JP3783616B2 - Genetic diagnostic equipment - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a genetic diagnostic apparatus that can easily and accurately detect the presence or absence of a genetic abnormality.
[0002]
[Prior art]
All diseases involve genetic factors and environmental factors with various probabilities, but diseases such as congenital metabolic disorders, cancer, diabetes, hypertension, Alzheimer, autoimmune diseases, atopy, obesity, and alcoholism Hereditary factors account for a very large proportion. On the other hand, diseases that have a large contribution of environmental factors include diseases caused by infections and aftereffects of trauma.
[0003]
By the way, due to the rapid development of molecular biology in recent years, genetic elements, that is, gene involvement in various diseases have been elucidated fairly accurately, and attention has been focused on gene-targeted medicine. Yes. At present, what is attracting the most attention is SNPs (snips). This is an abbreviation for single nucleotide polymorphism and is generally translated as “single nucleotide polymorphism”, and is a general term for the difference of one code (one base) in genes between individuals.
[0004]
All life-form genes on earth, including humans (or the genome that means the entire collection of genes), are composed of four common bases, and various proteins are created by the sequence of these bases. Life activities are taking place. The four bases common to all organisms are adenine (denoted as A), guanine (denoted as G), thymine (denoted as T), and cytosine (denoted as C). Human genes are said to be composed of about 3 to 3.2 billion base sequences, but each individual has a base that is different from other people at a rate of about one in several hundred to 1000 bases. Exists. Usually, this single base change is present at a frequency of 1% or more in the total population of a certain human population, and is called SNPs.
[0005]
Therefore, it is said that there are 3 to 10 million SNPs in all genes (genome), and the search for SNPs is currently continued all over the world. The reason why SNPs are attracting attention is that it is considered that the morbidity rate for many diseases, which are said to be statistically related to the genes of each individual, can be estimated by the classification of SNPs. For example, taking breast cancer as an example, SNPs common to those who are likely to have breast cancer can be identified by comparing SNPs between a group of patients with breast cancer and a normal group. At the time of a health examination, it becomes possible to find a person who has investigated the gene and has the SNPs, that is, a person who is normal but is likely to have breast cancer in the future.
[0006]
With this diagnosis, a person with a constitution that is likely to have breast cancer can be treated very early, and the possibility of survival can be improved even if he / she suffers from cancer. The same can be said for lifestyle-related diseases such as diabetes and hypertension, and it is possible to accurately diet and guide living before the onset of diseases that many people around the world suffer.
[0007]
In addition, SNPs are expected to play an important role regarding drugs used for treatment of diseases. Drugs used during treatment are not equally effective for everyone. In general, it is well known that drugs are effective for a certain proportion of people but not for others at all, and may cause adverse results due to side effects. Incidentally, it is well known that drug side effects are at the top of the causes of death in the United States. The effect of the drug is deeply related to the constitution of the person, and the constitution is said to be distinguishable by the classification of SNPs. That is, the classification based on the analysis of SNPs makes it possible to predict the effect and sensitivity of a certain drug in advance and make an appropriate prescription, and the optimal drug administration and risk of side effects according to the individual patient's constitution. Avoidance is expected. Such medical care is called tailor-made medical care or tailor-made medical care, and future practical use is surely expected.
[0008]
In addition, it is known that cancer is caused by a mutation occurring at a specific site on a gene that plays an important role in normal cells, for example, by the action of ultraviolet rays or mutagenic substances. By reading a mutation on a specific gene, it becomes possible to diagnose from an early stage whether or not a cell is cancerous. SNPs are also effective in identifying criminals in criminal investigations, determining ambiguous parent-child relationships, and identifying whether or not they are individuals. As described above, there are 3 to 10 million SNPs in each individual, and since there are separate SNPs from parents, there are absolutely no humans on the planet who have the same SNPs even if they are parents and siblings. It is said not to. This is the reason why complete identification of individuals is possible.
[0009]
In this manner, observing a mutation that occurs at one base in a specific gene may greatly contribute to various matters including medical treatment. However, at present, the method of observing a mutation of a specific gene requires a very complicated operation or an expensive apparatus as described below, and the running cost is very high, so that it has not yet been widely used. is there.
