Source: https://patents.google.com/patent/EP1542009B1/en
Timestamp: 2019-02-16 07:50:28
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EP1542009B1 - Method of detecting nucleic acid by using dna microarrays and nucleic acid detection apparatus - Google Patents
Method of detecting nucleic acid by using dna microarrays and nucleic acid detection apparatus Download PDF
EP1542009B1
EP1542009B1 EP20020755911 EP02755911A EP1542009B1 EP 1542009 B1 EP1542009 B1 EP 1542009B1 EP 20020755911 EP20020755911 EP 20020755911 EP 02755911 A EP02755911 A EP 02755911A EP 1542009 B1 EP1542009 B1 EP 1542009B1
EP20020755911
EP1542009A1 (en
EP1542009A4 (en
2002-08-12 Application filed by Hitachi High Technologies Corp filed Critical Hitachi High Technologies Corp
2002-08-12 Priority to PCT/JP2002/008212 priority Critical patent/WO2004017068A1/en
2005-06-15 Publication of EP1542009A1 publication Critical patent/EP1542009A1/en
2007-06-13 Publication of EP1542009A4 publication Critical patent/EP1542009A4/en
2009-11-25 Publication of EP1542009B1 publication Critical patent/EP1542009B1/en
US 2001/0034030 A1 discloses a technology for detecting nucleic acids in a sample, with which the invention has the features recited in the pre-characterising first part of each independent claim in common. The document discloses an array of elements having probes which bind to the nucleic acids of interest. The change of dielectric properties of the elements resulting from the binding is electronically detected. Switches made of field effect transistors are used to electrically address desired ones of the elements. The probes of different elements may bind to different nucleic acid sequences, but redundant elements for the same sequence may also be provided.
US 2002/0098496 A1 discloses a method for detecting the variants of polymorphic nucleic acids with a DNA array having probes selectively binding to the variants and probes with substitutions of nucleotides serving as internal controls. The array is scanned and the ratios of the responses for the different probes are taken as a quality measure.
It is an object of the invention to provide an apparatus and a method for detecting a nucleic acid by use of a DNA microarray, which allow a specific nucleic acid contained in a sample to be quantified with high accuracy.
This object is solved by the method of claim 1 and the apparatus of claim 5. The dependent claims relate to preferred embodiments of the invention.
2) The DNA microarray is formed by immobilizing the nucleic acid probe on the surface of gate insulator directly or via a carrier and comprises a plurality of insulated gate field effect transistors corresponding to the plurality of nucleic acid probe parts; and outputs from the insulated gate field effect transistors are monitored.
3) In the step of allowing the sample containing nucleic acids to interact with the DNA microarray, nucleic acid amplification is carried out on the DNA microarray using the nucleic acid as a template.
4) The nucleic acid amplification is carried out by an isothermal amplification method.
6) The DNA microarray is formed by immobilizing the nucleic acid probe on the surface of gate insulator directly or via a carrier and comprises a plurality of insulated gate field effect transistors corresponding to the plurality of nucleic acid probe parts; and the detecting units monitor outputs from the insulated gate field effect transistors.
7) The DNA microarray comprises a plurality of sections having the plurality of nucleic acid probe parts; and time taken for hybridization between the specific nucleic acid and the nucleic acid probe is different in each section.
9) The DNA microarray comprises detecting units that are arranged so as to correspond to the plurality of nucleic acid probe parts and detect hybridization between the specific nucleic acid and the nucleic acid probe.
10) The detecting units are insulated gate field effect transistors.
11) The DNA microarray comprises a plurality of sections having the plurality of nucleic acid probe parts, wherein time required for hybridization between the specific nucleic acid and the nucleic acid probe is different in each of the plurality of sections.
12) The DNA microarray comprises the plurality of sections having the plurality of nucleic acid probe parts, wherein a density of the nucleic acid probe in the nucleic acid probe part differs in each of the plurality of sections.
13) The DNA microarray comprises the plurality of sections having the plurality of nucleic acid probe parts, wherein an area of the nucleic acid probe part differs in each of the plurality of sections.
