Abstract:
Method and apparatus for constructing a cDNA library by hybridizing mRNA with oligo (dT) on a support and treating with a reverse transcriptase to immobilized complementary DNA, or for constructing a gDNA library by ligating a double-stranded chromosomal DNA library with an oligonucleotide on a support having a restriction enzyme site and then immobilizing the gDNA library.

Description:
REFERENCE TO RELATED APPLICATIONS 
     The present application is the national stage under 35 U.S.C. 371 of international application PCT/JP00/03000, filed May 10, 2000, which designated the United States, and which application was not published in the English language. 
     FIELD OF THE INVENTION 
     The present invention relates to molecular biology and biochemical gene technology, protein technology, and cell technology. More particularly, the present invention relates to a method for construction of a plurality of supports on which a DNA library is immobilized using a micro amount of DNA test material, a method for constructing the supports, and supports on which DNA is immobilized. 
     BACKGROUND OF THE INVENTION 
     DNA is a very important test material. In conventional methods for working with DNA, DNA is amplified using a Polymerase Chain Reaction (hereinafter, PCR) and divided to small groups. The DNA test material is preserved at remarkably low temperatures in a freezer. Conventionally, a DNA library is produced in a solution so that a replica of the DNA library cannot be produced. Accordingly, one must work very carefully with a DNA library in solution obtained from tissue or cells in order to search and identify the genes therein. 
     One purpose of the present invention is to provide a method for constructing a DNA library support, i.e., an original support, on which A DNA library is immobilized using a micro amount of the DNA library test material. Another purpose of the present invention is to provide a method for constructing the necessary number of replica supports. Furthermore, another purpose of the present invention is to provide a support on which a replica of DNA is immobilized. 
     SUMMARY OF THE INVENTION 
     The method for constructing a cDNA (complementary DNA) library according to the present invention comprises, hybridizing oligo (dT) n  on a support, and thereafter messenger RNA (hereinafter, mRNA), and reacting it with reverse transcriptase (hereinafter, RT) in order to immobilize complementary DNA. 
     In a method for constructing a cDNA library according to the present invention, mRNA is dehybridized from a cDNA library immobilized on a support. The method comprises immobilizing the same cDNA library by using dehybridized mRNA. 
     In a method for constructing a genomic DNA (hereinafter, gDNA) library according to the present invention, a double stranded gDNA is ligated to an oligo nucleotide on a support with restrictive enzyme portion. 
     A single stranded gDNA library according to the present invention is produced by ligating double stranded gDNA with respect to an oligonucleotide on a support having a restrictive enzyme portion which ligates the double stranded gDNA. 
     Another method for a single stranded gDNA library according to the present invention comprises dehybridizing an anti-sense portion of the gDNA library obtained above, and synthetically immobilizing a sense portion of the gDNA on a support using the anti-sense portion. 
     In any of the processes according to the present invention the support has been previously chemically modified with a nucleotide or an oligo nucleotide. 
     The substrate of the present invention comprises a support on which a DNA library is immobilized by any one of the methods described above. In another embodiment of the present invention, a single stranded DNA library is immobilized. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic view of a device for constructing a cDNA library support according to the present invention. 
         FIG. 2  is a schematic view of a device for constructing a gDNA library according to the present invention. 
         FIG. 3  is a flow chart for explaining a process for constructing a cDNA library support according to the present invention. 
         FIG. 4  is a schematic view for explaining a process for constructing a cDNA library support according to the present invention. 
         FIG. 5  is a flow chart for explaining a process of constructing a gDNA library support according to the present invention. 
         FIG. 6  is a schematic view for showing a process for constructing a gDNA library support according to the present invention. 
         FIG. 7  is an enlarged view of support f. 
         FIG. 8  is an enlarged view of support e. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A chemically modified original support which has been chemically modified with respect only to a nucleotide or oligonucleotide and a plurality of supports for producing replicas are prepared so as to produce an original support and replica supports. The supports are introduced into a replica constructing device. 
     A device for constructing a DNA library support according to the present invention will be explained with reference to the drawings.  FIG. 1  is a schematic view of a device for constructing a cDNA library support.  FIG. 2  is a schematic view of a device for constructing a cDNA library support.  FIG. 3  is a flow chart explaining a process of constructing a cDNA library support.  FIG. 5  is a flow chart for explaining a gDNA library support. 