[0010]
The most commonly used method for examining SNPs at present is a method called sequencing (determining the base sequence) in which the base sequence of DNA is directly read from the end. Since a gene is a unit of DNA having base sequence information for forming one kind of protein, SNPs can be elucidated by reading the base sequence from the end. There are several reports on the sequencing method, but the most commonly performed method is dideoxy sequencing (Sanger method) described below. All of these methods, including this method, are based on a technique that can separate and identify the difference in length of one base length by denaturing polyacrylamide gel electrophoresis or capillary electrophoresis with high resolution.
[0011]
The dideoxy sequencing (Sanger method), also called enzymatic sequencing, uses DNA polymerase to synthesize complementary strands of single-stranded template DNA, and then produces four special types of dideoxynucleotides artificially created. The feature is to use. As a sequencing operation, a synthetic base sequence that complements the 3 'end of the single-stranded DNA to be sequenced is used as a primer, and deoxynucleotides added equally from the primer to DNA polymerase are extended by an enzymatic reaction. At this time, four reaction vessels were prepared at the same time, and each did not have a hydroxyl group at the 3 'end of the ATGC four bases, and therefore dideoxynucleotide, a base analog that could not continue the DNA extension reaction any more. Mix a small amount separately. As a result, DNA synthesis was stopped when dideoxynucleotide was added to the end of the extending DNA, and each reaction vessel had various lengths, but the ends were always added base analogs. Stranded DNA is formed. The reaction vessel is reacted with S1 endonuclease to digest all the single-stranded DNA into only double-stranded DNA. If the DNA strands of the four reaction vessels thus obtained are subjected to gel electrophoresis or capillary electrophoresis, and the separated DNA is read in order from the shorter one (moved faster), the base sequence of the DNA complementary to the template strand will be Recognize. At this time, the electrophoresis results are identified by, for example, radioactively labeling the added dideoxynucleotide phosphorus or sulfur or binding a fluorescent chemical substance. In the case of a radioactive label, detection is performed by exposure to a film. In the case of a fluorescent chemical substance, fluorescence is detected by irradiating a laser beam. Recently, a method has been developed in which four types of base analogs A, T, G, and C are labeled with four types of reagents having different fluorescence wavelengths, and the four colors of fluorescence are simultaneously detected.
[0012]
In this way, conventionally, the exact base sequence of a gene isolated and purified from a subject is determined by this dideoxy sequencing (Sanger method) or other base sequence determination method, and compared with a normal or standard base sequence. Confirmation of the presence of SNPs and mutations, identification of individuals, etc. Further, there are cases where diagnosis is made only by the quantitative ratio between normal DNA and abnormal DNA, such as electrophoresis.
[0013]
[Problems to be solved by the invention]
As described above, genetic diagnosis and identification of individuals by gene using conventional gene sequencing methods are performed by isolating the target gene, then amplifying and purifying it, and using a device for determining the base sequence of the gene. Since it was performed by reading the base sequence of the target gene, the experiment required a huge amount of work, a very long time, and a great running cost. In addition, an automated apparatus for determining a base sequence is very expensive, occupies a large space, and requires a large amount of expensive reagents.
[0014]
Therefore, in order to solve such a conventional problem, the present inventors have proposed a detection method using electrophoresis. That is, a DNA sample is separated from a DNA sequence by bonding a base sequence capable of hydrogen bonding and a high molecular compound, and a separation DNA conjugate is prepared. From the difference in binding power of the separation conjugate to normal DNA and abnormal DNA, a DNA sample is obtained. Is separated into normal DNA and abnormal DNA. For this reason, the separation DNA conjugate and the DNA sample are filled in the buffer solution filled in the capillary tube, and the DNA sample and the separation DNA conjugate in the capillary tube are electrophoresed by applying a constant voltage. The detection unit detects the ratio of normal DNA to abnormal DNA by measuring the passing amount of normal DNA and abnormal DNA as ultraviolet absorbance.
[0015]
This method can separate and measure SNPs and abnormal DNA very easily and accurately, but has a problem that electrophoresis cannot be performed when bubbles or the like are mixed in the capillary tube. In this case, it is difficult to grasp the situation inside the capillary tube, and in order to continue the measurement without noticing the inclusion of bubbles or the like, it is often the case that the measurement value is abnormal during measurement or after the end of the measurement and noticed again. I had to make a very inefficient measurement.
