Patent Application
20070037199
1. Field of the Invention
The present invention relates to a method of discriminating individual using DNA sequence information, and further, relates to an array, an apparatus and a system for an individual discriminating test.
2. Description of the Related Art
By almost complete decoding of the nucleotide sequence of the human genome, a sequence having a difference between individuals and between races, and the frequency thereof has been made clear. The difference between individuals is utilized in studies for elucidating the relationship between the effect and the side effects of a drug in the medical field.
The difference in a genome nucleotide sequence is also utilized in individual identification in criminal investigation. Currently, the main method used in individual identification is a method based on a difference in a repetition time of a repeat sequence on a DNA sequence, a representative of which is Short Tandem Repeat (STR) and Variable Number of Tandem Repeat (VNTR). For example, a method which has been adopted by the police since 2003 is a method of amplifying a region of nine places of STR and one place of VNTR by a polymerase chain reaction (PCR), subjecting the amplification product to a capillary electrophoresis method, and determining the repetition number from mobility thereof.
However, in a sequence such as STR, VNTR and the like, a constant sequence of a few nucleotide units appears repeatedly, and regarding the aforementioned ten places, a full length of the repeat sequence is about 30 to 600 bp. In order to measure this repetition time, it is required that an objective region is retained in a sample to be tested without being cleaved. However, it is easily considered that a body liquid and a blood trace left at the scene of sample collecting for criminal investigation, or a sample left at the scene of a terrible disaster such as fire, explosion, accident and the like has been deteriorated. In such the case, there is a possibility that a DNA sequence has been fragmented, and an entire objective region has not been retained. Like this, in a discriminating method with a repeat sequence requiring a relatively long sequence, there is a problem that measurement is difficult in some cases, and therefore, an as small as possible DNA region necessary for a test is preferable.
Then, a method using single nucleotide polymorphism for individual identification which has been elucidated in recent years is contemplated. For example, JP-A 2004-239766(KOKAI) describes that it is possible to use single nucleotide polymorphism in a DNA probe of a microarray for discriminating an individual. However, its specific method is not disclosed. In addition, Lee H Y, et. al. (Selection of twenty-four highly informative SNP markers for human identification and paternity analysis in Koreans.; Forensic Aci Int. 2005 Mar. 10; 148(2-3):107-12.) discloses 24 nucleotide polymorphisms which can be used in identification of an individual and parentage test in the Korean population.
However, there are a huge number of single nucleotide polymorphisms which are found now, and it is not practical to test all of them. However, as to what single nucleotide polymorphism should be tested, there is no powerful determination criteria now.
An object of the present invention is to provide a method which allows for simple and rapid individual discrimination by selecting single nucleotide polymorphism which is advantageous for individual discrimination. Also the invention provides an array, a test apparatus and a system for use in an individual discriminating test.
According to the present invention, there is provided an individual discriminating method for determining consistency between a nucleotide sequence possessed by an individual and a nucleotide sequence possessed by a sample, comprising a step of selecting a plurality of single nucleotide polymorphism from a group of single nucleotide polymorphisms present in a population to which a subject individual to be discriminated belongs, and a step of determining genotypes in the selected plurality of single nucleotide polymorphisms in the nucleotide sequence possessed by a subject individual and the nucleotide sequence derived from a sample, and discriminating an individual by comparing the genotypes, wherein in the selecting step, single nucleotide polymorphism satisfying any one of the following conditions (i) to (iii) is selected:
(i) in case of a 2-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of two possible nucleotides are X and Y, X+Y=1, and Y≦X;
0.5≦X≦0.7,
(ii) in case of a 3-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of three possible nucleotides are X, Y and Z, X+Y+Z=1, and Z≦Y≦X;
1/3≦X and (1-X)/2≦Y and X+Y<1, and
(a) Y≦1/2·X and X<2/3, or (b) 1/2·X<Y and XY<2/9, and
(iii) in case of a 4-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of four possible nucleotides are X, Y, Z and W, X+Y+Z+W=1, and W≦Z≦Y≦X;
1/4≦X and (1-X)/3≦Y and X+Y<1, and
(a) Y≦1/2·X and X<2/3, or (b) 1/2·X<Y and XY<2/9.
Herein, the X is preferably 0.55≦X<0.7, more preferably 0.6≦X<0.7, and further preferably 0.65≦X≦0.68.
Also, according to another aspect of the present invention, there is provided an array in which nucleic acid probes are fixed to a substrate, for use in an individual discriminating test for determining consistency between a nucleotide sequence possessed by an individual and a nucleotide sequence possessed by a sample, characterized in that the nucleic acid probes have a sequence complimentary to a target sequence containing single nucleotide polymorphism, and the single nucleotide polymorphism is selected from a group of single nucleotide polymorphisms present in a population to which a subject individual to be discriminated belongs, and satisfies any one of the following conditions (i) to (iii):
(i) in case of a 2-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of two possible nucleotides are X and Y, X+Y=1, and Y≦X;
0.5≦X≦0.7,
(ii) in case of a 3-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of three possible nucleotides are X, Y and Z, X+Y+Z=1, and Z≦Y≦X;
1/3≦X and (1-X)/2≦Y and X+Y<1, and
(a) Y≦1/2·X and X<2/3, or (b) 1/2·X<Y and XY<2/9, and
(iii) in case of a 4-nucleotide substitution type single nucleotide polymorphism, assuming that respective allele frequencies of four possible nucleotides are X, Y, Z and W, X+Y+Z+W=1, and W≦Z≦Y≦X;
1/4≦X and (1-X)/3≦Y and X+Y<1, and
(a) Y≦1/2·X and X<2/3, or (b) 1/2·X<Y and XY<2/9.
Herein, the X is preferably 0.55≦X<0.7, more preferably 0.6≦X<0.7, further preferably 0.65≦X<0.68.
According to one aspect of the present invention, an individual discriminating method is provided. In the present specification, individual discrimination means determination of consistency between a nucleotide sequence possessed by an individual and a nucleotide sequence possessed by a sample. For example, the method can be used for specifying whether an article left such as a remaining blood trace and a hair is of a suspect or a victim or not in a criminal investigation. Therefore, a nucleotide sequence possessed by a sample may be a sequence of a nucleic acid contained in these articles left, being not limited thereto.
As used herein, the term “nucleic acid” is a term comprehensively indicating a nucleic acid and a nucleic acid analogue such as a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), a peptide nucleic acid (PNA), a methylphosphonate nucleic acid, a S-oligo, a cDNA and a cRNA, as well as any oligonucleotide and polynucleotide. Such the nucleic acid may be naturally occurring, or may be artificially synthesized.
Herein, a nucleotide sequence is preferably a genome DNA sequence, or may be a fragmental sequence which does not ensure a whole genome. In addition, individual discrimination of the present invention can be also used in person identification for confirming a person himself, parentage test or the like, being not limited thereto.
In the method of the present invention, single nucleotide polymorpholism (SNP) is used for individual discrimination, and particularly, SNP which is advantageous in individual discrimination is selected and used. This selection of SNP is performed by referring to its allele frequency.
Herein, SNP refers to a difference in a genome DNA sequence by one nucleotide, and this is recognized in a specified population usually at a frequency of 1% or more. In almost cases, in one place SNP, substitution occurs between two nucleotides, and for example, in an individual, A (adenine) is taken, and in another individual, G (guanine) is taken. Such the SNP is called 2-nucleotide substitution type, and in this case, there are three ways of genotypes in one place SNP. In the above example, there are A/A homo, G/G homo, and A/G hetero.
However, rarely, a 3-nucleotide substitution type or 4-nucleotide substitution type SNP is also present. In these SNPs, there are six or ten ways of genotypes, so only at one SNP site, there can be a number of genotypes.
An appearance frequency of these genotypes can be obtained from an allele frequency of each nucleotide. An allele frequency refers to each ratio of a nucleotide which can be taken at a certain SNP in a population. That is, when certain SNP in a population takes any nucleotide of A and T, the case of A in the population is a ratio of 70%, and the case of T is a ratio of 30%, an allele frequency X of A is expressed to be 0.7, and an allele frequency Y of T is expressed to be 0.3. Herein, since X+Y=1, and this is 2-nucleotide substitution, 0<X, and 0<Y.
This allele frequency is determined by testing a considerable number of individual genotypes contained in a population, and obtaining its distribution. Currently, allele frequencies in various populations are published by a plurality of databases, and populations according to various classifications such as a race and people have been investigated, respectively. As database listing a position and a frequency of SNP, for example, there are HAPMAP, NCBI Entrez SNP, JSNP, TSC and the like.
In the present specification, an individual group classified by these classifications, for example, race, people, nation, residence region, sex, age and the like is referred to as population. The number of individuals investigated for determining an allele frequency is suitable, and if highly reliable, an allele frequency published by any database may be used, or investigation of an allele frequency may be performed independently.