       FIG. 1  is a schematic view of a device for automatically constructing and duplicating a cDNA library support. A device for constructing a DNA library support as shown in  FIG. 1  comprises liquid feeding means  105  for feeding reaction solution to a container, a liquid feeding switch means  220  for stopping the flow of reaction solution and feeding new reaction solution, nozzle driving means  100  for driving a nozzle  101  for inleting/outleting test material in a front-rear and right-left direction in a plane and an upper-lower direction, solution temperature controlling means  30  for heating or cooling the reaction solution in the container, test material container holding means  20  for holding containers into which each test material or solution is set for constructing respective immobilized DNA library supports, and test material control means for maintaining the test material container holding means at a predetermined temperature, etc. It is preferable that the container holding means  10  hold  96  or more test material containers to be connected to a PCR device and/or a PCR analysis device for subsequent use. It is preferable that the test material container holding means comprise at least four test material container inserting holes for replica supports. It is preferable that the number of container inserting holes provided at the test material container holding means  20  be ten holes or more, for subsequent connection to the PCR device and/or the PCR product analysis device. It is preferable that the container holding means  10  and the test material container holding means  20  be an aluminum block with good conductivity for thermally controlling the solution temperature controlling means  30  and the test material container temperature control means  40 . 
     It is preferable that a support for construction a DNA library support be a plate shape, a ball shape, a cube shape or a grain shape in the both the original support and replica supports. Although the material of which the support is made is not specified, it is preferable that the material not react with reaction solution or that the material does not deposit harmful material when exposed to a DNA immobilization reaction. For example, plastic, glass, silicon, and metals are preferred materials. A plate shape, a ball shape, a cube shape and on the like are preferred. Particularly, a support made of diamond or carbon atoms including diamond is preferred. 
     Production of Original Support and its Replica Support with cDNA Library 
     Referring to  FIGS. 1 and 3 , a process for constructing an original support and replica supports on which a cDNA library is immobilized and its will be explained. First, a necessary number (T 1 –Tn) of supports of 3 mm×3 mm×0.1 mm chemically modified with respect only to (dT)n, are prepared before a DNA library is immobilized on these supports. A supports modified with oligo (dT)n and is used because it is easy to hybridize mRNA in total RNA that has been chemically modified. These supports are inserted into containers CB 1  to CBn and the containers are set into the container holding means  10 . In such a case, it is preferable to insert one support into a first container in view of constructing an original support as an immobilized cDNA library and its replica support using a micro amount of mRNA obtained from a small amount of test material. Regarding an order of setting containers CB 1 –CBn into which a chemically modified support is inserted at the container holding means, the container CB 1  an original support in which a chemically modified is set is inserted into a first inserting portion HT 1 . The necessary number of containers (CB 2 –CBn) for replica supports into which the corresponding number of chemically modified supports (T 2 –Tn) is set, respectively, are inserted into a second inserting hole HT 2 , . . . to the n th  inserting hole HTn in order. 
     Production of Original Support 
     Reaction solution  203 , which includes purified total RNA solution  201 , RT enzyme solution  202  and nucleotide (dT, dA, dG, dC) is set into the test material container holding means  20  controlled at a predetermined temperature (i.e., 4° C.). Tris ethylene diamine tetraacetic acid (hereinafter, TE) solution  204  for cleaning/eluting DNA (buffer solution including TE) and a waste solution tank  210  and others are provided. A capillary tube  230  is provided as a liquid feeding path by connection to the liquid feeding switch means  220  for feeding the respective solution. It is preferred that the capillary tube  230  be a corrosion resistant stainless tube for liquid chromatography. It is preferred that the connection between the test material inlet nozzle  101  and the liquid feeding switch means  220  through the liquid feeding means  105  be a silicone tube  231 . Then the test material inlet nozzle  101  is moved to a position of the hole HT 1  in the container holding means  10  so as to insert the nozzle  101  into the container CB 1  in which the first support (T 1 ) is set. The liquid feeding switch means  220  is provided at a side of the reaction solution, and the reaction solution is fed to the container CB 1  by driving the liquid feeding means  105 . The liquid feeding switch means  220  is shifted to the total RNA solution  201  and the predetermined amount of the solution  201  is inleted by the liquid feeding means  105 . After passing a predetermined time (for example, 20 to 30 minutes) at a temperature equal to or lower than the predetermined temperature (for example 20° C.), the liquid feeding switch means is shifted to RT enzyme solution (enzyme  1 )  202  so as to introduce a predetermined amount of the RT solution  202  by driving the liquid feeding means  105 . After the test material inlet nozzle  101  is removed from the container CB 1 , the temperature of the container holding means  10  is set at the predetermined temperature (for example, 42° C.), and the RT enzyme reaction for constructing cDNA from mRNA proceeds for a predetermined time (for example 30 to 60 minutes). After the temperature of the container holding means  10  is set at temperature equal to or lower than the predetermined temperature (for example, 20° C.), the liquid feeding switch means  220  is shifted to the waste liquid tank  210  so as to discharge reaction solution in container CB 1  to the waste liquid tank  210  by driving the liquid feeding means  105 . The liquid feeding switch means  220  is shifted to the TE solution  204  so as to introduce a predetermined amount of the TE solution  204  into the container CE 1  by driving the liquid feeding means  105 . The container holding means  10  is heated to a predetermined temperature (for example, 90° C.) by driving the solution temperature control means  30 , at which temperature mRNA is hybridized. Then, the liquid feeding switch means  220  is shifted to container  206  for temporarily preserving mRNA, and dehybridized mRNA solution is moved to container  206  for temporarily preserving mRNA by driving the liquid feeding means  105 . 
