is originally a heterojunction type with respect to the target region by prior examination on the subject, Step A, Step C, and Step E can be omitted. In this case, since the user can omit the preparation of the reference sample by purchasing and using the standard sample stock, there is also an effect that the user can save labor.
[0059]
Third, it has been confirmed in advance that the standard sample stock has a heterozygous type or a similar composition. Therefore, if two peaks are not detected when this sample is analyzed by the SSCP method in step K, it means that there is some problem with the analysis method, and this information can be used to control the quality of the analysis method. effective. If the composition ratio of the standard sample stock and the peak intensity ratio of the migration pattern are significantly different from each other, it may mean a large change in the analysis conditions, which enables quality control of the analysis method. There is also an effect that the user can be alerted and the cause investigation and countermeasures can be promoted.
[0060]
Example 4
FIG. 5 is a flowchart showing an outline of the LOH determination procedure according to the fourth embodiment of the present invention. The procedure of Example 4 is similar to the procedure of Example 3, but at each stage of the calculation of the signal intensity and concentration of the electrophoretic pattern, the alleles are not calculated independently for the allele-derived fragments. The difference is that the calculation is performed using the ratio value for the two fragments derived from. Specifically, in step J ′, instead of obtaining the concentrations of fragments 1 and 2 as C1 and C2, respectively, the concentration ratio C _1,2 Ask for. Similarly, in step F ″, instead of the signal strengths S1 and S2, the signal strength ratio S is calculated by (Equation 6). _1,2 And F is calculated from (Equation 7). Similarly, in step G ′, instead of the signal intensities P1 and P2 of the fragment 1 and the fragment 2 of the test cell, the signal intensity ratio P _1,2 And T is obtained from (Equation 9).
[0061]
[Equation 5]
C _1,2 = C2 / C1 (Expression 5)
[0062]
[Formula 6]
S _1,2 = S2 / S1 (Expression 6)
[0063]
[Expression 7]
F = S _1,2 / C _1,2 ... (Equation 7)
[0064]
[Equation 8]
P _1,2 = P2 / P1 (Equation 8)
[0065]
[Equation 9]
T = P _1,2 / F (Equation 9)
The special effect of the fourth embodiment is that the amount of information to be handled can be reduced to about half, so that the storage capacity for the computer is small, the effort of numerical input is reduced for the user, and the calculation flow is easier to understand. It is.
[0066]
(Example 5)
Example 5 is the same as Example 2, except that the DNA fragments to be separated and quantified in Step C and Step D are not fragments derived from the forward chain, but fragments derived from the reverse chain. Changes in the procedure include obtaining a migration pattern around 20 minutes after the start of electrophoresis, a fragment labeled with a ROX derived from a reverse chain derived from a healthy cell, and a fragment labeled with a FAM derived from a reverse chain derived from a test cell Analysis was performed. Except that the observed peak was a fragment derived from the reverse chain, the same results as in Example 2 were obtained by the same procedure as in Example 2.
[0067]
The analysis conditions at this time were SBI conditions recommended by ABI, that is, a polymer concentration of 3% and a temperature of 30 ° C. In this case, the separation Rs of the peaks of the two fragments derived from the allele of the reverse chain (assuming that the peak shape of the migration pattern is a Gaussian distribution type, the peak interval is divided by the bandwidth, ie, 4 sigma. Defined) was about 0.8 to 1.0.
[0068]
Next, the polymer concentration was examined. When preparing a migration polymer of 6% or more, a migration polymer was prepared based on a solution obtained by concentrating a GeneScan Polymer solution (7%) (manufactured by ABI) to about 15% by centrifugal concentration. As a result, it was found that the separation improved linearly as the concentration increased in the polymer concentration range up to 9%. Separation of fragments derived from the reverse chain at a concentration of 9% gave a high separation of about 2.3.
[0069]
When the polymer concentration was 10% or more, the reproducibility of the electrophoresis results was lowered. This is thought to be because the viscosity of the polymer solution increased and the polymer solution could not be uniformly filled into the capillary. Therefore, from the viewpoint of obtaining high separation, the optimum value of the polymer concentration is considered to be about 9%. The condition of the polymer concentration of 9% in Example 1 was also set based on the knowledge obtained from the above examination.