[0016]
Accordingly, an object of the present invention is to provide a genetic diagnosis apparatus that can quickly detect that electrophoresis cannot be performed due to bubbles or the like and can perform measurement with high efficiency.
[0017]
[Means for Solving the Problems]
In order to solve the above problems, a genetic diagnosis apparatus of the present invention is provided in a sealed channel, a sealed channel in which a buffer solution containing a linear polymer and a DNA binding control agent is filled and communicated between the first container and the second container. A detection unit for detecting the amount of DNA sample passing through the interior, and further, in the sealed channel, a base sequence capable of hydrogen bonding to the DNA sample and a polymer compound are bound in a buffer solution , A DNA conjugate for separation that separates the DNA sample into normal DNA and abnormal DNA from the difference in binding force due to hydrogen bonding and the DNA sample are filled, and the DNA sample in the sealed channel is electrically charged by applying a predetermined voltage. are electrophoresed, a genetic diagnostic device for detecting unit measures each passage of normal DNA and abnormal DNA, the closed flow path, to measure at least two or more wavelengths of the ultraviolet absorbance bubbles in the flow path Wherein the bubble detector for detecting is provided by the.
[0018]
Thereby, it is possible to quickly detect that electrophoresis cannot be performed due to bubbles or the like, and measurement can be performed with high efficiency.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
The invention described in claim 1 includes a first container in which a buffer solution is stored and the first electrode is immersed, a second container in which the buffer solution is stored and the second electrode is immersed, a first container, and a second container. Provided with a sealed channel that is filled with a buffer containing a linear polymer and a DNA binding control agent between the containers, and a detection unit that is provided in the sealed channel and detects the amount of DNA sample passing through the interior, and is further sealed In the flow path, a base sequence capable of hydrogen bonding to a DNA sample and a polymer compound are bound in a buffer solution, and the DNA sample is separated into normal DNA and abnormal DNA from the difference in binding force due to hydrogen bonding. Diagnosis, in which a DNA conjugate for use and a DNA sample are filled, a DNA sample in an airtight flow path is electrophoresed by applying a predetermined voltage, and a detection unit measures the amount of normal DNA and abnormal DNA passing through, respectively Device, dense A bubble detection unit for detecting bubbles is provided in the closed channel, and the bubble detection unit detects the bubbles in the sealed channel by measuring ultraviolet absorbance of at least two wavelengths or more. Because it is a genetic diagnostic device characterized by the above, it can quickly detect the occurrence of air bubbles in the sealed channel when a DNA sample is electrophoresed and can immediately notify it, thus repeating unnecessary electrophoresis Without stopping, electrophoresis can be immediately stopped and the liquid in the sealed channel can be exchanged for liquid without bubbles. The fact that electrophoresis cannot be performed due to bubbles or the like can be quickly detected, and measurement can be performed with high efficiency.
[0020]
( Reference example for explaining the present invention )
FIG. 1 is an external view of a genetic diagnostic apparatus in a reference example for explaining the present invention, FIG. 2 is an external view of the genetic diagnostic apparatus with an open top, and FIG. 3 is an apparatus configuration diagram including an electrophoresis unit of the genetic diagnostic apparatus, FIG. 4 is a main part diagram of the control circuit of the genetic diagnosis apparatus, and FIG. 5 is an introduction state diagram of the DNA conjugate for separation and the DNA sample in the closed flow path of the genetic diagnosis apparatus.
[0021]
In FIG. 1, 1 is a power switch of the genetic diagnosis apparatus, 2 is an operation button for performing various operations of the apparatus, 3 is a display panel, 4 is an upper lid, and 5 is a casing of the apparatus. 2, 3, and 4, 6 is an electrophoresis unit for performing electrophoresis, 7 is a support base that supports the electrophoresis unit 6, 8 is a control for performing electrophoresis, a suction pump 20 described later, and detection. It is a control calculation part comprised by the board | substrate which controls the part 15 and calculates the detected data. Reference numeral 9 denotes a power supply box, and 9a denotes a power supply unit provided in the power supply box 9. Since this gene diagnostic apparatus has optimum applied voltage values that differ depending on the type, concentration, and processing conditions of DNA, the most suitable electrophoresis can be performed for separating abnormal DNA and normal DNA from a DNA sample B described later. The control calculation unit 8 controls the power supply unit 9a to apply a voltage. 10 is adjusted to a predetermined temperature for separating normal DNA and abnormal DNA, and a separation unit for separating the DNA into three DNAs of normal DNA, abnormal DNA, and other noise DNA in this portion, and 13 is a separation unit 10. It is a temperature controller that adjusts the temperature of the.