     Production of Replica Supports 
     Next, will be described a method for constructing replica supports by re-using dehybridized mRNA from the original cDNA library support produced by the above—described method. First, after removing the test material inlet/outlet nozzle  101  from container CB 1 , the nozzle  101  is moved to container CB 2  containing a replica support (T 2 ), temporarily preserved, inlet to the container CB 2  by reversely driving the liquid feeding means  105 . Then, steps described above for the production of the original support are repeated. However, the step for introducing the Total RNA solution  201  can be omitted. The liquid feeding switch means  220  is provided at a side of the reaction solution  203  of container CB 2 . The reaction solution is introduced into container CB 2  by driving the liquid feeding means  105 . After container CB 2  has been maintained for a predetermined time (for example, 20 to 30 minutes) at a temperature equal to or lower than a predetermined temperature (for example, 20° C.), the liquid feeding switch means  220  is shifted to the RT enzyme solution (enzyme  1 )  202  so as to introduced a predetermined amount of the solution by driving the liquid feeding means  105 . The test material inlet/outlet nozzle is removed  101  from the container CB 2 , and the temperature of the container holding means  10  is maintained at a predetermined temperature (for example, about 42° C.) for a predetermined time (for example, 30 to 60 minutes). The a temperature of the container holding means  10  is set a temperature equal or lower than a room temperature (20° C.), and the liquid feeding switch means  220  is shifted to the waste liquid tank  210 . The test material inlet/outlet nozzle  101  is inserted into container CB 2 , reaction solution in the container CB 2  is discharged to the waste solution tank  210  by driving the liquid feeding means  105 . The liquid feeding switch means  220  is shifted to the TE solution  204  so as to introduce a predetermined amount of the TE solution  204  into container CB 2  by driving the liquid feeding means  105 . Then, the liquid feeding switch means  220  is shifted to the waste liquid tank  210  so as to discharge the TE solution in container CB 2  into the waste liquid tank. By repeating the above process several times (preferably at least 5 times), a first replica support is produced which is a duplicate of the original cDNA library support. The necessary numbers of replica supports are produced by repeating the cyclic operation for constructing the replica support the required number of times. 
     The device for constructing a DNA library support as shown in  FIG. 2  comprises a liquid feeding means  105  for liquid feeding reaction solution etc. to a container, a liquid feeding switch means  220  for switching the liquid feeding of the reaction solution, a nozzle, a nozzle driving means  100  for driving a test material inlet/outlet nozzle  101  in a front-rear direction and left-right direction in a plane and an upper-lower direction, a container holding means  10  for holding a container in which a support is set, a container solution temperature control means  30  for heating or cooling the reaction liquid in the container, a test material container holding means  20  for holding containers in which test materials and test solutions for duplicating an immobilized DNA library support are set, respectively, and a test material container temperature control means  40  for controlling the test material container holding means at a predetermined temperature. It is preferred that at least 96 material test containers can be inserted into the container holding means  10  in view of connecting to a PCR device and/or a PCR product analysis device for subsequent operations. Preferably, the test material container holding means  20  contains at least four holes for a test material container in order to produce replica supports. It is preferred that the number of the test material holes provided at the test material container holding means  20  be at least 10 for subsequent connection to a PCR device and/or a PCR product analysis device. It is preferred that the container holding means  10  and the test material container holding means  20  be made of aluminum with good thermal conductivity in order to thermally control container liquid temperature control means  36  with Peltier element. 