As described above, a genotype frequency is obtained from an allele frequency. In the case of the above SNP, as the genotype, there are AA homo, AT hetero and TT homo, and a frequency of each genotype is obtained from (X+Y)2=XX+2XY+YY. That is, AA:AT:TT=0.49:0.42:0.09.
In the method of the present invention, proper SNP is selected depending on these allele frequency and genotype frequency. In a nucleotide sequence of an individual and a nucleotide sequence of a sample, genotypes in the selected SNPs are determined, and they are compared. If both genotypes are entirely consistent, it can be determined that the sample is derived from that individual.
In addition, in the present specification, an individual may be any entity such as an animal and a plant in addition to a human, as far as the entity can be discriminated by a genome DNA sequence. A preferable subject is a human, and additionally, a livestock and a pet, or a cultivating plant or a wild plant may be included.
Meanwhile, it is said that around ten millions of SNPs are present in a human genome DNA. Although when as many as possible, SNPs are tested, a test precision is apparently elevated, in view of simplicity, rapidness and economy, it is apparent that a small number of SNPs to be tested is better.
Then, the present inventors found that SNP which is advantageous in individual discrimination can be selected according to the following conditions, and this allows for a minimum number of SNPs necessary for individual discrimination. Conditions (i) to (iii) for selecting SNP will be successively explained below. (i) First, a method of selecting SNP in the case of a 2-nucleotide substitution type will be explained. Letting nucleotides being substituted to be A and B, a relationship between an allele frequency and a genotype frequency is shown in
Extraction of a maximum genotype frequency and a minimum genotype frequency in this
Herein, what a genotype frequency possessed by SNP is desirable in individual discrimination will be considered. In certain SNP, it is desirable that a frequency of a genotype having a maximum genotype frequency is as low as possible. This is because when a maximum genotype frequency is high, this leads to many individuals having the genotype in the population, and the ability to discriminate an individual of the SNP is reduced. Therefore, in order to ensure lowest limit of discrimination ability, it is preferable that a frequency of a genotype having a maximum genotype frequency is 0.5 or less.
In addition, it is desirable that a frequency of a genotype having a minimum genotype frequency is as low as possible. When this genotype frequency is low, it can be said that the genotype is rare, and it can be said that the SNP has extremely high discriminating ability, and is useful SNP.
Herein, respective allele frequencies of nucleotides A and B are expressed by X and Y. It is provided that X+Y=1, and Y≦X. In addition, from definition, 0<X and 0<Y, and from these conditions, 0.5≦X. Thereupon, in
Herein, as described above, in order to decrease a maximum gene frequency, it is desirable that a value of X approaches 0.66. However, in order to decrease a minimum genotype frequency, it is preferable that the value of X is greater. In view of these conditions, a further preferable range is a range of 0.55≦X<0.7, more preferably a range of 0.6≦X<0.7, most preferably a range of 0.65≦X≦0.68.
SNP having X (allele frequency) of the aforementioned range has better balance in discriminating ability, and such the SNP can be preferably used in the method of the present invention.
(ii) Then, a method of selecting SNP in the case of a 3-nucleotide substitution type will be explained. There are six ways of genotypes in the 3-nucleotide substitution type SNP as described above. Therefore, discriminating ability by one SNP is improved, and this is advantageous for use in discrimination of an individual.
Further, in the 2-nucleotide substitution type, a minimum genotype frequency which can be taken by a maximum genotype frequency is 4/9. Therefore, in the 3-nucleotide substitution type, it is possible to select SNP more effectively in individual discrimination by selecting SNP having a smaller genotype frequency.
Herein, it is provided that nucleotides being substituted are A, B and C, and allele frequencies are X, Y and Z, respectively. Herein, X+Y+Z=1, and Z≦Y≦X. Thereupon, 1/3≦X, (1-X)/2≦Y, and X+Y<1.
Meanwhile, genotypes in the 3-nucleotide substitution type are X2, Y2, Z2, 2XY, 2YZ, and 2ZX. Among them, genotypes in which a genotype frequency can be maximal are X2 or 2XY. (a) In the case of 2XY≦X2, that is, Y≦1/2·X, a maximum genotype is X2. Herein, as described above, since a genotype frequency smaller than 4/9 is desirable, X2<4/9. Therefore, a desirable allele frequency is Y≦1/2·X and X<2/3, and such the SNP is selected.
Or, (b) in the case of 2XY>X2, that is, 1/2 X<Y, a maximum genotype is 2XY. Herein, as described above, since a genotype frequency smaller than 4/9 is desirable, 2XY<4/9. Therefore, a desirable allele frequency is 1/2·X<Y and XY<2/9, and such the SNP is selected.
A range of X and Y in conformity with the above conditions (a) and (b) is shown as a region of an oblique line in
(iii) Then, a method of selecting SNP in the case of a 4-nucleotide substitution type will be explained. There are ten ways of genotypes in the 4-nucleotide substitution type SNP as described above. Therefore, discriminating ability by one SNP is greatest, and this is advantageous for use in individual discrimination. In the 4-nucleotide substitution type SNP like the 3-nucleotide substitution type SNP, by selecting SNP having a genotype frequency in which a maximum genotype frequency is smaller than 4/9, it is possible to select SNP more effectively in individual discrimination.
Herein, it is provided that nucleotides being substituted are A, B, C and D, and allele frequencies are X, Y, Z and W, respectively. Herein, X+Y+Z+W=1, and W≦Z≦Y≦X. Thereupon, 1/4≦X, (1-X)/3≦Y, and X+Y<1.
Genotypes in the 4-nucleotide substitution type in which a genotype frequency can be maximum are X2 and 2XY.
(a) In the case of 2XY≦X2, that is, Y≦1/2·X, a maximum genotype is X2. Herein, as described above, since a genotype frequency smaller than 4/9 is desirable, X2<4/9. Therefore, a desirable allele frequency is Y≦1/2·X and X<2/3, and such the SNP is selected.
Or, (b) in the case of 2XY>X2, that is, 1/2·X<Y, a maximum genotype is 2XY. Herein, as described above, since a genotype frequency smaller than 4/9 is desirable, 2XY<4/9. Therefore, an desirable allele frequency is 1/2·X<Y and XY<2/9, and such the SNP is selected.
A range of X and Y in conformity with the above (a) and (b) is shown as an oblique line region in
SNP shown in the above (ii) and (iii) can be searched, for example, from SNP registered in SNP database of National Center for Biological Information (NCBI).
It is desirable that SNP selected so that the conditions (i) to (iii) are satisfied is selected from different chromosomes in a genome. When using SNPs present on the same chromosome, it is desirable to use SNPs being clearly not linked to each other, or SNPs having a low probability thereof.
In addition, it is desirable that a combination of a plurality of SNPs selected according to the above conditions is selected so that, in a genotype frequency calculated from an allele frequency, each of selected i single nucleotide polymorphisms, letting a highest genotype frequency in a nth single nucleotide polymorphism to be (Max)n and a lowest genotype frequency to be (Min)n, 1/π(Max)i and 1/π(Min)i become suitable values, respectively.
For example, letting the number of individuals constituting the population to be U, SNP can be also selected so that each of 1/π(Max)i and 1/π(Min)i is in an appropriate range when expressed as a ratio to U. 1/π(Max)i represents that, in a combination of selected SNPs, an individual having a combination of a highest probability is present at a ratio of one person per 1/π(Max)i persons. When a value of 1/π(Max)i is low, many individuals having the combination of SNPs are present in a population, and it can be said that the combination of SNPs has low individual discriminating ability.
In addition, 1/π(Min)i represents that, in a combination of selected SNPs, an individual having a combination of a lowest probability is present at a ratio of one person per 1/π(Min)i persons. When a value of 1/π(Min)i is low, an individual having the combination of SNPs is rarely present, and it can be said that the combination is a combination of SNPs having high individual discriminating ability.
A number i of SNPs selected for use in individual discrimination in the invention may be appropriately determined depending on necessary discriminating ability, and may be determined in view of simplicity, rapidness, economy, and reliability of determination.
In an individual discriminating method using SNPs selected according to the aforementioned conditions, further, by calculating a frequency of a genotype possessed by a subject individual from an allele frequency, multiplying a genotype frequency regarding all selected SNPs, and taking a reciprocal, a probability that a combination of genotypes possessed by a subject individual is present in a population can be calculated. To explain using
For example, in the case of a genotype possessed by one person per ten persons in a population of 100 millions, even when genotypes of a subject individual and a sample are consistent, reliability that they are the same is decreased. Conversely, in the case of a genotype, when a genotype possessed by a subject individual is a genotype which is rarely present, reliability of a determination of is increased, therefore, it is also important upon individual discrimination to determine a probability of existence in a population, and make clear reliability of determination of whether a nucleotide sequence possessed by an individual and a nucleotide sequence of a sample are consistent or not. By calculating an existence probability by this step, reliability of the result of individual discrimination can be determined.