[0070]
Next, the temperature at which electrophoresis was performed was examined. In the prior art-1, 20 ° C. is adopted as the temperature condition for electrophoresis of p53 exon4. In general, the SSCP method has a tendency of higher separation at lower temperatures, so electric transfer at lower temperatures was attempted. However, in the study of the present invention using capillary electrophoresis, the separation was hardly improved even under the condition of a temperature of 20 ° C. On the contrary, when electrophoresis was performed under conditions higher than 30 ° C., surprisingly, a phenomenon was found that the separation of the fragments derived from the reverse strand at about 46 ° C. to 48 ° C. was specifically improved. .
[0071]
FIG. 6 is a diagram showing an example of the temperature dependence of separation (Rs) of the fragment derived from the reverse chain of p53exon4 by the SSCP method, which is Example 5 of the present invention. FIG. 6 shows the change in separation of the migration peak of the fragment derived from the wild type and the mutant of the p53 exon 4 reverse chain when the temperature is changed from 30 ° C. to 60 ° C., that is, the separation temperature at a polymer concentration of 3%. A change 23 (◯ mark), a separation temperature change 24 (Δ mark) at a polymer concentration of 6%, and a separation temperature change 25 (□ mark) at a polymer concentration of 9% are shown.
[0072]
As is clear from FIG. 6, the separation (Rs) was almost constant regardless of the temperature in the range from 30 ° C. to 40 ° C., but the sharp separation (Rs) was observed in the narrow temperature range near 40 ° C. to 50 ° C. An improvement was seen. The highest values of separation (Rs) in this range were about 3.3, 4.8 and 6.0 at polymer concentrations of 3%, 6% and 9%, respectively. These values were about three times higher when compared to the values at 30 ° C.
[0073]
In Example 5, since 46 ° C. to 48 ° C., which is a specific temperature that gives such a large separation (Rs), was adopted as the electrophoresis condition, it is easy to separate and quantify peaks and to improve measurement accuracy. There is. The separation (Rs) is proportional to the effective length of the capillary (1/2), but the analysis (electrophoresis) time is proportional to the effective length of the capillary. Therefore, the separation (Rs) is three times that obtained by optimizing the temperature. If the improvement in the offset is offset by the decrease in separation (Rs) due to shortening of the capillary effective length, the same separation (Rs) can be achieved with a capillary effective length of 1/9, and thus the analysis (electrophoresis) time can be achieved with 1/9. .
[0074]
Therefore, there is an effect that the speed can be increased by about one digit in exchange for the improvement of the separation (Rs) three times obtained by the optimization of the temperature. Similarly, if the large separation (Rs) obtained by optimizing the temperature is offset by the decrease in separation (Rs) due to the decrease in polymer concentration, the speed can be increased.
[0075]
In addition, there are few reports of improvement of the separation temperature (Rs) at the specific temperature related to the separation (Rs) of the SSCP method as described above, particularly at a high temperature of 46 ° C. to 48 ° C., and the mechanism is still unclear. Therefore, in order to analyze the mechanism, calculation of the secondary structure of the DNA fragment was performed using public domain software RNAfold (Monatsheft for Chemie 125, 167-188, 1994). As the calculation algorithm, a calculation method using both a partition function and a pair probabilities was adopted.
[0076]
Parameters for DNA described in the literature (SantaLucia, Proc. Natl. Acad. Sci. USA 95, 1460-1465, 1998) were used for energy calculations. Of course, there are several other software for calculating the secondary structure of the DNA fragment, and the same calculation is possible with other software.
[0077]
FIG. 7 is a diagram showing the results of calculation of secondary structures at 30 ° C. and 46 ° C. of wild-type and mutant DNA fragments of the reverse strand of p53exon4 showing the above specific temperature, which is Example 5 of the present invention. It is. In FIG. 7, since the individual base sequences are difficult to see due to the scale, the position of the single nucleotide polymorphism (codon 72), which is the only difference between the wild type and the mutant type, is indicated by an arrow (←) and the type of the base. Is indicated by C (wild type) or G (mutant type). FIG. 7A shows a wild-type secondary structure at 30 ° C., and FIG. 7B shows a mutant-type secondary structure at 30 ° C. The secondary structures shown in FIGS. 7 (a) and 7 (b) both show a secondary structure with a lot of self-association, and the structures are slightly similar in the vicinity of the position of the single nucleotide polymorphism, but similar as a whole. It is understood that it is a secondary structure.