14) The DNA microarray comprises the plurality of sections having the plurality of nucleic acid probe parts, wherein a length of the nucleic acid probe differs in each of the plurality of sections.
Fig. 8 is a circuit diagram showing a case where the outputs from the plurality of nucleic acid probe parts are measured with the use of a reference field effect transistor,
Fig. 10 is a schematic cross sectional view of the essential part of the DNA microarray showing each step of nucleic acid amplification carried out on the DNA microarray;
Fig. 1 represents a schematic perspective view of a DNA microarray 1 used in the present method, and Fig. 2 represents a cross sectional view of an essential part of the DNA microarray 1. The DNA microarray 1 comprises, as shown in Fig. 1, a plurality of nucleic acid probe parts 2 arranged on a matrix and insulated gate field effect transistors 3 arranged so as to correspond to the plural probe parts 2. In the DNA microarray 1 of the present embodiment, the plural nucleic acid probe parts 2 are divided into two (called a first region 3a and a second region 3b, respectively). In the first region 3a, one polymorphism (hereinafter, sometimes called "first polymorphism") of the target gene is detected, and in the second region 3b, the other polymorphism (hereinafter, sometimes called "second polymorphism") of the target gene is detected.
In the DNA microarray 1 of the present embodiment, the nucleic acid probe 6 in the first region 3a has a sequence capable of hybridizing to the target gene of the first polymorphism and incapable of hybridizing to the target gene of the second polymorphism. On the other hand, the nucleic acid probe 6 in the second region 3b has a sequence capable of hybridizing to the target gene of the second polymorphism and incapable of hybridizing to the target gene of the first polymorphism.
In the DNA microarray 1 constructed as described above, following application of a sample to be examined, the target gene contained in the sample and the nucleic acid probe 6 are allowed to hybridize to each other under an appropriate reaction condition. That is, a complex of either one of the nucleic acid probe 6 in the first area 3a and the nucleic acid probe 6 in the second area 3b with the target gene is formed depending on the polymorphism of the target gene contained in the sample, or complexes of both of the nucleic acid probe 6 in the first area 3a and the nucleic acid probe 6 in the second area 3b with the target gene are formed.
The internal flow channel 21 in the housing 22 is provided inside the housing by making, for example, a hole of one millimeter diameter, and makes contact with the DNA microarray 1 at the approximate center of the housing 22. Here, the internal flow channel 21 is in contact with at least the first region 3a and the second region 3b in the DNA microarray 1. Further, the internal flow channel 21 is connected to the flow channel 13 by attaching bolts 28 with the flow channel 13 inside to bolt-mounting portions 29 in the housing 22. The bolt-mounting portions 29 in the housing 22 are threaded so as to be fitted with the bolts 28 to be attached.
The detection apparatus 10 constructed as described above makes it possible to identify polymorphisms of a target gene contained in a sample to be examined using the DNA microarray mounted in the flow cell 12. Specifically, first, the valve 16 is switched so as to supply only the hybridization solution 14 to the flow channel 13, and the pump 17 is driven. At the same time, the sample is supplied from the dispenser 18 to the flow channel 13 via the valve 16. This makes it possible to supply the hybridization solution 14 containing the sample to the flow channel 13 and the internal flow channel 21 in the flow cell 12, thereby allowing the sample to interact with the first region 3a and the second region 3b of the DNA microarray 1.
Thus, the nucleic acid probe 6 having a nucleotide sequence complementary to the target gene contained in the sample and the target gene undergo hybridization reaction in the first region 3a or the second region 3b, or in both of the first region 3a and the second region 3b. In other words, the target gene contained in the sample hybridizes only to the nucleic acid probe 6 having a nucleotide sequence complementary to the target gene among the nucleic acid probes 6 contained in the first region 3a and the second region 3b. After the reaction, the solution supplied to the flow channel 13 and the internal flow channel 21 is sent to the waste bottle 19 by the pump 17.
When the frequency distribution is determined using measured time as the horizontal axis and the number of pieces of the nucleic acid probe parts 2 whose outputs have been measured to exceed the threshold level as the vertical axis, a normal distribution is obtained, for example, as shown in Fig. 7. In this case, the signal processing circuit 20 determines the output time T3 as an average value (a maximum value in the normal distribution in this case) of the frequency distribution. The signal processing circuit 20 is able to determine the output time for the plural nucleic acid probe parts 2 contained in the first region 3a and the output time for the plural nucleic acid probe parts 2 contained in the second region 3b.