The individual discriminating method of the present invention which has been described in detail above is summarized. First, a plurality of SNPs satisfying any of the above (i) to (iii) are selected from a group of SNPs present in a population to which a subject individual to be discriminated belongs. Herein, genotypes in selected plurality of single nucleotide polymorphisms are determined, respectively, in a nucleotide sequence possessed by a subject individual and a nucleotide sequence derived from a sample. Then, determined genotypes of both of them are compared, and it is determined whether a nucleotide sequence of a subject individual and a nucleotide sequence of a sample are consistent or not based on the comparison results.
Herein, a method of determining genotypes in nucleotide sequences of a subject individual and a sample may be performed by the well-known method, for example, an appropriate method such as by a method of amplifying the SNP site by a polymerase amplification reaction, and investigating a genotype, and a method of analyzing a DNA sequence.
By using SNPs selected according to the present invention, an individual can be discriminated with a minimum number of SNPs within required reliability, a test can be performed simply and rapidly, and the cost can be suppressed.
Then, according to another aspect of the present invention, an array for use in an individual discriminating test is provided. The array in the present invention is such that nucleic acid probes having a sequence complementary with a target nucleic acid is fixed to a substrate. Herein, a target nucleic acid means a nucleic acid strand having a target sequence containing SNP selected from a group of SNPs present in a population to which a subject individual to be discriminated belongs so as to satisfy any one of the above conditions (i) to (iii). The nucleic acid probes fixed to a substrate can hybridize with a target nucleic acid under the appropriate condition.
As used herein, the terms “complementary”, “complementarity” and “complementation” are enough to be complementary in a range of 50% to 100%, preferably refer to 100% complementary.
The nucleic acid probes fixed to a substrate may be a single nucleic acid probe, or a plurality different nucleic acid probes. That is, nucleic acid probes having sequences which are complementary with target nucleic acids having target sequences containing different SNPs, respectively, may be fixed.
In addition, for example, in SNP of A and T 2-nucleotide substitution type, both of a nucleic acid probe complementary with a sequence in the case of A and a sequence in the case of T may be fixed to an array, or only a probe for one sequence may be fixed. Similarly, also in SNP of the 3-nucleotide substitution type and 4-nucleotide substitution type, probes corresponding to sequences in the case of respective nucleotides may be used, or only a probe corresponding to a sequence which is intended to be detected may be used. As a length of a nucleic acid probe, a length suitable for fixation to a substrate and hybridization may be appropriately selected, and the length may be shorter than that of a target nucleic acid. For example, the length may be about 3 to about 1000 bp, preferably about 10 to about 200 bp.
A target nucleic acid is a nucleic acid having a sequence consisting of sequences upstream and downstream of a site where SNP is present.
As a target nucleic acid which is subjected to hybridization with nucleic acid probes on an array, a test solution containing the nucleic acid may be used as it is, or a target sequence site may be amplified, for example, by PCR in advance, and excised, and used. Thereupon, a length of a target nucleic acid may be arbitrarily determined, but may be a length of, for example, around 30 to 500 bp by primer design. By adopting an appropriate length of a target nucleic acid, an efficacy of hybridization can be increased.
A substrate which can be used in the present invention may be a substrate to which a nucleic acid probe can be fixed, and may have a shape of a well, a plate having a groove or a planar surface, or a steric shape such as a sphere, made of a non-porous, hard or semi-hard material. The substrate can be manufactured, without limitation, of a silica-containing substrate such as silicon, glass, quartz glass and quartz, or a plastic or a polymer such as polyacrylamide, polystyrene, polycarbonate and the like.
In the array of the present invention, as a means for detecting the presence of a duplex generated as a result of hybridization between a nucleic acid probe fixed to a substrate and a target nucleic acid, an electrochemical method can be used, being not limited thereto.
Detection of a double-stranded nucleic acid by an electrochemical method may be performed using the known duplex recognizing substance. The duplex recognizing substance is, without limitation, Hoechster 33258, acridine orange, quinacrine, daunomycin, metallointercalater, bisintercalater such as bisacridine and the like, trisintercalater and polyintercalater can be used. Further, it is possible to modify these intercalaters with an electrochemically active metal complex, for example, ferrocene, biogen or the like. Alternatively, other known duplex recognizing substances may be used.
In a method of detecting a double-stranded nucleic acid by an electrochemical method, an electrode is provided on a substrate, and nucleic acid probes are fixed to this electrode. The electrode is not particularly limited, but can be formed of a carbon electrode such as graphite, glassy carbon, pyrolytic graphite, carbon paste, and carbon fiber, noble metal electrode such as platinum, platinum black, gold, palladium, and rhodium, an oxide electrode such as titanium oxide, tin oxide, manganese oxide, and lead oxide, a semiconductor electrode such as Si, Ge, ZnO, CdS, TiO2 and GaAs, titanium or the like. These electrodes may be covered with an electrical conductive polymer, or may be covered with a single molecule membrane, or may be treated with other surface treating agent, if necessary.
Fixation of a nucleic acid probe may be performed by the known means. For example, a nucleic acid probe may be fixed to an electrode via a spacer by fixing a spacer to an electrode, and fixing a nucleic acid probe to the spacer. Alternatively, a spacer is bound to a nucleic acid probe in advance, and the probe may be fixed to an electrode via the spacer. Alternatively, a spacer and a nucleic acid probe may be synthesized on an electrode by the known means. In addition, for fixing a nucleic acid probe with a spacer, the spacer may be directly fixed to a treated or non-treated electrode surface with a covalent bond, an ionic bond or physical adsorption. Alternatively, a linker agent which assists fixing of a nucleic acid probe via a spacer may be used. In addition, an electrode may be treated with a blocking agent for preventing nonspecific binding of a test nucleic acid to an electrode together with a linker agent. In addition, a linker agent and a blocking agent used herein may be a substance for advantageously performing electrochemical detection.
Nucleic acid probes having different nucleotide sequences may be fixed to different electrodes via a spacer, respectively.
Further, like other general electrochemical detecting method, an array may be provided with counter electrodes and/or reference electrodes. When reference electrodes are arranged, for example, general reference electrodes such as silver/silver chloride electrode and mercury/mercury chloride can be employed.
Detection of a target nucleic acid by an array can be performed, for example, as follows. A nucleic acid component is extracted as a sample nucleic acid from a sample collected from a subject such as an individual such as an animal including a human, a tissue and a cell. The resulting sample nucleic acid may be subjected to treatment such as reverse transcription, elongation, amplification and/or enzyme treatment, if necessary. The sample nucleic acid which has been pre-treated as necessary is contacted with a nucleic acid probe immobilized on a nucleic acid probe immobilizing substrate, and a reaction is performed under the condition allowing for appropriate hybridization. A person skilled in the art can appropriately select such the appropriate condition depending on various conditions such as a kind of a nucleotide contained in a target sequence, a kind of a spacer and a nucleic acid probe set on a nucleic acid probe immobilizing substrate, a kind of a sample nucleic acid, and the state thereof.
A hybridization reaction may be performed, for example, under the following condition. As a hybridization reaction solution, a buffer having an ionic strength in a range of 0.01 to 5 and a pH in a range of 5 to 10 is used. To this solution, may be added dextran sulfate which is a hybridization promoter, as well as a salmon spermatozoon DNA, a bovine thymus DNA, and a surfactant such as EDTA. A sample nucleic acid is added thereto, and this is thermally denatured at 90° C. or higher. Immediately after denaturation of a nucleic acid, or after rapid cooling to 0° C., a nucleic acid probe immobilizing substrate is inserted into this solution. Alternatively, it is also possible to perform a hybridization reaction by adding dropwise the solution to a substrate.
During the reaction, a reaction rate may be enhanced by a procedure such as stirring and shaking. A reaction temperature is, for example, in a range of 10° C. to 90° C., and a reaction time is not shorter than 1 minute to around overnight. After the hybridization reaction, an electrode is washed. As a washing solution, for example, a buffer having an ionic strength in a range of 0.01 to 5 and a pH in a range of 5 to 10 is used. When target nucleic acids containing a target sequence is present in a sample nucleic acid, it is hybridized with nucleic acid probes to generate double-stranded nucleic acids on a substrate.
Subsequently, detection of a double-stranded nucleic acid is performed by electrochemical procedure. In a usual procedure, a substrate is washed after hybridization and then a duplex recognizing entity is acted on a double-stranded part formed on an electrode surface, and a signal generated therefrom is measured electrochemically.