[0022]
Details in the separation unit 10 will be briefly described. In order to fill the capillary tube 19, at the start of measurement, as shown in FIG. 5, first, a linear polymer 18d and a buffer solution 18c containing a DNA binding control agent are introduced into the capillary tube 19, and then DNA for separation is introduced. Add conjugate A. At this time, it may be introduced by mixing with the linear polymer 18d, or the linear polymer 18d may be introduced after the DNA conjugate A for separation is introduced. In the case of FIG. 5, it is introduced in a separated state after introduction. Subsequently, a DNA sample A for separation and a DNA sample B diluted with a buffer solution 18c in a separated state are introduced and electrophoresed.
[0023]
In this reference example, since acrylamide and DNA are polymerized to prepare the separation DNA conjugate A, the difference in weight and structure from the DNA is large, and the migration speed of the separation DNA conjugate A (0.6 cm). / Min to 0.7 cm / min) is about 1/20 to 1/30 of the optimal migration speed of DNA (13 cm / min to 20 cm / min), and the movement is very slow and is relatively pseudo. A state that may be said to be fixed is realized. By performing the arrangement and voltage control, concentration adjustment, temperature control, and the like set in this way, the present gene diagnosis apparatus performs normal electrophoresis and abnormal DNA treatment by the action of the DNA conjugate A for separation while performing electrophoresis. Can be separated in several tens of minutes.
[0024]
Reference numeral 15 denotes a detection unit that measures the passing amount of the DNA sample to be electrophoresed. 15a is D ₂ lamp, 15b photodiode array, 15c preamplifier, 15d is A / D converter. Some of the capillary tube 19 to expose the glass portion is irradiated with ultraviolet rays having a wavelength of 260nm from D ₂ lamp 15a. The ultraviolet irradiation light obtained at this time is detected by the photodiode array 15b, and the absorbance is measured. The control arithmetic unit 8 controls the power unit 9a is emitting a D ₂ lamp 15a, a photodiode array detected current is amplified by a preamplifier 15c at 15b, A / D converter 15d control arithmetic unit 8 to the absorbance as a digital value in It is converted by. The control calculation unit 8 has a built-in timer (not shown), and can measure the elapsed time from the migration start time. The earlier the time, the longer the elapsed time, the noise DNA, the middle the elapsed time is abnormal DNA, and the shorter the elapsed time is normal DNA.
[0025]
Reference numeral 16 denotes an electrode that applies a positive potential during electrophoresis (second electrode of the present invention), and 17 denotes an electrode that applies a negative potential during electrophoresis (first electrode of the present invention). Controls the power supply unit 9a, applies a voltage between the electrode 16 and the electrode 17, and performs electrophoresis. In FIG. 3, 18 is a buffer solution for carrying charge during electrophoresis and stabilizing the pH of the DNA sample, 18 a is a cathode side container (first container of the present invention) containing the buffer solution 18, and 18 b is It is a container (second container of the present invention) on the anode side that accommodates the buffer solution 18. 19 is a capillary tube for electrophoresis of the DNA sample B during electrophoresis (sealed flow path of the present invention), and 20 is a suction pump for injecting a reagent such as a DNA sample or linear polymer into the capillary tube 19. is there. And, 21a denotes a current detector of the reference example for monitoring a current value changes to the presence of air bubbles in the buffer 18 (bubble detecting section of the present embodiment), 22 was detected that there is a bubble It is a notification unit for an alarm or the like that rings.