[0078]
FIG. 7C shows a wild type secondary structure at 46 ° C., and FIG. 7D shows a mutant type secondary structure at 46 ° C. The secondary structure in FIG. 7 (d) was basically similar to the secondary structure shown in FIG. 7 (b) except that the ends were partially dissociated. On the other hand, the secondary structure of FIG. 7C is greatly different from the secondary structure of FIG. 7A, and many self-association structures are dissociated, resulting in many single-stranded regions.
[0079]
The secondary structure calculation results described above are considered to correspond to the fact that the separation characteristics of both wild type and mutant fragments in the SSCP method are greatly different at 30 ° C. and 46 ° C. That is, at 30 ° C., both wild type and mutant fragments have a secondary structure with many self-associations, but their structures are similar to each other. Therefore, even if electrophoresis is performed by the SSCP method, separation is not good because there is no significant difference in structure. When the temperature is raised to 46 ° C., both the wild type and the mutant type fragments have different secondary structures so that there is almost no common point between them, so that high separation (Rs) can be obtained by electrophoresis using the SSCP method.
[0080]
The electrophoretic characteristics at 46 ° C. can be considered in further detail as follows. Wild-type dissociates most of the self-association structure and has a high proportion of single-stranded parts, so it is flexible with few forbidden points. Then, the dynamic radius when moving between electrophoresis gels decreases, in other words, the mobility increases. On the other hand, the change in mobility is small because the mutant has little structural change even when the temperature is raised to 46 ° C. Therefore, it is considered that the migration time of the wild type is relatively shortened compared with the mutant type and the separation is improved.
[0081]
The SSCP method has been proposed to be the mechanism of separation by changing the higher order structure of the electrophoretic fragment due to changes in the base sequence such as single nucleotide polymorphism, but there are few examples of analyzing the separation characteristics in association with the higher order structure. . In particular, as an SSCP method of about 46 ° C., an example of successful analysis of the phenomenon of improvement of specific and steep separation (Rs) at a high temperature and corresponding to the higher order structure The present invention is considered to be the first time. Conventionally, the SSCP method requires trial and error as to whether separation can be obtained. In other words, in some cases, it was a technique that relied on intuition, experience, and luck that separation would not be achieved or abandoned. The above examination of Example 5 shows the possibility that the relationship between the secondary structure of nucleic acid and SSCP separation can be theoretically associated.
[0082]
Therefore, before attempting separation by the SSCP method, the secondary structure is predicted by calculation in advance, whether or not fragments having single nucleotide polymorphisms can be expected, and what is the temperature with the best separation (Rs)? It was shown in Example 5 that a method of predicting whether or not the degree is appropriate by calculation and optimizing the conditions of the SSCP method based on the result is effective.
[0083]
Conditions of the SSCP method that can be optimized by this method include electrophoresis temperature, fragment arrangement, and the like. Even if the position of the target single nucleotide polymorphism has been determined, the sequence of the PCR-amplified fragment can be changed by changing the position of the primer sandwiching it, and the separation characteristics in that case are predicted by the same procedure as above. , Can be optimized. Naturally, it is also effective in Example 5 to design a primer based on the optimization result. In addition, a simulation for improving separation by adding an arbitrary sequence to the 5 ′ end of the primer is also possible.
[0084]
The peculiar effect of Example 5 is that a single nucleotide polymorphism of the gene locus of exon4codon72, which has been reported to be related to the susceptibility to cervical cancer caused by papillomavirus, on the p53 gene, which is attracting attention as a tumor suppressor gene. When separation is detected, an extremely high separation (Rs) of 3 times or more compared to the conventional method can be obtained by the SSCP method, the reason can be explained by theoretical analysis, and the separation of the SSCP method can be simulated.
[0085]
(Example 6)
Example 6 is the same as Example 2, except that in Step B, tissue-derived epithelial cells are collected and used from peripheral blood instead of urinary bladder-derived cells.
[0086]
For selection and collection of epithelial cells from blood, a method described in a literature using a cell sorter (Racila et al., Proc. Natl. Acad. Sci. USA 95, 4589-4594 (1998)) was adopted. Specifically, after mixing magnetic particles bound with an antibody against EPCAM (epithelial cell adhesion molecule) and peripheral blood, B / F separation is performed using a magnet, and a fraction containing cells bound to the magnetic particles is obtained. Obtained.