A concentration of a duplex recognizing entity is different depending on a kind thereof, and generally, the entity is used in a range of 1 ng/mL to 1 mg/mL. Thereupon, a buffer having an ionic strength in a range of 0.001 to 5 and a pH in a range of 5 to 10 may be used.
Electrochemically measurement can be performed, for example, by applying a potential which is not lower than the potential at which a duplex recognizing entity reacts electrochemically, and measuring a reaction current value derived from the duplex recognizing entity. Thereupon, a potential may be scanned at a constant rate, or may be applied in a pulse manner, or a constant potential may be applied. Upon measurement, a current and a voltage may be controlled using a device such as a potentiostat, a digital multimeter and a function generator. Further, based on the resulting current value, a concentration of a target nucleic acid may be calculated from a calibration curve.
A sequence of a target nucleic acid detected by the hybridization reaction using the aforementioned array is a nucleotide sequence possessed by an individual to be tested, and a sample. Thereby, genotypes of selected SNPs in a nucleotide sequence contained in each sample can be determined.
After respective genotypes of an individual to be discriminated and a sample are determined, they are compared, and it is determined whether both are consistent or not. Further, based on a genotype frequency of used SNP, an existence ratio of genotype of the individual or the sample is calculated, and reliability of determination may be determined.
According to another aspect of the present invention, an individual discriminating test apparatus provided with the aforementioned array is provided. In the apparatus, a substrate-like array is suitably used, and is also referred to as chip herein. Further, according to another aspect of the present invention, a system for implementing an individual discriminating test by the individual discriminating test apparatus is provided.
The individual discriminating test apparatus of the present invention is provided with the array according to the present invention, a flow channel which is provided on a substrate of the array, and is provided along a flow direction of a drug solution or the air, a working electrode which is provided at a plurality of number on the substrate along the flow channel, and on which the probe is immobilized, counter electrodes for imparting an electric potential difference between the working electrodes, which are provided on an internal circumferential surface of the flow channel corresponding to the working electrodes, each being arranged so as to be situated on a first surface facing the substrate surface, reference electrodes for feed-backing a detected voltage to the working electrodes, which are provided on an internal circumferential surface of the flow channel corresponding to the working electrodes, each being arranged so as to be situated on a second surface facing the substrate surface, an inlet port which is opened in the flow channel, and flows a drug solution or the air in the flow channel from an upstream side of the flow channel, an outlet port which is opened in the flow channel, and flows out a drug solution or the air from the flow channel to a downstream side of the flow channel, and an injecting port for injecting test solution into the flow channel.
In addition, the individual discriminating test system according to the present invention is provided with the individual discriminating test apparatus, a first piping which is communicated with the inlet port, and supplying a drug solution or the air into the flow channel via the inlet port, a supply system provided with a first valve for controlling a flow rate of a drug solution or the air of the first piping, a second piping which is communicated with the outlet port, and discharges a drug solution or the air from the flow channel via the outlet port, a second valve for controlling a flow rate of a drug solution or the air of the second piping, a discharge system which is provided in the second piping and is provided with a pump for drawing up a drug solution or the air from the flow channel, a measuring system provided with a voltage applying unit for imparts an electric potential difference between the working electrode and the counter electrode, a temperature control system for controlling a temperature of the array, a control mechanism which controls the first valve of the supply system, the second valve and the pump of the discharge system, the voltage applying unit of the measurement system, and the temperature control system, detects an electrochemical reaction signal from the working electrode or the counter electrode, and stores this electrochemical reaction signal as measurement data, and a computer which imparts a control condition parameter to the control mechanism to control the control mechanism, and at the same time, executes nucleotide sequence analyzing treatment based on the measurement data, and determines consistency between a nucleotide sequence possessed by an individual and a nucleotide sequence possessed by a sample.
One embodiment of the individual discriminating test apparatus and system of the present invention will be explained below by referring to the drawings.
These measurement system 12, flow system 13 and temperature control system 14 are controlled by a control mechanism 15. A control mechanism 15 is electrically connected to a computer 16, and a control mechanism 15 is controlled by a program provided in this computer 16. In the present embodiment, the chip cartridge 11, the measurement system 12, the flow system 13 and the temperature control system 14 are referred to as a measurement unit 10.
A printed board 22 packaged with a chip 21 on which a nucleic acid probe is immobilized is attached to a chip cartridge 11, which is used. A nucleic acid probe is immobilized on a working electrode of a chip 21. A sample (test solution) which is introduced into a cell of a chip 21 contains a nucleic acid to be tested. The individually discriminating test apparatus of this embodiment determines whether a target nucleic acid is contained in a sample (test solution) or not by hybridizing a target nucleic acid with a nucleic acid probe, and monitoring the presence or the absence of the reaction after introduction of a buffer and an intercalator.
Two openings are provided on a side part of a chip cartridge upper lid 112, and an interface part 113a is connected to one of openings, and an interface part 113b is connected to the other opening. These interface parts 113a and 113b function as an interface for a flow system 13 and a chip cartridge 11. In an interior of these interface parts 113a and 113b, flow channels 114a and 114b are provided, respectively. A drug solution or the air from an upper stream side of a flow system 13 is flown into an interior of a chip cartridge 11 via a flow channel 114a. A sample, a drug solution and the air in a hip cartridge 11 are flown out to a downstream side of a flow system 13 via a flow channel 114b.
In
A chip cartridge upper lid 112 is situated on an upper side of a cell 115 provided with an inlet port 116a and an outlet port 116b. A flowing port in 116a penetrates from a side surface to a bottom surface of a chip cartridge upper lid 112, and is opened on a bottom surface of a chip cartridge upper lid 112 at a cell pore part 115a. A outlet port 116b penetrates from another side surface to a bottom surface of a chip cartridge upper lid 112, and is opened on a bottom surface of a chip cartridge upper lid 112 at a cell pore part 115b. By connection of an inlet port 116a to a flow channel 114a, and of an outlet port 116b to a flow channel 114b, a flow channel 114a and a cell 115, and a flow channel 114b and a cell 115 are communicated.
An electric connecter 22a is provided at a position which is a printed board 22 surface and is spaced from a cell 115. An electric connector 22a is electrically connected to a lead frame of a substrate body of a printed board 22. In addition, this lead frame of a substrate body is electrically connected to various electrodes of a chip 21 with a lead. By connecting a terminal of a measurement system 12 to this electric connector 22a, an electric signal obtained in a chip 21 can be outputted to a measurement system 12 via a predetermined terminal provided at a predetermined position of a printed board 22, and further, via an electric connector 22a.
As shown in
As a temperature control system 14 for regulating a temperature of a chip 21, for example, a Peltier element is used. Thereby, temperature control of ±0.5° C. is possible. A reaction of a nucleic acid is generally performed at a temperature range relatively near room temperature. Therefore, temperature control only with a heater is poor in stability. In addition, since it is necessary to control a reaction of a nucleic acid by a temperature profile, another cooling mechanism becomes to be necessary. In this respect, in a Peltier element, since any of heating and cooling is possible by changing a direction of a current, the element is optimal.
An electric connected 22a is disposed at an end of a printed board 22. A working electrode 501 of a chip 21 and an electric connector 22a are electrically connected with a lead frame provided on a surface of a printed board 22. Various electrodes of a chip 21 and a measurement system 12 can be electrically connected to an electrical connector 22a by connecting a signal interface of a measurement system 12.
A cell 115, when seen from an upper side, has a shape in which an elongate flow channel 601 is arranged as shown in
In
An outlet port 116b extends to a predetermined height in a direction approximately vertical to a cell bottom surface, and further, is bent approximately orthogonally in a direction far away from a center of a cell 115, and is branched into two pathways at the bending position. One pathway penetrates to a surface of a chip cartridge upper lid 112, and is communicated with an injecting port 119. Thereby, a sample injected through an injecting port 119 is introduced into a cell 115 through an outlet port 116b. A central axis of an injection port 119 and that of an outlet port 116b are approximately consistent, and an aperture diameter of an injection port 119 is set to be greater than an aperture diameter of a flowing port 116b. In addition, it is provided in vicinity of an injection port 119, and an injection port 119 can be covered with a lid 120. Thereby, without utilizing an injection port 119, when a drug solution is circulated in a flow channel 114b from a flow channel 114a to a flow channel 114b via a cell 115, a drug solution can be prevented from flowing out through an injection port 119, and a pathway of a drug solution can be maintained. In addition, a sealing material 121 is provided on a lid 120, and by sealing an injection port 119, slight leakage of a drug solution can be prevented. In an example of
By the above construction, a drug solution can be flown in an order of a flow channel 114a, an inlet port 116a, a cell 115 (flow channel 601), an outlet port 116b and a flow channel 114b in a direction shown by an arrow in
A bottom surface of a detection flow channel 601a is defined by a chip 21. Each one of a working electrode 501 is formed on each bottom surface of a detection flow channel 601a. A side surface of a detection flow channel 601a is defined by a flow channel-like convex part 112a which is provided in a convex manner from a chip cartridge upper lid 112, and a sealing material 24a. A reference electrode 503 is fixed on a side surface of this flow channel, that is, a side surface of a flow channel-like convex part 112a, respectively, to a predetermined height from a flow channel bottom. Like this, a plurality of reference electrodes 503 are situated on a plane which is parallel to a chip surface and faces with a chip surface and the plane is situated on a plane higher than a plane on which a working electrode 501 is provided. An upper side of a detection flow channel 601a is defined by a bottom surface of a chip cartridge upper lid 112 on which a flow channel-like convex part 112a is not provided. Each counter electrode 502 is fixed on an upper side of this flow channel. Like this, a plurality of counter electrodes 502 are situated on a plane which is parallel to a chip bottom and faces with a chip surface, and the plane is situated on a plane higher than a plane on which a working electrode 502 or a reference electrode 503 is provided. Like this, a working electrode 501, a counter electrode 502 and a reference electrode 503 are three-dimensionally arranged on different planes, respectively.