[0026]
As shown in FIG. 4, the electrode 17 is immersed in the buffer solution 18 of the container 18a, and the electrode 16 is immersed in the buffer solution 18 of the container 18b. The buffer 18 in the container 18a and the container 18b is communicated with the buffer in the capillary tube 19. Further, the buffer solution contains a linear polymer 18d and a DNA binding control agent, and the buffer solution 18 contained in the containers 18a and 18b or the capillary tube 19 is Tris-Borate (pH 7.2 to pH 8). About) It is appropriate to use a buffer or the like. Among these, the DNA binding control agent mixed in the buffer accommodated in the capillary tube 19 includes a binding promoter such as magnesium chloride for promoting the binding of DNA to the DNA conjugate for separation, and a releasing agent such as urea for promoting the separation. There are two types. These two types of DNA binding control agents can control various migration speeds for DNA by selecting two types of mixing ratios and material substances (for example, other electrolytes as binding promoters). .
[0027]
When a voltage is applied between the electrode 16 and the electrode 17, electrophoresis is induced in the capillary tube 19, and the separation DNA conjugate A and the DNA sample B are negatively charged. Therefore, the separation from the container 18a to the container 18b is performed. DNA conjugate A and DNA sample B move, but if air bubbles are present inside capillary tube 19 at this time, the conductor in capillary tube 19 is insulated, and electrophoresis becomes impossible. Therefore, it is necessary to prevent bubbles from being mixed into the capillary tube 19, but if bubbles are mistakenly mixed, it is necessary to detect as soon as possible, interrupt electrophoresis, and wash the capillary tube 19. is there. In order to detect this, the gene diagnosis apparatus of the reference example detects the current value flowing out from the electrode 17 during electrophoresis by the current detection unit 21a and monitors it by the control calculation unit 8, and the current value is a predetermined threshold value 0.01 μA. In the following cases, it is detected that bubbles are generated in the capillary tube 19.
[0028]
Next, the operation of the gene diagnostic apparatus of this reference example will be described. First, a capillary tube 19 into which a DNA sample or each solution has been introduced is set in the genetic diagnosis apparatus, and the buffer solution 18 in the containers 18a and 18b is immersed at both ends as shown in FIGS. A predetermined voltage is applied between the electrode 16 and the electrode 17 by the power supply unit 9 a in the control calculation unit 8. Since the DNA in the capillary tube 19 is negatively charged and proceeds to the anode side, the power supply unit 9a needs to apply the electrode 16 so as to have a positive potential and the electrode 17 have a negative potential. Electrophoresis is started by application of a predetermined voltage from the power supply unit 9a. However, when the control calculation unit 8 has a current value of 0.01 μA or less after electrophoresis, the genetic diagnosis apparatus of this reference example While controlling the entire system so as to stop the migration, the notifying unit 22 can notify the measurer of abnormality due to bubbles or the like.
[0029]
(Embodiment 1 )
Next, the genetic diagnostic apparatus according to Embodiment 1 of the present invention will be described. The genetic diagnosis apparatus of the first embodiment uses a photodiode array as a bubble detection unit. Figure 6 is a control circuit main portion view of genetic diagnosis apparatus in the first embodiment of the present invention, FIG. 7 is a graph showing the relationship between the bubbles and the absorbance of the genetic diagnosis apparatus according to the first embodiment of the present invention. Since the genetic diagnostic apparatus of Embodiment 1 has basically the same configuration as the genetic diagnostic apparatus of the reference example , the same reference numerals are given to the same members, and the description thereof is omitted.
[0030]
In FIG. 6, reference numeral 15b denotes a photodiode array for detecting bubbles, which is a bubble detector in the genetic diagnosis apparatus of the first embodiment.
[0031]
The bubble detection unit of the first embodiment measures ultraviolet absorbances of at least two wavelengths, preferably 200 nm and 260 nm, at an initial position where the liquid is injected into the capillary tube 19, and the two absorbances are below a predetermined threshold. When they are simultaneously inserted, it is detected that bubbles are generated in the capillary tube 19. Detection accuracy can be increased when measuring absorbance of three wavelengths or more. Note that the ultraviolet absorbance of two or more wavelengths can be seen by separating the wavelengths from one D ₂ lamp 15a using the photodiode array 15b.