[0087]
This fraction was treated with a solution containing an anti-cytokeratin antibody conjugated with phycoerythrin and an anti-CD45 antibody labeled with peridinin chlorophyll. After B / F separation and purification with a magnet, measurement was performed with a cell sorter. As described in the above document, the number of CD45 antibodies was small, and epithelial cells reacted with cytokeratin antibodies at a high concentration were sorted. At this time, the number of sorted cells was recorded.
[0088]
After epithelial cells were selected and collected from the peripheral blood in this way, DNA extraction and amplification were performed by the same procedure as in Step B of Example 2. For the other steps, the same steps as in Example 2 were employed to determine the LOH of nucleic acid derived from epithelial cells in blood.
[0089]
As described in the above document, cells with a small number of CD45 antibodies and cytokeratin antibodies reacted at high concentrations are epithelial cell lines, and most epithelial cells present in peripheral blood are carcinoma cells. Therefore, most of the cells sorted by the cell sorter are considered metastatic cancer cells. However, epithelial cells other than cancer cells may be mixed into the peripheral blood due to contamination of epithelial cells by the needle at the time of collecting peripheral blood or various traumas and inflammations. If the diagnosis of cancer is based only on the number of cases, false positives may occur, and there is a problem that accuracy is low.
[0090]
In Example 6, in addition to sorting and counting the epithelial cells in the peripheral blood, the LOH of the genomic DNA of the cells is obtained to evaluate whether or not the cells are derived from cancer cells with high accuracy. Is possible. Therefore, Example 6 has a feature that it can provide a highly accurate diagnostic material with few false positives. In addition, it can detect and diagnose not only bladder cancer but also metastatic carcinoma in general using peripheral blood, so it has a wide range of applications, is less invasive to subjects, and is an effective screening method for early detection of various cancers. It has the feature that it can contribute to early treatment and improvement of survival rate.
[0091]
(Example 7)
Example 7 is the same as Example 6, except that cell sorting is not performed and cell separation is performed only by immunization. Specifically, after mixing magnetic particles bound with an antibody against EPCAM (epithelial cell adhesion molecule) and peripheral blood, B / F separation is performed using a magnet, and a fraction containing cells bound to the magnetic particles is obtained. Obtained.
[0092]
After breaking the bond between EPCAM and magnetic particles, B / F separation is performed using a magnet, unbound fraction and magnetic particles bound with anti-cytokeratin antibody are mixed, and B / F separation is performed using a magnet. To obtain a fraction containing cells bound to magnetic particles. From this fraction, DNA extraction and amplification were performed by the same procedure as in Step B of Example 2. For the other steps, the same steps as in Example 2 were employed to determine the LOH of nucleic acid derived from epithelial cells in blood.
[0093]
In Example 7, since not only immune reaction but also DNA LOH is used as a judgment material, even if a small amount of white blood cells are mixed, the influence is small. Therefore, there is an effect that it is possible to provide a judgment material capable of highly accurate diagnosis with a simple device and method.
[0094]
In the above description, the magnetic immunization method has been described as an example of the cell separation method by the immunization method, but of course, cell separation methods by various immunization methods not using magnetism are also applicable. For example, by using an immunobead method in which B / F separation is performed by centrifugation using a carrier such as an inorganic or organic bead bound to an antibody, or a column packed with an inorganic or organic bead or fibrous carrier bound to an antibody. An immunochromatography method for performing B / F separation is also applicable. In the above description, in order to perform two kinds of immune reactions against EPCAM and cytokeratin both by the magnetic immunization method, a step of cutting the bond with the magnetic particles in the middle is adopted.
[0095]
However, when the subsequent immune reaction is performed by a method other than magnetic immunization, such as immunochromatography, this magnetic particle removal step is unnecessary, and B / F separation is directly performed by a column having the second antibody. Be put on. Cells bound to the carrier in the column can be cleaved once and recovered as cells, followed by subsequent DNA extraction. Alternatively, it is possible to extract by extracting a nucleic acid by adding a reagent that lyses the cell while the cell is bound in the column.