A sealing material 24a is immobilized on a flow channel-like convex part 112a of a chip cartridge upper lid 112 with printing in advance. Therefore, when a cell 115 is assembled, a chip cartridge upper lid 112 integrated with a sealing material 24a is pushed against a chip 21 in a direction shown by an arrow of
As shown in
In an example of
Like this, each one of a working electrode 501, a counter electrode 502 and reference electrode 503 are provided as one set of three electrodes in the same cross-sectional shape flow channel along a direction of flow of a drug solution or the air, and a construction is such that a positional relationship of these three electrodes is the same, and a flow channel shape is the same. When seen from a working electrode 501, directions to a flow channel bottom surface, a side surface and an upper surface relative to a working electrode 501, and positional relationships from a working electrode 501 to corresponding counter electrode 502 and reference electrode 503 are the same. Thereby, uniformity of property of an electrochemical signal detected by each of three electrodes is improved. As a result, detection reliability is improved.
Herein, a counter electrode 502 and a reference electrode 503 are arranged so as to be separated relative to a corresponding working electrode 501, respectively, being not limited thereto. A counter electrode 502 or a reference electrode 503 may have a construction that pluralities of electrodes are connected in any case. In that case, a region nearest each working electrode in each electrode functions as a counter electrode or a reference electrode. In addition, a cross-sectional shape of a flow channel is not limited to the aforementioned construction of
Then, a process for manufacturing the aforementioned chip 21 and printed board 22 will be explained in line with a step cross-sectional view of
Then, a photoresist film 210 is removed by oxygen ashing (
Then, a photoresist 215 is coated on a whole surface, and this is patterned so as to open an electrode part and a bonding pad (
Then, chipping is performed by dicing finally in order to clean an electrode surface, treatment with a CF4/O2 mixed plasma is performed. Thereby, a chip 21 is obtained. This chip 21 is mounted on a printed board 22 packaged with an electric connecter 22. A bonding pad of a chip 21 and a lead wiring on a printed board 22 are connected by wire bonding. Thereafter, a wire bonding part is protected using a sealing resin 23. By the above step, a printed board 22 packaged with a chip 21 can be manufactured.
A top view of a manufactured chip 21 is shown in
Then, one example of a specific construction of a flow system 13 is explained using
A Milli Q water supply source 411 accommodating Milli Q water as one of drug solutions is connected to a piping 414. On a downstream side of this Milli Q water supply source 411, a check valve 412 for preventing reverse flow of a drug solution or the air other than Milli Q water to a Milli Q water supply source 411 is provided, and on a further downstream side, a three-way electromagnetic valve 413 (Vwa) is provided. This three-way electromagnetic valve 413 switches between communication of a piping 404 with a piping 415, and communication of a piping 414 and a piping 415. That is, at turn-off of a three-way electromagnetic valve 413, a piping 404 is communicated with a piping 415, and at turn-on, a piping 414 is communicated with a piping 415. Thereby, supply of the air and Milli Q water to a piping 415 can be switched.
A buffer supply source 421 accommodating a buffer as one of drug solutions is connected to a piping 424. On a downstream side of this buffer supply source 421, a check valve 422 for preventing reverse flow of a drug solution other thane a buffer or the air to a buffer supply source 421 is provided, and on a further downstream side, a three-way electromagnetic valve 423 (Vba) is provided. This three-way electromagnetic valve 423 switches between communication of a piping 424 with a piping 425, and communication of a piping 415 with a piping 425. That is, at turn-off of a three-way electromagnetic valve 423, a piping 415 is communicated with a piping 425, and at turn-on, a piping 424 is communicated with a piping 425. Thereby, supply of a buffer to piping 425, and supply of the air or Milli Q water can be switched.
An intercalator supply source 431 accommodating an intercalator as one of drug solutions is connected to a piping 434. On a downstream side of this intercalator supply source 431, a check valve 432 for preventing reverse flow of a drug solution other than a intercalator or the air to an intercalator supply source 431 is provided, and on a further downstream side, a three-way electromagnetic valve 433 (Vin) is provided. This three-way electromagnetic vale switches between communication of a piping 434 with a piping 435, and communication of a piping 425 with a piping 435. That is, at turn-off of a three-way electromagnetic valve 433, a piping 425 is communicated with a piping 435, and at turn-on, a piping 434 is communicated with a piping 435. Thereby, supply of an intercalator to a piping 435, and supply of the air, Milli Q water or a buffer can be switched.
As described above, in an air or drug supply system, by controlling a two-way electromagnetic valve 403 and three-way electromagnetic valves 413, 423 and 433, supply of the air, Milli Q water, and a drug solution such as a buffer and an intercalator to a chip cartridge 11 via a piping 435 can be switched, and a flow rate of the air or these drug solutions to be supplied can be controlled.
With an upstream side of a piping 435, the aforementioned three-way valve 433 is communicated, and with its downstream side, a three-way electromagnetic 441 (Vcbin) is communicated. By a three-way electromagnetic valve 441, a piping 435 can be branched into a piping 440 and a bypass piping 446. A three-way electromagnetic valve 441 switches as follows: at turn-off, a piping 435 is communicated with a bypass piping 466, and at turn-on, a piping 435 is communicated with a piping 440. In addition, a three-way electromagnetic valve 445 switches as follows: at turn-off, a bypass piping 446 is communicated with a piping 450, and at turn-on, a piping 440 is communicated with a piping 450. By these three-way electromagnetic valves 441 and 445, supply of various drug solutions and the air can be switched between a bypass piping 446 and a piping 440.
In a piping 440, in an order towards a downstream side which is seen from a three-way electromagnetic valve 441, a two-way electromagnetic valve 442 (Vlin), a chip cartridge 11, a liquid sensor 443, a two-way electromagnetic valve 444 (Vlout), and a three-way electromagnetic valve 445 (Vcbout) are provided. With a two-way electromagnetic valve 442, a flow channel 114a corresponding to a flowing in system of a chip cartridge 11 is communicated, and with a two-way electromagnetic valve 444 side, a flow channel 114b corresponding to a flowing out system of a chip cartridge 11 is communicated. Thereby, a drug solution or the air is supplied to a flowing in system of a chip cartridge 11 via a piping 440, and these drug solution and air can be discharged from a flowing out system of a chip cartridge 11. In addition, by two-way electromagnetic valves 442 and 444, a flow rate of a drug solution or the air in this passway for a flowing solution and a discharging solution can be controlled. In addition, by a liquid sensor 443, a flow rate of a drug solution which is flown into a chip cartridge 11, or a flow rate of a drug solution discharged from a chip cartridge 11 can be monitored.
In a piping 450, in an order toward a downstream side which is seen from a three-way electromagnetic valve 445, a two-way electromagnetic valve 451 (Vvin), a reduced pressure region 452, a two-way electromagnetic valve 453 (Vout), a flowing pomp 454, and a three-way electromagnetic valve 455 (Vww) are provided. Two-way electromagnetic valves 451 and 453 prevent reverse flow of a drug solution or the air in a passway around a reduced pressure region 452. A flowing pomp 454 consists of a tube pump, and is characterized in that it is provided in a discharge system on a flowing out side (downstream side) when seen from a chip cartridge 11. That is, since a drug solution is not contacted with a mechanism other than a tube wall by using a tube pump, this is preferable from a viewpoint of pollution prevention. In addition, by performing supply to a drug solution or the air to a chip cartridge 11 and discharge from a drug solution or the air from a chip cartridge 11 by suction action, not only replacement between a drug solution and the air in an interior of a chip cartridge 11 can be performed smoothly, but also, even when a piping is loosened accidentally, or when a chip cartridge 11 is slipped from a piping 440, liquid leakage does not occur. Thereby, stability of apparatus mounting is improved.
