Abstract:
The present invention relates to apparatus for measuring the genetic sequence of electrically charged biopolymers by means of hybridization. The measuring apparatus comprises a container that contains a biopolymer and can be removed from the apparatus, and electrodes that apply an electric field to the container and are electrically insulated from the container. When an electric field is applied to the container, suspended biopolymeric molecules are attracted toward the positive electrode, thus increasing the speed of hybridization.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to apparatus for measuring the genetic sequence of biopolymers, such as DNA and protein. 
     2. Description of the Prior Art 
     The DNA chip for hybridization described in the published Japanese translation of patent application Ser. No. 512,605 of 1999 is configured in such a manner that a large number of electrodes are formed on a substrate and a current source is connected to each of these electrodes. There are 100 to 10,000 electrodes on the substrate and, in normal applications, a different type of DNA segment is fixed to each electrode. 
     By flowing unknown DNA segments onto the substrate to which known DNA segments are fixed for hybridization as described above, it is possible to have an unknown DNA segment combine with its corresponding DNA segment. If the unknown DNA segment is labeled with a fluorescent reagent, the sequence of the unknown DNA segment that has combined with any known DNA segment can be determined. 
     The prior art cited above will now be described in further detail. As shown in  FIG. 1 , a positive voltage is applied to an electrode  1  to which a known DNA segment indicated by  2  is fixed. Since DNA is negatively charged, an unknown DNA segment indicated by  3  is attracted toward the electrode  1  where the known DNA segment  2  is fixed, as shown in  FIG. 1   b . As a result, hybridization that used to take several hours to finish is completed in only several tens of seconds. 
     The combining force is weak in case known and unknown DNA segments with different sequences wrongly combine with each other, as shown in  FIG. 2   a . Thus means the segment pair can be separated by applying a weak negative voltage to the electrode  1  after hybridization, as shown in  FIG. 2   b . Thus, it is possible to precisely measure even a single-base difference as seen in such DNA samples as SNPs (single nucleotide polymorphisms). 
     Such a DNA chip as described above is housed in a cartridge together with, for example, a fluid system combined with a detection system. 
     This DNA chip has had the following problems, however: 
     1) Cartridges are designed to be disposable, as a rule. On the other hand, the prior art cartridge requires a large number of electrodes and electrical connection terminals to be formed thereon and, therefore, is costly. Furthermore, electrodes and their processing circuits are also required for a reading unit used with the cartridge. Thus, the cost of a total system also is high.
 
2) Since the electrical connections of the DNA chip are made of electrodes, metal surfaces in contact with a solution are liable to cause electrochemical noise or fluctuations. Furthermore, electrical contact with the terminals of the reading unit tends to become defective.
 