     Production of Original Support of gDNA Library and its Replica Supports 
     Referring to  FIGS. 2 and 5 , the production of an original support and replica supports on which immobilized a gDNA library will be explained. The necessary number (T 1 ˜Tn) of supports (for example, 3 mm×3 mm×0.1 mm) of supports chemically modified with oligonucleotide sense portion having a restrictive enzyme portion are prepared. With respect to the original support, T 1 , oligo nucleotide (anti-sense portion) is hybridized and treated with restrictive enzyme so as to prepare a complete restrictive enzyme portion. The original support T 1  is set in a container CB 1 . Replica supports T 2 –Tn are chemically modified with oligigonucleotide (sense portion) having restrictive enzyme portion are set in containers CB 2 –CBn. These containers are set in the solution holding means  10 . With respect to a setting order, the container CB 1  in which an original support T 1  is set is inserted into a first inserting hole HT 1  initially, and a second container and successive containers CB 2 –CBn, in which each replica support is set, is inserted in order. Reaction solution  303 , including purified gDNA library solution  306  treated with restrictive enzyme, DNA ligase solution (enzyme  1 )  305 , DNA Polymerase solution (enzyme  2 )  302  and nucleotide (dT, dA, dG, dC)  303  are set into a test material solution holding means  20  in which the temperature is maintained at a predetermined temperature (24° C.). TE solution for cleaning/eluting DNA  304  and a waste liquid tank  310  are provided. A capillary tube  330  for the respective solutions is connected to a liquid feeding switch means  220 . It is preferred that the capillary tube  230  be a corrosion-resistant stainless tube for liquid chromatography. 
     The liquid feeding switch means  220  and the test material inlet/outlet nozzle  101  and others are connected to the front end of the capillary tube  230  through the liquid feeding means  105 . A silicone tube  231  is preferred for this connection. A test material inlet/outlet nozzle  101  is moved to a location of the hole HT 1  of the container holding means  10  so as to insert the nozzle  101  into the container CB 1  in which the first support, T 1 , is set. The liquid feeding switch means  220  is shifted to the reaction solution  303  so as to inlet a predetermined amount (for example, 17 μL) of the reaction solution  303  by driving the liquid feeding means  105 . The liquid feeding switch means  220  is shifted to the gDNA library solution  306  treated with restrictive enzyme so as to introduce a predetermined amount (for example, 2 μL) of the solution  306  by driving the liquid feeding means  105 . After maintaining the container CB 1  at a temperature equal to or lower than a predetermined temperature (for example, 20° C.) for a predetermined time (for example, 20 to 30 minutes), the liquid feeding switch means  220  is shifted to DNA ligase solution (enzyme  1 )  305  so as to introduce a predetermined amount (for example, 1 μL) of the solution  305  into the container CB 1  by driving the liquid feeding means  105 . After removing the test material inlet/outlet nozzle  101  from container CB 1 , the temperature of the container holding means is maintained at a predetermined temperature (for example, 37° C.). After maintaining container CB 1  at this temperature for a predetermined time (for example, 30 to 60 minutes), the gDNA library immobilized with DNA ligase is produced on the support T 1 . After maintaining the temperature of the container holding means  10  at a predetermined temperature (for example, equal to or less than 20° C.), the liquid feeding switch means  220  is shifted to the waste liquid tank  310  so as to insert the test material inlet/outlet nozzle  101  into container CB 1  and discharge the reaction solution in container CB 1  by driving the liquid feeding means  105 . The liquid feeding switch  220  is shifted to the TE solution  304 , a predetermined amount (for example, 500 μL) of the TE solution is introduced into container CB 1  by driving the liquid feeding means  105 . Then, the liquid feeding switch means  220  is shifted to the waste liquid tank  310  so as to discharge the TE solution in the container CB 1 . By repeating the process several times (for example, at least five times), the immobilized support T 1  is cleaned. After the immobilized support T 1  has been cleaned, the liquid feeding switch means  220  is shifted to the reaction solution  303  so as to introduce a predetermined amount (for example, 19 μL) of the reaction solution  303  into container CB 1  by driving the liquid feeding means  105 . By maintaining the temperature of the container holding means  10  at a predetermined temperature (for example, 90° C.), and maintaining the container CB 1  at this temperature for a predetermined time (for example, 10 to 20 minutes), anti-sense portion is dehybridized from the immobilized gDNA library. Then, the liquid feeding switch means  220  is shifted to container  306  in which a gDNA library (anti-sense portion) is temporarily preserved so as to remove the gDNA library (anti-sense portion) solution from container CB 1 . This produces the first gDNA library (sense portion) support (original support). 