The detection apparatus 10 is capable of judging the polymorphisms of the target gene based on each output time from the first region 3a and the second region 3b. That is, when the target gene is a homotype consisting only of a first polymorphism, the output time can be obtained only from the first region 3a. In contrast, when the target gene is a homotype consisting of only a second polymorphism, the output time can be obtained only from the second region 3b. Further, when the target gene is a heterotype consisting of the first polymorphism and the second polymorphism, the output time can be obtained from both of the first region 3a and the second region 3b. As described above, the polymorphism of the target gene can be judged from the output times from the first region 3a and the second region 3b using the detection apparatus 10.
The detection apparatus 10 allows the polymorphism of the target gene to be determined not only by obtaining the output time by the signal processing circuit 20 as described above, but also by the following way. The signal processing circuit 20 computes proportions of the insulated gate field effect transistors 4 that have output signals after a predetermined elapsed time for each of the first region 3a and the second region 3b and compares them to each other. As the result, when the proportion for the first region 3a is larger compared to that for the second region 3b, the target gene can be judged to be a homotype consisting of only the first polymorphism. Conversely, when the proportion for the second region 3b is larger compared to that for the first region 3a, the target gene can be judged to be a homotype consisting of only the second polymorphism. Further, when the proportion for the first region 3a and that for the second region 3b are comparable, the target gene can be judged to be a heterotype consisting of the first polymorphism and the second polymorphism.
It should be noted that when the presence of the first polymorphism, for example, is desired to be promptly detected, the first polymorphism of interest may be judged to be present at the time when an output from at least one nucleic acid probe part 2 among the nucleic acid probe parts 2 contained in the first region 3a has been obtained. This way of judging makes it possible to judge promptly whether or not the first polymorphism is contained in the sample.
Furthermore, the detection apparatus 10 may use the DNA microarray 1 provided with a first section consisting of the first region 3a and the second region 3b, a second section consisting of a third region 3c and a fourth region 3d, a third section consisting of a fifth region 3e and a sixth region 3f, and a fourth section consisting of a seventh region 3g and an eighth region 3h. The first region 3a to the eighth region 3h are provided with the plural nucleic acid probe parts 2, respectively. The nucleic acid probe 6 that hybridizes to the first polymorphism of the target gene is immobilized in the first region 3a, the third region 3c, the fifth region 3e, and the seventh region 3g. The nucleic acid probe 6 that hybridizes to the second polymorphism of the target gene is immobilized in the second region 3b, the fourth region 3d, the sixth region 3f, and the eighth region 3h.
In addition, the time required for hybridization between the nucleic acid containing the target gene and the nucleic acid probes 6 is adjusted so as to be mutually different in the first, second, third, and fourth sections. Specifically, the time required for hybridization in each section becomes different by making mutually different the immobilization densities of the nucleic acid probe 6 in the first section, the nucleic acid probe 6 in the second section, the nucleic acid probe 6 in the third section, and the nucleic acid probe 6 in the fourth section. Further, the time required for hybridization in each section becomes different by making different the areas of the nucleic acid probe parts 2 in the first section, the nucleic acid probe parts 2 in the second section, the nucleic acid probe parts 2 in the third section, and the nucleic acid probe parts 2 in the fourth section. Still further, the time required for hybridization in each section becomes different by making different the lengths (nucleotide lengths) of the nucleic acid probe 6 in the first section, the nucleic acid probe 6 in the second section, the nucleic acid probe 6 in the third section, and the nucleic acid probe 6 in the fourth section.
It should be noted that when the presence of the first polymorphism is desired to be promptly detected also in the case of using the DNA microarray 1 shown in Fig. 9, the first polymorphism may be judged to be present at the time when an output from at least one nucleic acid probe part 2 among the nucleic acid probe parts 2 contained in any one of the regions consisting of the first region 3a, the third region 3c, the fifth region 3e, and the seventh region 3g has been obtained. When the DNA microarray 1 shown in Fig. 9 is used, it is possible to judge promptly whether or not the first polymorphism is contained in the sample.