[0096]
Next, the effect of the present invention, that is, the ability to perform highly accurate LOH determination will be described. Here, a comparative method for comparison with the present invention will be described. This comparative method for comparison with the present invention is a method in which some of the conditions of the prior art-1 method are changed. Prior art-1 and Example 1 of the present invention are as follows: (1) The target DNA fragments of healthy cells and test cells are labeled with the same fluorescent dye in prior art-1 and with different fluorescent dyes in the present invention; 2) In electrophoresis, the conventional technique-1 uses a slab gel, and in the present invention, a capillary is used. (3) The target DNA fragments of healthy cells and test cells are separated in the conventional technique-1 by another electrophoresis path. Then, it is basically different in that it migrates with the same capillary.
[0097]
In the comparative method, the target DNA fragment of the healthy cell and the test cell is labeled with the same fluorescent dye as in the prior art-1, and the target DNA fragment of the healthy cell and the target DNA fragment of the test cell are separated by one. Electrophoresis using a capillary. Since the target DNA fragments of healthy cells and test cells are labeled with the same fluorescent dye, electrophoresis is performed twice. The comparative method is a method in which the method of Prior Art-1 is applied to electrophoresis using a single capillary.
[0098]
FIG. 8 is a diagram illustrating the results of an accuracy comparison example of LOH determination according to the procedure of the first embodiment of the present invention and the procedure of the comparative example. FIG. 8 shows the results of LOH determination obtained by repeatedly performing experiments on four different samples (experiment numbers 1, 2, 3, and 4). In addition, raw data (measurement data) S1, S2, P1, and P2 according to the procedure of Example 1 and the LOH value (ratio of deviation (deviation) from 1 of T in%) are shown as analysis results.
[0099]
In FIG. 8, the measurement data S1, S2, P1, and P2 according to the procedure of Example 1 are used as they are, and the target DNA fragment data S1 and S2 from healthy cells are used as n (n = 1, 2, ...) The measurement data of the second time is used as the data P1, P2 of the target DNA fragment from the test cell, and the analysis data is analyzed using the (n + 1) th measurement data of each experiment number. Simulated. The rightmost column in FIG. 8 shows the LOH value according to the procedure of the comparative example.
[0100]
In the present invention, in any of the samples of Experiment No. 1 to Experiment No. 4, the LOH values were all within ± 10%, and it was determined that the test cells had no LOH (no abnormality). Since the samples used were normal cells of healthy subjects and test cells, all the judgment results are correct. On the other hand, in the comparative example, similar results were obtained for most of the samples with the experiment numbers. However, in the first measurement of the sample with the experiment number 2 and the second measurement of the sample with the experiment number 3, the LOH value was −. 10.7% and + 13.8%, which exceeded the range of ± 10%. Therefore, the test cell was erroneously found to have LOH (abnormal).
[0101]
The average of the sample of experiment number 3 exceeds the range of ± 10%. As shown in FIG. 8, in comparison, half of the samples of Experiment No. 1 to Experiment No. 4 gave erroneous results.
[0102]
The above results are comparison results when a single capillary device is used. Even when a multicapillary device having a plurality of capillaries is used, LOH determination can be similarly performed with high accuracy. When the present invention is applied to a multicapillary, target DNA fragments of healthy cells and test cells labeled with different fluorescent dyes are mixed and injected into different capillaries for each sample, and fragments relating to a plurality of samples are placed in each capillary. Simultaneously separate by electrophoresis. In contrast to this, all target DNA fragments are labeled with the same fluorescent dye, the target DNA fragment of healthy cells is injected into one capillary, the target DNA fragment of test cells is injected into another capillary, A method is conceivable in which two capillaries are used and fragments relating to a plurality of samples are electrophoresed and separated simultaneously in each capillary.
[0103]
This comparative method is not affected by fluctuations in measurement conditions over time, as in the method of the present invention. However, in this comparative example, a subtle difference in conditions between capillaries affects the measurement results. In the method of the present invention, target DNA fragments of healthy cells and test cells are migrated with the same capillary using the same capillary. Since they are separated, it is not affected by the difference in conditions between capillaries, and it can be understood that the method of the present invention is more accurate than the comparative example and the prior art-1.
[0104]
As is clear from the above comparison, the first embodiment of the present invention can determine LOH with high accuracy as compared with the comparative example, and has an effect of providing data suitable for high-accuracy diagnosis. The same applies to other embodiments of the present invention.