     Production of Replica Supports 
     The test material inlet/outlet nozzle  101  is removed from container CB 1  and moved to the next container, CB 2 , in which a replica support (T 2 ) is set so as to introduce the gDNA library (anti-sense portion) solution  306  temporarily preserved into container CB 2 . In order to produce a replica support, the above-described cyclic operation is repeated as necessary so as to produce the required number of replica supports. However, in the second embodiment described below, it is noted that DNA polymerase solution (enzyme  2 )  302  is selected during a replica constructing process so as to introduce a predetermined amount (for example, 1 μL) of solution  302  into container CB 2  by driving the liquid feeding means  105 . 
     Embodiments 
     Embodiment 1 
     This embodiment, referring to  FIGS. 1 ,  3 , and  4 , explains production of an original support immobilized a cDNA library, and production of replica supports. The test material is prepared by (1) breaking cells and tissues and purifying total RNA (see Step S 1  in  FIG. 3 ). For pre-treating a support immobilized with a cDNA library, a piece of rat liver tissue of about 5 mm×5 mm is homogenized in a test material kit (for example, ISOGEN sold by K.K. Nippon Gene) and the total RNA is purified in accordance with its protocol. Ten supports (T 1 ˜T 1 O) of 3 mm×3 mm×0.1 mm are chemically modified with oligo (dT) n  (wherein n is from 15 to 30), as shown in Step  2  in  FIG. 3 . The supports T 1 –T 10 , immobilized with an amino group on their surfaces, are treated with activating dihydric carbonic solution. After being cleaned with ethanol and distilled water in that order, the supports are maintained in the oligo (dT)n solution for ten minuets. Each support is inserted into respective containers CB 1 ˜CB 10 , individually. The containers are set into the temperature controlling aluminum block  10 . Reaction solution  203  including purified total RNA solution  201 , RT enzyme solution  202  and nucleotide (dT, dA, dG, dC) are set into a low temperature test material aluminum block  20  in which the temperature is maintained at 4° C. TE solution (buffer liquid including tris-ethylene diamine tetraacetic acid) for cleaning DNA and the waste liquid tank  210  are provided at an exterior side of the low temperature test material aluminum block  20 . The total RNA solution  201 , the RT enzyme solution  202 , the reaction solution  203 , the TE solution  204 , and the waste liquid tank  210  are connected to an automatic 8-way switching valve  220  through capillary tubes  230 , respectively. A test material inlet/outlet capillary needle  101  is moved to the location of the inlet hole HT 1  of the temperature control container aluminum block  10  so as to insert the capillary needle  101  into container CB 1  in which the first support T 1  (original support) is set. The automatic 8-way switching valve  220  is shifted to reaction solution  203  so as to introduce 17 μL of the reaction solution into container CB 1  by driving a peristaltic pump  105 . The automatic 8-way switching valve  220  is shifted to the total RNA solution  201 , and 2 μL of solution  201  is pumped by the peristaltic pump  105 . In order to hybridize oligo (dT) immobilized on a surface of the support and mRNA in the total RNA solution, the solutions are maintained at 20° C. for 20 minutes (see step S 3  in  FIGS. 3 and 4   a ). After this time, the automatic 8-ways switching valve  220  is shifted to RT enzyme solution  202  1 μL of the RT enzyme solution  202  by means of the peristaltic pump  105 . After the test material inlet/outlet capillary needle  101  is removed from the container CB 1 , the container holding means  10  is maintained at 42° C. for 30 minutes so as to produce a cDNA library immobilized on support T 1 ) by the RT enzyme (see a step S 4  in  FIGS. 3 and 4   b ). After the container holding means is cooled to 10–20° C., the automatic 8-ways switching valve  220  is shifted to the waste liquid tank  210  to insert the test material inlet/outlet capillary needle  101  into container CB 1 . The reaction solution in container CB 1  is discharged to the waste liquid tank  210  by driving the peristaltic