[0032]
FIG. 7 is a relationship diagram between the elapsed time and the absorbance when the elapsed time from the start of reagent and sample filling is plotted on the horizontal axis and the absorbance is plotted on the vertical axis. The solid line indicates the absorbance at 200 nm, and the broken line indicates the absorbance at 260 nm. In FIG. 6, the portion where the absorbance decreased at both 200 nm and 260 nm is a portion indicating that bubbles are mixed in the capillary tube 19. In the first embodiment, as shown in FIG. 6, the glass portion is exposed at the initial stage of sample injection of the capillary tube 19, and ultraviolet rays including two or more wavelengths from the D ₂ lamp 15a, 200 nm and 260 nm in the first embodiment. The ultraviolet ray irradiation light obtained at this time is separated and detected by the photodiode array 15b, and the absorbance of each is measured. The control arithmetic unit 8 controls the power unit 9a is emitting a D ₂ lamp 15a, a photodiode array detected current is amplified by a preamplifier 15c at 15b, A / D converter 15d control arithmetic unit 8 to the absorbance as a digital value in It is converted by. The control calculation unit 8 monitors two wavelengths, and controls the entire system to stop electrophoresis when the absorbance falls from a predetermined threshold value for both wavelengths. The measurer can be notified.
[0033]
As described above, when air bubbles are mixed in the capillary tube 19 of the genetic diagnosis apparatus according to the first embodiment, the air bubbles are detected, the electrophoresis is immediately stopped, and the sample in the capillary tube 19 is washed. it can.
[0034]
【The invention's effect】
As described above, according to the present invention, the following advantageous effects can be obtained.
[0035]
Since the genetic diagnosis apparatus of the present invention can notify that bubbles are generated in the closed flow path when electrophoresis a DNA sample, the electrophoresis is immediately stopped without repeating unnecessary electrophoresis. It becomes possible to replace the liquid in the sealed flow path with a liquid without bubbles. The fact that electrophoresis cannot be performed due to bubbles or the like can be quickly detected, and measurement can be performed with high efficiency.
[0036]
By measuring the UV absorbance of at least two wavelengths, it is possible to quickly detect and notify immediately, and without repeating unnecessary electrophoresis, the electrophoresis is immediately stopped and the liquid in the sealed channel is bubbled. It is possible to replace it with a liquid without any water.
[Brief description of the drawings]
FIG. 1 is an external view of a genetic diagnostic apparatus in a reference example for explaining the present invention. FIG. 2 is an external view of the genetic diagnostic apparatus with an open top. FIG. 3 is an apparatus configuration diagram including an electrophoresis unit of the genetic diagnostic apparatus. FIG. 4 is a main part diagram of the control circuit of the genetic diagnosis apparatus. FIG. 5 is an introduction state diagram of the DNA conjugate for separation and the DNA sample in the closed flow path of the genetic diagnosis apparatus. FIG. 7 is a main part diagram of the control circuit of the genetic diagnosis apparatus according to the first embodiment. FIG. 7 is a diagram showing the relationship between bubbles and absorbance of the genetic diagnosis apparatus according to the first embodiment of the present invention.
1 Power switch 2 operation button 3 display panel 4 upper lid 5 housing 6 electrophoresis unit 7 detecting unit 15a D ₂ support table 8 control arithmetic unit 9 power box 9a power unit 10 separation unit 13 temperature controller 15 lamp 15b photodiode array 15c Preamplifier 15d A / D converter 16, 17 Electrode 18, 18c Buffer 18a, 18b Container 18d Linear polymer 19 Capillary tube 20 Suction pump 21a Current detection unit 22 Notification unit A DNA conjugate for separation B DNA sample

Claims (1)

A first container in which a buffer solution is contained and the first electrode is immersed; a second container in which a buffer solution is contained and the second electrode is immersed; and a linear polymer and DNA bond between the first container and the second container A sealed channel that is filled with a buffer containing a control agent and communicated, and a detection unit that is provided in the sealed channel and detects a passing amount of a DNA sample passing through the interior; A DNA conjugate for separation in which a base sequence capable of hydrogen bonding to a DNA sample and a polymer compound are bound in a buffer solution, and the DNA sample is separated into normal DNA and abnormal DNA from the difference in binding force due to hydrogen bonding ; the DNA sample and are filled, the DNA sample of the closed flow path by applying a predetermined voltage is electrophoresis, gene diagnosis device the detecting unit measures each passage of normal DNA and abnormal DNA In Te, the said closed passage, the bubble detecting section is provided for detecting the air bubbles, which the bubble detecting section detects by measuring the bubble at least 2 or more wavelengths of the ultraviolet absorbance of the closed flow path genetic diagnosis and wherein the at.

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