In the second embodiment, a DNA microarray in which the 5' terminus of the nucleic acid probe 6 having a nucleotide sequence complementary to the target gene to be detected in the 3' terminal portion is immobilized on the surface of the gate insulator 2 is used for the DNA microarray 1.
Next, nucleic acid amplification is carried out on the DNA microarray 1. As specifically shown in Figs. 10A to 10D, when a DNA fragment 38 having the target gene is contained in the solution containing human chromosomes, the nucleic acid amplification is performed using the nucleic acid probe 6 as a primer. That is, first as shown in Fig. 10A, hybridization (annealing) between the nucleic acid probe 6 and a portion of the target gene in the DNA fragment 38 takes place by controlling the gate insulator 2 to a predetermined temperature. Then, as shown in Fig. 10B, an extension reaction is carried out from the 3' terminus of the nucleic acid probe 6 using the DNA fragment 38 as the template with the aid of an enzyme (DNA polymerase, etc.) included in the reagents necessary for the nucleic acid amplification by controlling the gate insulator 2 to a predetermined temperature. Controlling the gate insulator 2 to a predetermined temperature after the extension reaction induces heat denaturation to allow the DNA fragment 38 to be dissociated. Then again, by controlling the gate insulator 2 to the predetermined temperature, hybridization (annealing) between the unreacted nucleic acid probe 6 and the portion of the target gene in the DNA fragment 38 takes place as shown in Fig. 10C. Subsequently, as shown in Fig. 10D, the extension reaction takes place from the 3' terminus of the nucleic acid probe 6 using the DNA fragment 38 as the template with the aid of the enzyme (DNA polymerase, etc.) included in the reagents necessary for the nucleic acid amplification by controlling again the gate insulator 2 to the predetermined temperature.
In this way, the extension reaction from the 3' terminus of the nucleic acid probe 6 using the DNA fragment 38 as the template is carried out in turn by controlling the temperature of the gate insulator 2. When the DNA fragment 38 having the target gene is contained in the sample to be measured, the progress of the extension reaction in the nucleic acid probe part 2 results in a change of charge density in the vicinity of the gate insulator 5 of the insulated gate field effect transistors 4 corresponding to the nucleic acid probe part 2, and the surface potential of the gate insulator 5 is changed. This change acts like a gate voltage change in a conventional insulated gate field effect transistor, resulting in a change of electric conductivity of the channel. Therefore, the progress of the extension reaction in each nucleic acid probe part 2 can be detected as a change in drain current flowing between the source 8 and the drain 9 by using the detection apparatus 10.
In a first specific example, the following nucleic acid probe 6 with the 5' terminus modified to have an amino group was immobilized on the gate insulator 5 of the insulated gate field effect transistor 4 in order to examine the presence or absence of hepatitis B virus DNA.
HBV probe (i); 5'-GCG GAT CCG TGG AGT TAC TCT CGT TTT TGC-3'
The nucleic acid 33 having a sequence different from the HBV probe (i), e.g. the nucleic acid 33 having a length of 30 nucleotides composed exclusively of A, was immobilized on the gate insulator of the reference field effect transistor 31. Here, the nucleic acid 33 may not be immobilized on the gate insulator of the reference field effect transistor 31, but may be modified to have an amino group on the 5' terminus.
The sample used was Hepatitis B virus DNA extracted from serum that is available in a kit (Qiagen viral DNA kit). One hundred microliter of a sample solution containing the Hepatitis B virus DNA, IX PCR buffer (Mg2+ plus) (product of Takara Co., Ltd.), 0.4 µM dNTP, and 5 units of Taq polymerase (Takara) was introduced into the flow cell 12 from the dispenser 18, and the gate insulator 5 was heated and cooled according to the following steps (1) to (3):
(1) Heat denaturation; 94°C for 3 min
(2) A cycle of 94°C for 30 sec for heat denaturation, 55°C for 30 sec for annealing, and 72°C for 30 sec for extension reaction is repeated 40 cycles.