pump  105 . The automatic 8-way switching valve  220  is shifted to the TE solution  204 . Five hundred μL of TE solution  204  is introduced into container CB 1  by driving the peristaltic pump  105 . Then the automatic 8-way switching valve  220  is shifted to the waste liquid tank  210  so as to discharge the TE solution in the container CB 1  to the waste solution tank  210 . By repeating this operation several times (at least five times), immobilized support T 1  is cleaned (see step S 5  in  FIG. 3 ). After immobilized support T 1  is cleaned, the automatic 8-way switching valve  220  is shifted to the reaction solution  203 , 19 μL of reaction  203  is introduced into container CB 1  by activating the peristaltic pump  105 . Container holding means  10  is heated to 90° C., mRNA is dehybridized from the immobilized cDNA library after holding the support at this temperature for ten minutes (see step S 6  in  FIG. 3  and  FIG. 4   d ). Next, the automatic 8-way switching valve  220  is shifted to container  206  in which mRNA is temporarily preserved, dehybridized mRNA solution is eluted and removed from container CB 1  and temporarily preserved in container  206  (see a step S 7  in  FIG. 3 ). In accordance with the above steps, the first cDNA library support, the original support on which a cDNA library is immobilized, is produced (see step S 8  in  FIGS. 3 and 4   c ). After the test material inlet/outlet capillary needle  101  has been removed from container CB 1 , capillary needle  101  is moved to container CB 2  in which a replica support, T 2 , is set. Replica support (T 2 ) has been previously chemically modified. The automatic 8-way switching valve  220  is shifted to container  206  for temporarily preserving mRNA, and 19 μL of temporarily preserved mRNA is introduced to container CB 2  by driving peristaltic pump  104  (see arrow R as shown in  FIGS. 4   d  to  4   a ). In order to hybridize immobilized oligo (dT) and mRNA, container CB 2  is maintained at 20° C. for 20 minutes. The automatic 8-way switching valve  220  is shifted to the RT enzyme solution  202  and 1 μL of RT enzyme solution  202  is introduce into container CB 2  by driving the peristaltic pump  105  (see step S 9  in  FIG. 3 ). After the test material inlet/outlet capillary needle  101  has been removed from container CB 2 , the temperature of the container holding means  10  is controlled at 42° C. for 30 minutes so as to produce a cDNA library support immobilized on support T 2  by RT enzyme (see a step S 10  in  FIGS. 3 and 4   b ). After the container holding means is cooled to 10 to 20° C., the automatic 8-way switching valve  220  is shifted to the waste liquid tank  210 . The test material inlet/outlet capillary needle  101  is inserted into container CB 2  so as to discharge the reaction solution in container CB 2  to the waste liquid tank  210  by driving the peristaltic pump  105 . The automatic 8-way switching valve  220  is shifted to the TE solution  204 , 500 μL of TE solution  204  is introduced into container CB 2  by driving the peristaltic pump  105 . Then, the automatic 8-way switching valve  220  is shifted to the waste liquid tank  210  so as to discharge the TE solution in container CB 2 . By repeating the above operation five times, immobilized support T 2  is cleaned (see step S 11  in  FIG. 3 ). After immobilized support T 2  is cleaned, the automatic 8-way switching valve  220  is shifted to reaction solution  203 . Then 19 μL of reaction  203  is introduced into container CB 2  by driving the peristaltic pump  105 . In the next step, the container holding means  10  is heated to 90° C. and maintained at that temperature for 10 minutes so as to dehybridize mRNA from the immobilized cDNA library see step S 12  in  FIG. 3 ). In the next step, the automatic 8-way switching valve  220  is shifted to the container  206  for temporarily preserving mRNA, dehybridized mRNA solution is separated and eluted from container CB 2  by driving the peristaltic pump  105  (see  FIG. 4   d ) and temporarily preserved in container  206  (see step S 13  in  FIG. 3 ). In accordance with the above steps, a replica cDNA library support (replica support) is produced (see step S 14  in  FIGS. 3 and 4  ( c ). 