3) The DNA chip requires electrodes and their electrical taps to be formed on the cartridge, thereby increasing the size of the cartridge. The reading unit also requires such elements as terminals and voltage supply circuitry.
 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to solve the above-mentioned problems, by providing inexpensive and highly reliable apparatus that uses small containers and measures the genetic sequence of biopolymers. 
     In order to attain the above-described object, according to claim  1  of the present invention, an apparatus is provided for measuring the genetic sequence of electrically charged biopolymers by means of hybridization, wherein the measuring apparatus comprises a container that contains a biopolymer and can be removed from the apparatus and electrodes that apply electric fields to the container and are electrically insulated therefrom. 
     This measuring apparatus configuration provides the following advantageous effects: 
     When electric fields are applied to the container, suspended biopolymeric molecules are attracted toward the positive electrode, thereby increasing the speed of hybridization. The container requires neither electrodes nor electrical connection terminals and is therefore inexpensive. Furthermore, a reading unit used with the container requires no more than one pair of an electrode structure and processing circuitry. Thus, the system as a whole is also inexpensive. Since the container has no electrode structure, electrochemical noise or fluctuations are very unlikely and, therefore, failures due to defective electrical contact with the terminals of the reading unit will never occur. The measuring apparatus requires neither electrodes nor their electrical taps to be formed on the container, thereby reducing the size of the container. The reading unit can also be downsized since the unit requires no more than one pair of an electrode structure and voltage source circuitry. 
     According to a feature of the present invention, the above-mentioned measuring apparatus may be provided with means for altering the direction of an electric field applied through electrodes, so that wrongly hybridized segment pairs are easily separated. 
     According to another feature of the present invention, the container may be formed using a film. If the container is made of a film, sites and electrodes can be easily brought close to each other, thus, the electric field can be positioned with higher precision and the cost of the container can be reduced. 
     According to a further feature of the present invention, the electrodes may be provided with protrusions formed at spatial positions corresponding to sites where biopolymeric molecules within the container gather. This configuration is advantageous since electric field strength can be made intensive at specific locations. 
     According to another feature of the present invention, conductive members may be formed at positions corresponding to sites where biopolymeric molecules within the container gather. 
     According to a further feature of the present invention, the electrodes may be placed in mechanical contact with the container. 
     According to another feature of the present invention, the electrodes may be of transparent type. 
     According to a further feature of the present invention, the transparent electrodes may be formed using an ITO film. 
     According to another feature of the present invention, the biopolymer may be either DNA, RNA, PNA or electrically charged protein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic view explaining how DNA segments are attracted toward an electrode. 
         FIG. 2  is a schematic view explaining how combined DNA segments are separated once again. 
         FIG. 3  is a schematic view showing one embodiment of the measuring apparatus according to the present invention. 
         FIG. 4  is a schematic view explaining how DNA segments are observed through an objective lens. 
         FIG. 5  is a schematic view showing another embodiment of the measuring apparatus according to the present invention. 
         FIG. 6  is a schematic view showing yet another embodiment of the measuring apparatus according to the present invention. 
         FIG. 7  is a schematic view showing yet another embodiment of the measuring apparatus according to the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Preferred embodiments will now be described in detail with reference to the accompanying drawings.  FIG. 3  is a schematic view showing one embodiment of the measuring apparatus according to the present invention. In  FIG. 3   a , a numeral  11  indicates a container which is made of an insulator and into which a solution containing DNA is injected (corresponds to a conventional cartridge and, therefore, may also be referred to as a cartridge), and numerals  12  and  13  denote positive and negative electrodes, respectively. The electrodes  12  and  13  are arranged so as to sandwich the container. Note that the cartridge  11  and the electrodes  12  and  13  are supported by means of known mechanisms. However, the mechanisms are excluded from the explanation of the embodiment. 
     A numeral  14  indicates a voltage source for generating voltage to be applied to the electrodes  12  and  13 . 
     Hermetically sealed within the cartridge  11  is a solution containing known and unknown DNA segments, wherein the known DNA segments are fixed onto the inner wall (bottom side in the figure) of the cartridge  11 . 
     When a voltage is supplied by the voltage source  14  to the electrodes  12  and  13 , an unknown DNA in suspension is attracted toward the positive electrode  12 , as shown in  FIG. 3   b , since the unknown DNA segment is negatively charged. Thus, the unknown DNA segment approaches toward a known DNA segment. As a result, the speed of hybridization is increased. 
     By reversing the electrode polarity after hybridization, it is possible to separate wrongly hybridized DNA segment pairs. Consequently, it is possible to precisely measure even a one-base difference as seen in SNPs. 
     It is possible to label DNA segments with a fluorescent reagent and observe the segments in the same way as practiced in the prior art. Preferably, the electrodes  12  and  13  are configured so that the electrodes can be removed from the cartridge  11  after hybridization. Removing the electrodes  12  and  13  from the cartridge  11  after hybridization will make it easy to observe the cartridge  11  through an objective lens  21 , as shown in  FIG. 4 . 
     It should be noted that the present invention is by no means limited to the above-described embodiment. The invention may be embodied in other ways without departing from the spirit and essential characteristics thereof. Accordingly, it should be understood that all modifications falling within the spirit and scope of the present invention are covered by the appended claims. 
     For example, the positive electrode  12  may be alternatively configured in such a manner that a protrusion  121  extending toward the cartridge  11  is formed on the electrode at a position corresponding to each site (also referred to as a “spot”) of DNA, as shown in  FIG. 5 . By employing this electrode configuration, it is possible to make electric field strength intensive at the site. 
     Alternatively, a conductive member (conductor)  111  may be formed at the position of each site on the inner wall of the cartridge  11 , as shown in FIG.  6 . In this case, a known DNA segment is previously fixed onto the topside of the conductor  111 . 
     Alternatively, the present invention may be carried out by combining the embodiments of  FIGS. 5 and 6 . 
     Alternatively, the electrodes  12  and  13  may be placed in contact with the cartridge  11  if the cartridge  11  is formed using plastic or any other insulator. This configuration is possible since the only requirement in this case is that the electrodes  12  and  13  be electrically isolated from the cartridge  11 . Mechanical contact is advantageous in stabilizing the positional relationship between the electrodes and the cartridge. 
     Alternatively, transparent electrodes like those made of an ITO (Indium Tin Oxide) film may be used as the electrodes  12  and  13 , as shown in  FIG. 7 . Use of transparent electrodes is advantageous since fluorescence observation is still practicable without having to move the electrodes. 
     Note that fluorescence may be measured with the internal solution drained out of the cartridge  11  and the cartridge dried out. 
     Alternatively,the cartridge  11  may be made of a thin film. This configuration reduces the distance between sites and the electrode. Consequently, the configuration is advantageous in the case of the embodiments of  FIGS. 5 and 6  since the positional accuracy of an electric field can be improved. 
     Although the case where sites are made of DNA has been explained, the present invention is not limited to this case. Alternatively, the sites may be made of RNA, PNA (peptide nucleic acid), or electrically charged protein. 
     As described heretofore, the present invention has the following advantageous effects: 
     1) There is no need for forming electrodes and electrical connection terminals on a cartridge, thus reducing the cost thereof. Furthermore, a reading unit used with the cartridge requires no more than one pair of an electrode structure and processing circuitry. Thus, the system as a whole is also inexpensive.
 
2) Since the container has no electrode structure, electrochemical noise or fluctuations are very unlikely. Furthermore, electrical contact with the terminals of the reading unit never becomes defective.
 
3) There is no need for forming electrodes and their electrical taps on the container, thereby reducing the size of the container. The reading unit can also be downsized since the unit requires no more than one pair of an electrode structure and voltage source circuitry.