[0105]
【The invention's effect】
According to the present invention, LOH can be determined with high accuracy even if the signal of the electrophoresis peak fluctuates due to electrophoresis conditions and the like, and it is possible to provide highly reliable data for diagnosis.
[Brief description of the drawings]
FIG. 1 is a flowchart showing an outline of an LOH determination procedure according to the first embodiment of the present invention.
FIG. 2 is a diagram showing an example of an electrophoresis pattern according to the SSCP method, which is Embodiment 1 of the present invention.
FIG. 3 is a diagram showing an analysis example of the fragment length of PCR products using various polymerases, which is Example 2 of the present invention.
FIG. 4 is a flowchart showing an outline of an LOH determination procedure according to the third embodiment of the present invention.
FIG. 5 is a flowchart showing an outline of an LOH determination procedure according to the fourth embodiment of the present invention.
FIG. 6 is a graph showing an example of temperature dependence of separation by the SSCP method of a fragment derived from the reverse strand of p53exon4, which is Example 5 of the present invention.
FIG. 7 is a diagram showing the calculation results of the secondary structure at 30 ° C. and 46 ° C. of wild-type and mutant DNA fragments of the reverse strand of p53exon4, which is Example 5 of the present invention.
FIG. 8 is a diagram showing an example of comparison results of LOH determination according to the procedure of the first embodiment of the present invention and the procedure of the comparative example.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... DNA fragment corresponding to wild type, 2 ... DNA fragment corresponding to mutant type, 3 ... Step E, 4 ... No information, 5 ... Step F, 5 '... Step F', 5 "... Step F", 6 ... Step B, 7 ... Step D, 8 ... Step G, 8 '... Step G', 9 ... Step H, 10 ... No LOH (normal), 11 ... With LOH (abnormal), 12 ... Target DNA fragment derived from healthy cells Migration peak corresponding to the wild-type FAM-labeled fragment of forward chain, 13 ... migration peak corresponding to the forward-chain mutant FAM-labeled fragment of the target DNA fragment derived from healthy cells, 14 ... test cell Migration peak corresponding to the wild-type ROX-labeled fragment of the forward strand of the target DNA fragment derived from, 15 ... migration peak corresponding to the ROX-labeled fragment of the forward-chain variant of the target DNA fragment derived from the test cell , 16 ... eyes 115 nt fragment, 17 ... 115 nt longer, 18 ... 115 nt longer, 19 ... 115 nt shorter, 20 ... 115 nt shorter, 20 ... step I, 21 ... step J, 21 '... step J', 22 ... Process K, 23 ... Separation temperature change at 3% polymer concentration, 24 ... Separation temperature change at 6% polymer concentration, 25 ... Separation temperature change at 9% polymer concentration, 100 ... Process A, 200 ... Process C.

Claims (3)

Labeling a DNA fragment to be analyzed and a heterozygous reference DNA fragment as a reference for comparison with the DNA fragment to be analyzed with different fluorescent dyes,
Separating the DNA fragment to be analyzed and the reference DNA fragment by electrophoresis simultaneously in the same capillary by the SSCP method using capillary electrophoresis ;
Correcting the peak signal intensity of the DNA fragment to be analyzed of the electrophoresis pattern using the peak signal intensity of the reference DNA fragment of the electrophoresis pattern obtained in the separating step;
Wherein the result of the correction to process, possess the determining step whether the loss of heterozygosity of the analyte DNA fragments,
The reference DNA fragment is a 3'- end smooth PCR product using genomic DNA derived from a healthy cell of a heterozygous healthy person as a template, and the DNA fragment to be analyzed is a genomic DNA derived from a test cell of a subject. 3 'end is smooth PCR product, and the reference DNA fragment and the analyte DNA fragments, genetic polymorphism analysis method which is characterized in that which was PCR amplified using identical primers with. Claims, characterized in that using the signal intensity ratio of two peaks corresponding to each of the mutant and wild-type reference DNA fragments of the heterojunction, correcting the signal strength of the peak of the analyte DNA fragments 2. The gene polymorphism analysis method according to 1.   The gene polymorphism analysis method according to claim 1, wherein the PCR product is a product of a PCR reaction performed using Tsp polymerase.

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