     With respect to the containers CB 3 –CB 10  in which supports T 3 –T 1 O are inserted, respectively, a similar process is conducted. By repeating the above steps S 9  through S 14  in order, eight replica supports are produced. By utilizing supports T 1 –T 1 O immobilized with a cDNA library of rat liver tissue, a gene is amplified by a PCR device with respect to 18S rRNA. AN electrophoresis device is used to confirm that the cDNA Library is immobilized. Thus, it can be confirmed that the original support T 1  and the replica supports T 2 –T 1 O are produced as normal cDNA library supports. 
     Embodiment 2 
     Production of an original support on which is immobilized with a gDNA library and its replica supports are explained with reference to  FIGS. 2 ,  5 , 6 , and  7 . Test materials are prepared by (1) breaking cells and tissues and (2) purifying and treating gDNA with restrictive enzyme (see step S 21  in  FIG. 5 ). Ten supports chemically modified with a sense portion of oligo nucleotide a having base sequence of a target restrictive enzyme portion are prepared. The size of the support is about 3 mm×3 mm×0.1 mm (see step S 22  in  FIG. 5 ). A concept of the support is shown as support f surrounded with a broken line in  FIG. 6   d .  FIG. 7  is an enlarged view of the portion. By using one chemically modified support, an anti-sense portion of the oligo nucleotide is hybridized. The support is treated with restrictive enzyme so as to produce one support, T 1 , having a complete restrictive enzyme cut portion (see step S 23  in  FIG. 5  and an arrow (1) in  FIGS. 6   d  to  6   a ). A concept of the support is shown as a support e surrounded with a broken line in  FIG. 6   a .  FIG. 8  is an enlarged view of this portion. 
     Support TI is inserted into container CB 1 , and nine supports chemically modified with a sense portion of oligonucleotide having the restrictive enzyme portion are introduced into the container holding means  10 . As shown in  FIG. 2 , the reaction solution  303 , including purified gDNA library solution  306  treated with restrictive enzyme, DNA solution (enzyme  1 )  305 , DNA Polymerase solution (enzyme  2 )  302  and nucleotide (dT, dA, dG, dC) are set into a low temperature test material aluminum block  20  in which the temperature is controlled at 4° C. TE solution  304  for cleaning/eluting DNA and a waste liquid tank  310  are provided at an exterior side of the low temperature test material aluminum block  20 . As shown in  FIG. 2 , reaction solution  303  including the GDNA (genomic DNA) library solution  306 , the DNA Ligase solution (enzyme  1 )  305 , the DNA polymerase solution (enzyme solution  2 )  302  and nucleotide (dT, dA, dG, dC), TE solution  304  for cleaning/eluting DNA and the waste liquid  310  are connected to an automatic 8-way switching valve  220  through a capillary tube  230 , respectively. The test material inlet/outlet capillary needle  101  is moved to a location of the hole HT 1  of the temperature control container aluminum block  10  so as to insert the capillary needle  101  into the container CB 1  in which the first support T 1  (original support) is set. The automatic 8-way switching valve  220  is shifted to the reaction solution  303 , and 17 μL of reaction solution  303  is introduce into container CB 1  by driving the peristaltic pump  105 . The automatic 8-way switching valve  220  is shifted to the gDNA library solution  306  treated with restrictive enzyme, 2 μL of this solution is pumped by the peristaltic pump. After the has been maintained at 20° C. for 20 minutes, the automatic 8-way switching valve  220  is shifted to DNA ligase solution (enzyme  1 )  305  so as to introduce 1 μL of solution  305  to the container CB 1  by driving the peristaltic pump  105 . After removing the test material inlet/outlet capillary needle  101  from the container CBI, the temperature of the container holding means  10  is maintained at 37° C. for 30 minutes so as to produce the gDNA library immobilized by DNA ligase on the support T 1  (see step S 25  in  FIG. 5  and an arrow  2  in  FIGS. 6   a  and  6   b ). After the container holding means is cooled to 10 to 20° C., the automatic 8-way switching valve  220  is shifted to the waste liquid tank  310  so as to discharge the reaction solution in the CB 1  by driving the peristaltic pump  105 . The automatic 8-way switching valve  220  is shifted to the TE solution  304  and 500 μL of TE solution is introduced into container CB 1  by driving the peristaltic pump  105 . By repeating these steps five times or more, support T 1  is cleaned (see step S 26  in  FIG. 5 ). After cleansing the support T 1 , the automatic 8-way switching valve  220  is shifted to the reaction solution  303 . Nineteen μL of reaction solution is introduced into container CB 1  by driving the peristaltic pump  105 . The temperature control container aluminum block  10  is heated to 