Of course, a pump is provided in a piping on an upstream side of a chip cartridge 11, and this pump can be configured to pump the air or a drug solution to a chip cartridge 11. A pump is not limited to a tube pump, but a syringe pump, plunger pump, a diaphragm pump, and a magnetic pump may be used.
A three-way electromagnetic valve 455 switches so as to communicate a piping 450 with a piping 461 at turn-off, and communicate a piping 450 with a piping 463 at turn-on. A waste tank 462 is provided in a piping 461, and an intercalator waste tank 464 is provided in a piping 463. Thereby, a drug solution such as Milli Q water and a buffer other than an intercalator can be guided to a waste tank 462 by switching with a three-way electromagnetic valve 455, and an intercalator can be guided to an intercalator waste tank 464. Thereby, it becomes possible to fractionation-recover an intercalator.
In addition, electromagnetic valves may be connected with a piping such as a teflon tube, and the present embodiment can be configured of a manifold structure in which an electromagnetic valve and a flow channel are integrated on an upper stream side and a downstream side relative to a chip cartridge 11, respectively. Thereby, since a volume in a piping is reduced, an amount of a necessary drug solution can be considerably decreased. In addition, since drug solution flow is stabilized in a piping, reproductivity and stability of detection result are improved.
A flowing step using a flow system 13 shown in
Parallel with this hybridization reaction, a drug solution line is started (s22). Specifically, by controlling three-way electromagnetic valves 441 and 445, a bypass piping 446 side is utilized, and by turning on a three-way electromagnetic valve 433, an intercalator is supplied from an intercalator supply source 431, for example, for around 10 seconds. A three-way electromagnetic valve 455 is turned on, and an intercalator from a piping 450 is accommodated in an intercalator waste tank 464. Then, an intercalator and the air are alternately introduced into a bypass piping 446 from a piping 435, repeatedly, for example, for 5 seconds. Then, only the air is introduced into a bypass piping 446 from a piping 435. At this stage, a waste is switched to a waste tank 462. A buffer is introduced into a bypass piping 446 from a buffer supply source 421. Thereafter, Milli Q water and the air are alternately introduced into a bypass piping 446 from a piping 435, repeatedly, for example, for each 5 seconds.
When starting of this drug solution line is completed, and a hybridization reaction is completed, piping washing is performed (s23). For piping washing, for example, a temperature of a printed board 22 is adjusted at around 25° C. with a temperature control system 14, a bypass piping 446 is purged with Milli Q water, and the air and Milli Q water are alternately introduced repeatedly, for example, for each 5 seconds. Then, a chip cartridge is washed (s24). For washing a chip cartridge, a drug solution introducing passway is switched from a bypass piping 446 to a piping 440, and the air and Milli Q water are alternately introduced into a piping 440 repeatedly, for example, for each 5 seconds. After it is confirmed by a liquid sensor 443 that a chip cartridge 11 has been filled with water, an introducing passway is switched to a bypass piping 446.
Then, purging in a piping is performed (s25). In purging in a piping, first, the air is introduced into a bypass piping 446 so as not to mix a buffer and Milli Q water. Then, the air and a buffer are alternately introduced into a bypass piping 446 repeatedly, for example, for each 5 seconds. It is confirmed by a liquid sensor 447 provided in a bypass piping 446 that a bypass piping 446 has been replaced with a buffer. Then, a buffer is injected into a chip cartridge (s26), in injection of a buffer into a chip cartridge, first, a bypass piping 446 is switched into a piping 440, the air and a buffer are alternately introduced into a chip cartridge 11 repeatedly, for example, for each 5 seconds. Then, a buffer is filled into a chip cartridge 11 (s27). In buffer filling, a buffer is introduced into a chip cartridge 11 while the state in a chip cartridge 11 is monitored with a liquid sensor 443, and an unnecessary sample is washed out by allowing to stand, for example, at 60° C. for 30 minutes (s28). After a step of washing out an unnecessary sample, a piping 440 is switched into a bypass piping 446, and washing in a piping is performed by introducing Milli Q water (s29). In this washing in piping, further, the air and Milli Q water are alternately introduced repeatedly, for example, for around each 5 seconds.
Then, washing in a chip cartridge is performed (s30). In washing in a chip cartridge, a bypass piping 446 is switched into a chip cartridge 11, and the air and water are alternately introduced repeatedly, for example, for around each 5 seconds. Thereafter, after it is confirmed by a liquid sensor 443 that Milli Q water has been filled into a chip cartridge 11, switching into a bypass piping 446 is performed. Then, measurement is initiated. In measurement, first, purging of a piping with an intercalator is performed (s31). In this purging of a piping with an intercalator, a waste is switched to an intercalator waste tank 464 while the air is introduced into a bypass piping 446. Then, the air and an intercalator are alternately supplied to a bypass piping 446 repeatedly, for example, for around each 5 seconds, and it is detected whether a bypass piping 446 has been replaced with an intercalator by using a liquid sensor 447.
Then, injection of an intercalator into a chip cartridge 11 is performed (s32). In this step, first, a bypass piping 446 is switched to a chip cartridge 11, and the air and an intercalator are alternately introduced repeatedly, for example, for around each 5 seconds. Then, under monitoring with a liquid sensor 443, an intercalator is filled into a chip cartridge 11 (s33). Thereafter, measurement is performed (s34). When measurement is complete, Milli Q water is introduced into a bypass piping 446, and then, the air and Milli Q water are alternately introduced, for example, for around each 5 seconds, and a piping is replaced with the air to perform washing in a piping (s35).
Finally, a bypass piping 446 is switched to a chip cartridge 11, the air and Milli Q water are alternately introduced, for example, for around each 5 seconds, and a chip cartridge 11 is further replaced with the air to perform washing in a chip cartridge (s36), thereby, a series of flowing steps are completed.
Like this, according to a step shown in
In addition, as a method of filling a drug solution into a cell 115, in the state where a two-way electromagnetic valve 444 as a chip cartridge outlet valve, after a pressure in a piping 440 on a chip cartridge downstream side is reduced (by controlling a two-way electromagnetic valve 451 in the state where a pump 454 is actuated, a pressure in a reduced pressure region 452 is reduced, and a two-way electromagnetic valve 453 is controlled to retain the reduced pressure state of a reduced pressure region 452), a two-way electromagnetic valve 44 is opened, thereby, a drug solution can be introduced into a chip cartridge reaction cell 115. In addition, timing of flowing shown in this
This
A voltage pattern generating circuit 510 is a circuit for converting a digital signal input from a control mechanism 15 into an analogue signal to generate a voltage pattern, and is provided with a DA converter. A resistor Rs is connected to a wiring 512b. A non-inversion input terminal of an inversion amplifier 512 is grounded, and a wiring 502a is connected to an output terminal. A wiring 512b on an inversion input terminal side of an inversion amplifier 512 and a wiring 502a on an output terminal side are connected with a wiring 512a. In this wiring 512a, a protecting circuit 500 consisting of a feedback resistor Rff and a switch SWf is provided. A wiring 502a is connected to a terminal C. A terminal C is connected to a counter electrode 502 on a chip 21. When a plurality of counter electrodes 502 are provided, terminals C are connected parallel to respective electrodes. Thereby, by one voltage pattern, a voltage can be applied to a plurality of counter electrodes 502 at the same time. In a wiring 502a, a switch SWo for controlling on-off of application of a voltage to a terminal C is provided.
A protecting circuit 500 provided in an inversion amplifier 512 is configured not to apply an excessive voltage to a counter electrode 502. Therefore, an excessive voltage is applied at measurement, and a solution is electrolyzed, thereby, stable measurement becomes possible without influencing on detection of an oxidation current of a desired intercalator. A terminal R is connected to a non-inversion input terminal of a voltage follower amplifier 513 (OPr). An inversion input terminal of a voltage follower amplifier is short-circuited with a wiring 513b and a wiring 513a connected to its input terminal. In a wiring 513b, a resistor Rf is provided, and this is connected between a resistor of a wiring 512b, and an intersection of a wiring 512a and a wiring 512b. Thereby, a voltage obtained by feed-backing a voltage of a reference electrode 503 to a voltage pattern generated by a voltage pattern generating circuit 510 is inputted in an inversion amplifier 512, and based on an output obtained by inversion-amplifying such the voltage, a voltage of a counter electrode 502 is controlled.