(3) Extension reaction; 72°C for 10 min
After completing the above steps (1) to (3), double-stranded nucleic acids were denatured to convert to single-stranded nucleic acids either by raising the temperature of the gate insulator 5 or introducing an alkaline solution into the flow cell 12 from the dispenser 18. Then, a washing solution was introduced into the flow cell 12 from the dispenser 18, thereby removing nucleic acids not immobilized on the substrate, unreacted nucleic acids, and various components from the DNA microarray 1. Subsequently, a buffer solution was introduced into the flow cell 12 from the dispenser 18, and output values from the insulated gate field effect transistors 4 were measured by the signal processing circuit 20.
In the first specific example, the output values from the signal processing circuit 20 were the ones resulting from the single-stranded nucleic acids extended from the 3' terminus of the HBV probe (i) by the nucleic acid amplification, and the detection was made possible by a larger number of nucleotides compared with that of the nucleic acid 33 immobilized on the gate insulator of the reference field effect transistor 31. Fig. 11 schematically depicts a state that single-stranded nucleic acids were extended from the nucleic acid probe 6 and the nucleic acid 33 immobilized on the gate insulator of the reference field effect transistor 31.
In a second specific example, the nucleic acid probe 6 having a tendency to assume a higher order structure in accordance with the progress of the amplification reaction that takes place by the presence of the target gene in the sample was immobilized on the gate insulator 5 of the insulated gate field effect transistor 4 in order to detect the presence or absence of hepatitis B virus DNA with high sensitivity. The nucleic acid probe 6 used in the second specific example had a nucleotide sequence complementary to Hepatitis B virus DNA, with its 5' terminus being modified to have an amino group. The nucleic acid probe 6 was as follows:
In the second specific example, the output values were the ones resulting from the single-stranded nucleic acids that extended from the 3' terminus of the HBV probe (ii) by the nucleic acid amplification and formed a higher order structure. The detection was made possible by a larger number of nucleotides compared with that of the nucleic acid 33 immobilized on the gate insulator of the reference field effect transistor 31 as well as by the difference in the higher order structure. Fig. 12 schematically depicts a state that single-stranded nucleic acids were extended from the nucleic acid probe 6 and the nucleic acid 33 immobilized on the gate insulator of the reference field effect transistor 31. In the second specific example, when Hepatitis B virus DNA was contained in the sample, the 3' terminus of the nucleic acid probe 6 assumes a higher order structure, i.e. a stem structure or a loop structure in this case, as the result of the progress of the extension reaction, as shown in Fig. 12. This stem structure is formed because the single-stranded nucleic acid synthesized by the extension reaction is complementary to the HBV probe (ii) from its 5' terminus up to the 30 nucleotides. Further, the loop structure is formed by the 19 nucleotides of the HBV probe (ii) on its 3' terminal side.
Next, as a third specific example, the following nucleic acid probes 6 modified to have an amino group at the 5' terminus were immobilized on the gate insulator 5 of the insulated gate field effect transistor 4 in order to detect the presence or absence of hepatitis B virus DNA with two kinds of the probes:
HBV probe (iii); 5'-GCA AGC TTT CTA ACA ACA GTA GTT TCC GG-3'
In the third specific example as well, the above described steps (1) to (3) were carried out under the same reaction conditions using the same reagents as those in the first specific example. Figs. 13A to 13D illustrate the nucleic acid amplification in the third specific example. In Figs. 13A to 13D, the HBV probe (i) is denoted by a nucleic acid probe 6a and the HBV probe (iii) is denoted by a nucleic acid probe 6b. When the sample to be studied contained hepatitis B virus DNA, hybridization (annealing) took place between the DNA fragment 38 containing hepatitis B virus DNA and the nucleic acid probe 6a by controlling the gate insulator 2 to the first predetermined temperature as shown in Fig. 13A. Next, as shown in Fig. 13B, an extension reaction was carried out from the 3' terminus of the nucleic acid probe 6a using the DNA fragment 38 as the template with the aid of an enzyme (DNA polymerase, etc.) included in the reagents necessary for nucleic acid amplification by controlling the gate insulator 2 to the second predetermined temperature. Controlling the gate insulator 2 to the third temperature induced heat denaturation to allow the DNA fragment 38 to be dissociated, and a portion of the region that extended from the 3' terminus of the nucleic acid probe 6a hybridized (annealed) to the nucleic acid probe 6b as shown in Fig. 13C. Then again, by controlling the gate insulator 2 to the second predetermined temperature, an extension reaction proceeded from the 3' terminus of the nucleic acid probe 6b using the region that extended from the 3' terminus of the nucleic acid probe 6a as the template, and finally nucleic acid double strand was formed between the nucleic acid probe 6a and the nucleic acid probe 6b as shown in Fig. 13D.