90° C. and maintained for 10 minutes, an anti-sense portion is dehybridized from a double stranded sense portion and the anti-sense portion of the immobilized DNA library (see step S 27  in  FIG. 5  and arrow ( 3 ) in  FIGS. 6   b  and  6   c ). The automatic 8-way switching valve  220  is shifted to a container  306  for temporary preservation so as to elute the anti-sense portion of the gDNA library solution from container CB 1  by driving the peristaltic pump  105  (see step S 29  in  FIG. 5  and arrow in  FIGS. 6   b  and  6   d ). The sense portion is only immobilized on support T 1 . Thus, the first support, that is, single stranded gDNA library support TI, original support, is produced (see step S 28  in  FIGS. 5 and 6   c . After removing the test material inlet/outlet capillary needle  101  from the container CB 1 , the capillary needle  101  is moved to container CB 2  which holds the support T 2 . The reaction solution including nucleotide is added to container CB 2 , which is maintained at 20° C. The gDNA library solution  306  including only anti-sense portion temporarily preserved is introduced into container CB 2  and maintained for 20 minutes (see step S 30  in  FIG. 5 ). In the next step, DNA polymerase is added, container CB 2  is heated to 37° C. and maintained at his temperature for one hour. As a result, a double stranded gDNA library, of which a sense portion is immobilized on the support T 2 , is produced (see step S 31  and arrow ( 4 ) in  FIGS. 6   d  and  6   b ). Container CB 2  in which the support T 2  is immobilized with the above double stranded gDNA library is maintained at 20° C. After shifting the automatic 8-way switching valve  220  to the waste liquid tank  310 , the test material inlet/outlet capillary needle  101  is introduced into container CB 2 . The reaction solution in container CB 2  is discharged by driving the peristaltic pump  105 . The automatic 8-way switching valve  220  is then shifted to the waste liquid tank  310 , and the TE solution in the container CB 2  is discharged. By repeating the above steps at least five times, support T 2  is cleaned (see a step S 32  in  FIG. 5 ). After support T 2  has been cleaned, the automatic 8-way switching valve  220  is shifted to reaction solution to  303  to introduce 19 μL of this reaction solution into container CB 2  by driving the peristaltic pump  105 . The aluminum block  10  is heated to 90° C. and maintained at that temperature for ten minutes to dehybridize the anti-sense portion from immobilized gDNA library with double stranded sense portion and the anti-sense portion (see step S 33  in  FIG. 5  and arrow ( 5 ) in  FIGS. 6   b  to  6   c ). The automatic 8-way switching valve  220  is shifted to a container for temporarily preservation so as to outlet the anti-sense portion of gDNA library solution from the container CB 2  (see step S 35  in  FIG. 5  and an arrow in  FIGS. 6   b  to  6   d . A second single stranded gDNA library support, that is, a replica support T 2  on which a sense portion is immobilized, is produced (see step S 34  in  FIGS. 5 and 6   c ). By repeating the above steps, a double stranded gDNA library is immobilized on a support. An anti-sense portion is dehybridized from the double stranded immobilized gDNA library so as to produce the remaining number (T 3 –T 1 O) of single stranded gDNA library supports (replica supports). That is, the process including steps as shown in  FIGS. 6   b  and  6   d  is repeated so that any number of supports on which the same single stranded gDNA library is immobilized can be produced. 
     The present invention produces a support immobilized with cDNA library from mRNA and a gDNA library treated with a restrictive enzyme of gDNA. Although it is impossible to produce replica supports duplicated from DNA library solution in the conventional art, the required number (until mRNA and gDNA are chemically or physically denatured) of replica supports can be produced by the method of the present invention. An immobilized DNA library support and its replica supports can be produced by collecting micro amounts of gene material from cultured cells or tissues of an important object to be detected. Various kinds of gene research and detection can be effected using the same kinds of test materials. By using the immobilized DNA library support and its replica supports, developing a new gene diagnosis technology can be more cheaply and easily than by using conventional techniques. If blood is collected from a patient or tissue is collected in a medical procedure at one time for gene diagnosis, the collected blood or tissue can be easily re-used with respect to preventive medical research, since a plurality of immobilized DNA library supports can be produced over a long period of time. The process of the present invention makes it possible to perform many diagnoses on a patient from sample, reducing mental and/or economic stress on a patient.