A terminal W is connected to an inversion input terminal of a trans impedance amplifier 511 (OPw) with a wiring 501a. A non-inversion input terminal of a trans impedance amplifier 511 is grounded, a wiring 511b connected to its terminal, and a wiring 501a are connected with a wiring 511a. In a wiring 511a, a resistor Rw is provided. Letting a voltage at an terminal O on an output side of this trans impedance amplifier 511 to be Vw, and a current to be Iw, then, Vw=Iw·Rw. An electrochemical signal obtained from this terminal O is outputted in a control mechanism 15. Since there are plural working electrodes 501, a plurality of terminals W and terminals O are provided corresponding to respective working electrodes 501. Outputs from a plurality of terminals O are switched with a signal switching part described later, and subjected to analog to digital conversion, thereby, an electrochemical signal from each working electrode 501 can be obtained as a digital value almost at the same time. Alternatively, a circuit such as a trans impedance amplifier 511 between a terminal W and a terminal O may be shared by a plurality of working electrodes 501. In this case, a wiring 501a may be provided with a signal switching part for switching a wiring 501a from a plurality of terminals W.
The effect of a measuring system 12 using this potentiostat 12a of
Then, one example of a measurement data analyzing procedure for performing signal analysis with a computer 16 based on measurement data will be explained. Herein, an analysis procedure of genotype determination of determining whether a nucleotide at a SNP position of a target nucleic acid is G type (homo type), T type (homo type) or GT type (hetero type) will be explained using a flowchart of
As assumption for performing this measurement data analysis, an example of the 4-nucleotide substitution type SNP will be explained. First, four kinds of A, G, C and T of nucleotides at a SNP position as a target nucleic acid to be detected are prepared, plural (per kind) nucleic acid probes having nucleotide sequences complementary to the target nucleic acid are immobilized on each working electrode 501. Separately, a plurality of nucleic acid probes (hereinafter, referred to as negative control) having nucleotide sequences different from these four kinds of nucleic acid probes are immobilized on other working electrode 501 (s61). A kind of a nucleic acid probe immobilized on one working electrode 501 is one, in principle.
Then, a sample containing a specimen target nucleic acid is injected in a chip on which the aforementioned nucleic acid probe is immobilized, to generate a hybridization reaction (s62), and via washing with a buffer, and an electrochemical reaction by introduction of an intercalator, a representative current value is calculated using a measurement system 12 (s63). A representative current value refers to a numerical value effective for quantitatively grasping occurrence of a hybridization reaction of each nucleic acid probe, and one example is a maximum of a current value of a signal to be directed (peak current value). A peak current value is obtained by measuring an oxidation current signal from an intercalator bound to a double-stranded nucleic acid hybridized with a nucleic acid probe immobilized on each working electrode 501, and obtaining a peak of the current value. It is desirable to perform detection of a peak current value by subtracting a background current other than an oxidation current signal from an intercalator. Of course, any value may be adopted as a representative current value depending on a precision and an object of signal treatment, and an example is an integrated value of an oxidation current signal. Of course, an example is not limited to a current value, but a voltage value, or a value obtained by performing numerical value analysis treatment on a current and a voltage may be adopted as a representative value.
Measurement data regarding a target nucleic acid where nucleotides at a SNP position are A, G, C and T types, that is, representative current values are defined as Xa, Xg, Xc and Xt, respectively, and a representative current value of a nucleic acid probe of a negative control is defined as Xn. In addition, since a plurality of representative current values are obtained depending on respective kinds, first Xa is defined as Xa1, second Xa is defined as Xa2, and so on in order to discriminate them from each other. In addition, the number of resulting representative current values of a target nucleic acid where nucleotides at a SNP position are A, G, C and T types is defined as na, ng, nc and nt, and the number of a representative current value obtained regarding a negative control is defined as nn.
Then, in order to exclude clearly abnormal data among resulting representative current values Xa, Xg, Xc, Xt and Xn, type determination filtering treatment is executed (s64). A flowchart of this type determination filtering treatment is shown in
Then, regarding these measurement data Xa1, Xa2, . . . Xana, a CV value (hereinafter, CV0) is calculated (s82). This CV0 is obtained by diving a standard deviation of measurement data Xa1, Xa2, . . . Xana by an average value. It is determined whether the resulting value CV0 is 10% or more, that is, 0.1 or more (s83). If 10% or more, a CV value of na-1 data set obtained by removing a minimum from measurement data (hereinafter, CV1) is calculated (s84). If less than 10%, it is determined that there is no clearly abnormal data, and the procedure progresses to type determination described later. After calculation of CV1, it is determined whether CV0≧2×CV1 is established or not (s85). If this inequality is established, a procedure goes to (s86), na-2 data set obtained by removing a minimum from measurement data is further defined as a data set newly, a procedure is returned to (s82), and abnormal data filtering is repeated. If an inequality is not established, it is determined that abnormal data is present not on a minimum side but on maximum side, and a CV value of na-2 data set obtained by removing a maximum from measurement data (hereinafter, CV2) is calculated (S87). It is determined whether CV0≧2×CV2 is established or not (S88). If established, na-3 data set obtained by removing a maximum from measurement data is further defined as a data set newly, a procedure is returned to (s82), and abnormal data filtering is repeated. If not established, it is determined that there is no clearly abnormal data, and a procedure goes to type determination described later. The aforementioned type determination filtering is performed also regarding Xg, Xc, Xt and Xn.
Then, type determination treatment is executed by use of the resultant type determination filtering result (s65). One example of this type determination treatment will be explained using a flowchart of
Then, regarding the resulting averages Ma, Mg, Mc, Mt and Mn, it is determined whether a maximum is an average Mg of a group G or not (s92). If a maximum, a procedure goes to (s93), and if not a maximum, a procedure goes to (s97). In (s97), regarding averages Ma, Mg, Mc, Mt and Mn, it is determined whether a maximum is an average Mt of a group T or not. If a maximum, a procedure goes to (s98), and if not a maximum, which results in that groups G and T are both not a maximum and a test is performed again due to disable determination. In (s93), it is determined whether there is a difference between group G measurement data Xg1, Xg2, . . . and group N measurement data Xn1, Xn2, . . . . For determining whether there is a difference or not, for example, 2 sample T-test is used. Specifically, a representative relationship between a probability P and a significance level ax obtained by a 2 sample T-test is compared, and this is determined as follows:
H0: if P≧α, there is not significant difference (null hypothesis)
H1: if P<α, there is significant difference (conflict hypothesis).
A significance level ax can be set by a user using a computer 16. In this example of (s93), a question H1 if there is a difference in a value between measurement data of a group G and measurement data of a group N is proposed, and against this question, hypothesis H0 postulating that there is no difference between these two groups is set. Provided that a difference between two groups are summarized in an average Mg of measurement data of a group G and an average Mn of measurement data of a group N, a probability is obtained. For calculating a probability, based on statistic values Xg1, Xg2, . . . of a group G, and statistic values Xn1, Xn2, . . . of a group N, a statistic constant t and a freedom degree φ are calculated, and a probability P is obtained from an integrated value of a probability density variable of a t distribution. Regarding the resulting probability P, if P≧α, H0 cannot be rejected, and determination is reserved. That is, it is determined that there is no difference. If P<α, H0 is rejected, and hypothesis H1 is adopted, and it is determined that there is a difference. When determination result is determined that “there is a difference” in this way, a procedure goes to (s94), and when it is determined “there is no difference”, a test is performed again due to disable determination.
In (s94), regarding a group G and a group A, it is determined whether there is a difference between two groups or not by using a 2 sample t-test as in (s93). If there is a difference, a procedure goes to (s95), and if there is no difference, a test is performed again due to disable determination. In (s95), regarding a group G and a group C, it is determined whether there is a difference between two groups or not by using the same 2 sample t-test as that of (s93). If there is a difference, a procedure goes to (s96), and if there is no difference, a test is performed again due to disable determination. In (s96), regarding a group G and a group T, it is determined whether there is a difference between two groups or not by using the same 2 sample t-test as that of (s93). If there is a difference, this is determined to be a group G type, because a group G type is a maximum average, and there is a difference between other measurement group. If there is no difference, this is determined to be a group GT type, because a group G type is a maximum average, but there is no difference in measurement result between a group G type and a group T type. In (s98), regarding a group T and a group N, it is determined whether there is a difference between two groups by using the same 2 sample t-test as that of (s93). If there is a difference, a procedure goes to (s99), and if there is not difference, a test is performed again due to disable determination. In (s99), regarding a group T and a group A, it is determined whether there is a difference between two groups or not by using the same 2 sample t-test as that of (s93). If there is a difference, a procedure goes to (s100), and if there is not difference, a test is performed again due to disable determination. In (s100), regarding a group T and a group C, it is determined whether there is a difference between two groups or not by using the same 2 sample t-test as that of (s93). If there is a difference, a group goes to (s101), and there is not a difference, a test is performed again due to disable determination. In (s101), regarding a group T and a group G, it is determined whether there is a difference between two groups or not by using the same 2 sample t-test as that of (s93). If there is a difference, this is determined to be a group T type, because a group T type is a maximum average, and there is a difference between other measurement groups. If there is no difference, this is determined to be a group GT type, because a group T type is a maximum average, but there is no difference in measurement result between a group T type and a group G type.