Next, the buffer solution was introduced from the dispenser 18 into the flow channel 13 through the valve 16 to remove the unreacted sample and the like on the DNA microarray 1. Then, a solution containing an intercalator 39 such as ethidium bromide or Hoechst 33258 was introduced from the dispenser 18 into the flow channel 13 through the valve 16, thereby inserting the intercalator 39 into the double stranded nucleic acid of the nucleic acid probe 6a and the nucleic acid probe 6b. The intercalator 39 reacts only with the double stranded nucleic acid to bind, and does not bind to single stranded nucleic acids. After inserting the intercalator 39 into the nucleic acid double strand, an output value was measured by the signal processing circuit 20 in a manner similar to that in the first specific example.
As a fourth specific example, the following nucleic acid probes 6 modified to have an amino group at the 5' terminus were immobilized on the gate insulator 5 of the insulated gate field effect transistor 4 in order to detect the presence or absence of hepatitis B virus DNA using two kinds of the probes and a DNA polymerase with strand displacement activity:
In the present example, one hundred microliter of a sample solution containing hepatitis B virus DNA, 0.1 mM dNTP, 0.5 mM MgCl2, and 32 units of Bst polymerase (product of New England BioLabs Inc.) was introduced from the dispenser 18 into the flow cell 12, and the gate insulator 5 was incubated at 65°C to subject to an isothermal amplification reaction. This isothermal amplification reaction proceeds in a manner similar to that shown in Figs. 10A to 10D. In the present example too, the output value was measured by the signal processing circuit 20 after inserting the intercalator 39 into the nucleic acid double strand in a manner similar to that in the third specific example.
<110> Hitachi High-Technologies Corporation
<120> A method of detecting a nucleic acid using DNA microarray and a nucleic acid detecting apparatus
<130> PH-1503-PCT
gcggatccgt ggagttactc tcgtttttgc 30
gcaagctttc taacaacagt agtttccgg 29
A method of detecting a specific nucleic acid by using a DNA microarray, comprising the steps of
allowing a sample containing nucleic acids to interact with the DNA microarray (1) comprising a plurality of nucleic acid probe parts (2) each having a nucleic acid probe (6) capable of hybridizing to the specific nucleic acid; and
monitoring outputs from the plurality of nucleic acid probe parts (2) due to hybridization between the nucleic acid probe (6) and the specific nucleic acid,
determining a distribution, per unit time, of the number of the nucleic acid probe parts (2) whose output exceeds a predetermined value; and
quantifying the specific nucleic acid contained in the sample based on a maximum value determined by normalizing said distribution.
The method of claim 1, wherein the DNA microarray (1) is formed by immobilizing the nucleic acid probe (6) on the surface of a gate insulator directly or via a carrier and comprises a plurality of insulated gate field effect transistors corresponding to the plurality of nucleic acid probe parts (2); and the outputs from the insulated gate field effect transistors are monitored.
The method of claim 1, wherein, in the step of allowing the sample containing nucleic acids to interact with the DNA microarray (1), nucleic acid amplification is carried out on the DNA microarray using the nucleic acid as a template.
The method of claim 3, wherein the nucleic acid amplification is carried out by an isothermal amplification method.
An apparatus for detecting a specific nucleic acid comprising:
a measuring unit to attach a DNA microarray (1) provided with a plurality of nucleic acid probe parts (2) each having a nucleic acid probe (6) capable of hybridizing to the specific nucleic acid; and
detecting units to detect outputs from the nucleic acid probe parts (2) of the DNA microarray attached on the measuring unit;
characterized by a computing unit (20) arranged to determine a distribution, per unit time, of the number of the nucleic acid probe parts whose output exceeds a predetermined level and quantify the specific nucleic acid contained in a sample based on a maximum number determined by normalizing the distribution.