The above determination results are displayed in a not shown display device provided in a computer 16 (s66). By using such the type determination algorithm, it becomes possible to determine a hetero type.
Although in FIGS. 19 to 21, a procedure for determining whether a type corresponds to any of G type, T type and GT type was shown, the procedure can be of course applied to determination of any two type of A type, G type, a C type and T type, or hetero of them. In addition, it is not necessarily required that measurement data is obtained regarding four kinds of A type, G type, C type and T type groups, or data may be obtained regarding only two groups with respect to possible to nucleotides of SNP, or one group of a negative control may be added to those two groups.
An automatic analyzing procedure for individual discrimination using the aforementioned individual discriminating test apparatus will be explained using a sequence view of
A flow system control parameter has an electromagnetic control parameter for controlling electromagnetic valves 403, 413, 423, 433, 441, 442, 444, 445, 451, 453 and 463 shown in
A temperature control parameter is given, in principle, accompanying with a flow system control parameter. That is, by setting a flow system control parameter, a temperature control parameter is set corresponding to motion of a flow system 13. Thereby, it becomes possible to control a temperature of a temperature control system 14 in conjunction with a flow system 13.
By execution of automatic analysis, an automatic analysis condition parameter is sent to a control mechanism 15 (s302). Among a received automatic analysis condition parameter, based on a measurement system control parameter, a control mechanism 15 controls a measurement system 12, and based on a flow system control parameter, the mechanism controls a flow system 13, and based on a temperature control system control parameter, the mechanism controls a temperature control system 14. In addition, a control mechanism 15 manages timing for controlling these measurement system 12, flow system 13, and temperature controlling system 14 based on control timing and control condition containing in each control parameter. Therefore, a control sequence can be freely determined by an automatic analysis condition parameter set by a user, but in this
Separately from this automatic analysis, a user prepares a chip cartridge 11. Thereupon, first, a printed board 22 on which an individual discrimination chip 21 is sealed, in which a desired nucleic acid probe is immobilized on a working electrode 501, is immobilized on a support 111 of a chip cartridge 11 with a substrate fixing screw 25, thereby, attachment to a chip cartridge 11 is performed (s401). A chip cartridge upper lid 112 integrated with a sealing material 24a with an upper lid fixing screw 117 and a support 111 are fixed, and this is prepared in the state where a cell 115 is formed (s402). A sample is injected into a chip cartridge 11 through an injection port 119 (s403). A chip cartridge 11 is mounted on an apparatus body, and an initiation procedure is performed, thereby, a hybridization reaction (s21) is initiated. It is desirable that a volume of a sample to be injected is slightly larger than an amount of a volume of a cell 115. Thereby, a cell 115 can be completely filled with a sample, leaving no air.
A control mechanism 15 initiates control of timing of a measurement system based on a measurement system control parameter received from a computer 16 (s303). In addition, a control mechanism 15 successively controls each element of a flow system 13 based on a flow system control parameter received from a computer 16 (s304). In addition, although not particularly shown in
A measurement system 12, based on this measurement instruction, performs measurement, for example, by generating a voltage pattern (s306), and the resulting measurement signal is input in a control mechanism 15 from a terminal O (s307). A control mechanism 15 processes a received measurement signal, and stores this as measurement data in a data memory 15b (s308). This measurement data is input in a computer 16 via a local bus 17 (s309). A computer 16 receives this measurement data (s310).
When necessary measurement data is obtained in this way, a computer 16 executes type determination filtering shown in
The above type determination is performed regarding each of an individual to be discriminated, and a sample, and data of results is stored in a computer 16. A computer 16 compares those results, and determines whether they are consistent or not. In addition, reliability of determination result is determined from pre-inputted data of an allele frequency and a genotype frequency, and this may be displayed with the determination result.
Allotment of processing between a computer 16 and a control mechanism 15 is not limited to the aforementioned allotment. For example, when a measurement system 12, a flow system 13, and a temperature control system 14 have a processor for interpreting an instruction from a computer 16 and executing each element, a control mechanism 15 may be omitted.
For managing timing of a measurement system 12, a flow system 13 and a temperature control system 14, when these measurement system 12, flow system 13 and temperature control system 14 have a processor for managing timing, each processing is executed based on timing managed by the processor. In this case, if a computer 16 sends an automatic analysis condition parameter to these measurement system 12, flow system 13 and temperature control system 14, it is not necessary to manage timing. Alternatively, a computer 16 may perform control of timing of a measurement system 12, a flow system 13, a temperature control system 14 and a control mechanism 15.
In addition, an example of an injection port 119 communicating with an outlet port 116b has been shown, but the port may be communicated with an inlet port 116a. In addition, a working electrode 501 and a bonding pad 221 on an individual discrimination chip 21 have been shown as a laminated structure of Ti or Au, but an electrode and a pad using other material may be used. In addition, arrangement of a working electrode 501 is not limited to that shown in
In addition, a flow system 13 is not limited to that shown in
In addition, flow channels 601a to 601d are not limited to arrangement shown in
According to the embodiment of the present invention as described above, an individual discriminating test and determination of the result can be performed automatically.
Table 1 shows an example of selected single nucleotide polymorphisms. For each single nucleotide polymorphism, an allele frequency of each nucleotide and a genotype frequency in the case of Asian (Chinese or Japanese) as a sample are described.
Table 2 shows estimated heterozygosity calculated from a genotype frequency of Table 1, and a maximum frequency (Max value) and a minimum frequency (Minimum value) in each genotype (C/C, T/T, C/T), and discriminating ability (power of identification) expressed by a reciprocal thereof (one person per how many persons). A lowest step of each column of discriminating ability is obtained by multiplying discriminating ability, respectively. Therefore, regarding 22 SNPs in Table 1, it is seen that, in even a highest frequency combination, one person per about 8.38×106 persons (about 8380 thousands persons) has the same genotype, and in a lowest frequency combination, one person per 5.22×1019 persons has the same genotype.
In Detailed Explanation of the present invention, as a method of calculating a probability that a combination of genotypes is present, there was described that frequencies of genotypes are multiplied, and thereafter, a reciprocal is taken. However, as in the present Example, a reciprocal for a genotype is pre-calculated as discriminating ability regarding each SNP, and thereafter, multiplication may be performed.
Then, among SNPs shown in the above Table, arbitrary five SNPs were selected, and an array on which nucleic acid probes for detecting the SNPs were immobilized was manufactured. Sequences of nucleic acid probes used are shown in Table 3.
-SH; thiol modification
A solution containing each nucleic acid probe (30 μg/mL) and NaCl (400 mM) was spotted on electrodes of an array, and this was allowed to stand for 1 hour. Thereafter, this was washed with distilled water, and air-dried to obtain a nucleic acid-immobilized chip.
As a target nucleic acid, a region which is amplified by PCR was synthesized, and this was used as a model sample. Table 4 shows sequences of synthetic oligos used as a target nucleic acid.
Each synthetic nucleic acid shown in Table 4 was dissolved in a 2×SSC solution to 1×1014 copy/mL, to obtain a target nucleic acid solution. Among synthetic nucleic acids shown in Table 4, a nucleic acid containing G at a polymorphism site was prepared as a sample 1, and a nucleic acid containing A at a polymorphism site was prepared as a sample 2. A manufactured array was immersed in 50 μL of a prepared target nucleic acid solution, to hybridize a nucleic acid probe on an array and a target nucleic acid in a target nucleic acid solution. The array was immersed in a 0.2×SSC solution (35° C.) for 40 minutes to wash away a non-specifically bound target nucleic acid. This was washed with ultrapure water, and air-dried. Hoechst 33258 (50 μM) was dissolved in a 20 mM phosphate buffer (100 mM NaCl), the air-dried chip was immersed in this solution, and after 5 minutes, electrochemical measurement was performed. A height of an oxidation peak of Hoechst 33258 was detected, and this was adopted as a peak current value.
A peak current value obtained from each electrode is shown in
On the other hand, a sample 2 shown in
As described above, according to the present invention, a plurality of single nucleotide polymorphisms which are advantageous for individual discrimination can be selected, and the number of single nucleotide polymorphisms necessary for individual discrimination can be minimized. Thereby, a simple, rapid and economic individual discriminating method can be provided.
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-188452 | Jun 2005 | JP | national |
This is a Continuation Application of PCT Application No. PCT/JP2006/313198, filed Jun. 27, 2006, which was published under PCT Article 21(2) in English. This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2005-188452, filed Jun. 28, 2005, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP06/13198 | Jun 2006 | US |
| Child | 11514894 | Sep 2006 | US |