An apparatus according to claim 5, further including said DNA microarray (1).
The apparatus according to claim 6, wherein the DNA microarray (1) is formed by immobilizing the nucleic acid probe (6) on the surface of gate insulator directly or via a carrier and comprises a plurality of insulated gate field effect transistors corresponding to the plurality of nucleic acid probe parts (2); and the detecting units monitor outputs from the insulated gate field effect transistors.
The apparatus according to claim 6, wherein the DNA microarray comprises a plurality of sections (3a,b, 3c,d, 3e,f, 3g,h) having the plurality of nucleic acid probe parts.
The apparatus of claim 8, wherein time required for hybridization between the specific nucleic acid and the nucleic acid probe (6) is different in each of the plurality of sections.
The apparatus of claim 8 or 9, wherein an area of the nucleic acid probe part (2) differs in each of the plurality of sections.
The apparatus of any of claims 8 to 10, wherein the DNA microarray comprises the detecting units that are arranged corresponding to the plurality of nucleic acid probe parts (2) and detect hybridization between the specific nucleic acid and the nucleic acid probe.
The apparatus of claim 11, wherein the detecting units are insulated gate field effect transistors.
The apparatus of any of claims 8 to 12, wherein a density of the nucleic acid probe (6) in the nucleic acid probe part differs in each of the plurality of sections.
The apparatus of any of claims 8 to 13, wherein a length of the nucleic acid probe (6) differs in each of the plurality of sections.
EP20020755911 2002-08-12 2002-08-12 Method of detecting nucleic acid by using dna microarrays and nucleic acid detection apparatus Expired - Fee Related EP1542009B1 (en)
PCT/JP2002/008212 WO2004017068A1 (en) 2002-08-12 2002-08-12 Method of detecting nucleic acid by using dna microarrays and nucleic acid detection apparatus
EP1542009A1 EP1542009A1 (en) 2005-06-15
EP1542009A4 EP1542009A4 (en) 2007-06-13
EP1542009B1 true EP1542009B1 (en) 2009-11-25
ID=31742920
EP20020755911 Expired - Fee Related EP1542009B1 (en) 2002-08-12 2002-08-12 Method of detecting nucleic acid by using dna microarrays and nucleic acid detection apparatus
US (1) US7273704B2 (en)
EP (1) EP1542009B1 (en)
JP (1) JP3980030B2 (en)
CN (1) CN100392097C (en)
DE (1) DE60234540D1 (en)
WO (1) WO2004017068A1 (en)
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2002-08-12 JP JP2004528806A patent/JP3980030B2/en active Active
2002-08-12 WO PCT/JP2002/008212 patent/WO2004017068A1/en active Application Filing
2002-08-12 EP EP20020755911 patent/EP1542009B1/en not_active Expired - Fee Related
2002-08-12 CN CN 02829357 patent/CN100392097C/en not_active IP Right Cessation
2002-08-12 DE DE2002634540 patent/DE60234540D1/en active Active
2002-08-12 US US10/522,991 patent/US7273704B2/en active Active
JPWO2004017068A1 (en) 2005-12-08
CN100392097C (en) 2008-06-04
EP1542009A1 (en) 2005-06-15
US7273704B2 (en) 2007-09-25
EP1542009A4 (en) 2007-06-13
US20060088839A1 (en) 2006-04-27
CN1703623A (en) 2005-11-30
JP3980030B2 (en) 2007-09-19
DE60234540D1 (en) 2010-01-07
WO2004017068A1 (en) 2004-02-26
Ipc: G01N 27/414 20060101ALI20070504BHEP
Ipc: G01N 33/543 20060101AFI20070504BHEP
Ipc: C12Q 1/68 20060101ALI20070504BHEP
Inventor name: MIYAHARA, YUJI
Inventor name: MATSUI, TAKUYA
Inventor name: HATTORI, KUMIKO
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