Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-23
2002-10-15
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C422S050000, C422S068100
Reexamination Certificate
active
06465190
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing a DNA chip (DNA microarray) in which several thousands to not less than ten thousands kinds of different types of DNA fragments are aligned and fixed as spots at a high density on a base plate such as a microscopic slide glass.
2. Description of the Related Art
The method for analyzing the genetic structure has been remarkably progressed in recent years. A large number of genetic structures represented by those of human gene have been clarified. The analysis of the genetic structure as described above uses a DNA chip (DNA microarray) in which several thousands to not less than ten thousands kinds of different types of DNA fragments are aligned and fixed as spots on a base plate such as a microscopic slide glass.
Those widely used as a method for forming the spots during the production of the DNA chip are based on a system such as the QUILL system, the pin & ring system, and the spring pin system in which a sample solution containing DNA fragments is supplied (stamped) onto the base plate by using a so-called pin. Even when any one of the foregoing methods is adopted, it is necessary to suppress the dispersion of the volume and the shape of each of the spots to be low so that the distance between the respective spots is maintained to be constant.
On the other hand, in order to realize a higher density, it is also greatly expected to develop a new method in which the shape control performance is satisfactory for the spot, and the productivity is excellent.
As shown in
FIG. 16
, when a spot is formed by dripping a sample solution onto a base plate
200
, the spot is hemispherical in accordance with the surface tension. In this procedure, a substantial amount of the sample immobilized on the base plate
200
resides in a slight portion
204
contacting with the base plate
200
. The amount is merely a part of the whole (spherical matter). The remaining portion
206
is not immobilized, and hence it is washed away during the washing step performed thereafter. As a result, a problem arises that a large amount of the sample solution is lost, and the efficiency of the use of the sample solution is low.
The cost for the production of the DNA chip is substantially determined by the amount of the sample solution. In the case of the procedure described above, almost all of the sample solution is washed away, and the procedure is disadvantageous in view of the production efficiency.
Several thousands to not less than ten thousands kinds of different types of sample solutions are dripped onto one base plate. However, the viscosity and the surface tension differ for each of the different types of the sample solutions. Therefore, in order to obtain an identical spot diameter, it is necessary to change the dripping amount of the sample solution depending on, for example, the viscosity and the surface tension.
However, in the case of the conventional technique, the sample solution adhered to a pin is allowed to physically make contact with the base plate together with the pin so that the sample solution is dripped. Therefore, the spot is formed on the base plate by means of one time of tripping. As a result, the following problem arises. That is, it is impossible to perform any delicate control of the dripping (control of the dripping amount and the dripping position), and any dispersion occurs in the spot diameter formed on the base plate.
In order to more reliably immobilize the DNA fragment in the sample solution onto the base plate, a method has been also developed, in which an organic or inorganic polymer is mixed in the sample solution to physically hold the DNA fragment in the polymer cross-link. However, in the case of this procedure, the following problem arises. That is, the viscosity of the sample solution is increased, and the sample solution tends to be dried, thickened, and solidified. The pot life of the sample upon the formation of the spot is shortened, and the amount of one time of dripping is increased.
SUMMARY OF THE INVENTION
The present invention has been made taking the foregoing problems into consideration, an object of which is to provide a method for producing a DNA chip, which makes it possible to improve the efficiency of the use of an expensive sample solution, improve the productivity of the DNA chip, and improve the yield.
Another object of the present invention is to provide a method for producing a DNA chip, which makes it possible to control the supply depending on the type of a sample solution supplied onto a base plate, realize a uniform spot diameter formed on the base plate, and improve the reliability and the quality of the DNA chip.
According to the present invention, there is provided a method for producing DNA chip including a large number of. spots of sample solutions arranged on a base plate, the method comprising the step of supplying the sample solutions onto the base plate; wherein the sample solution is supplied a plurality of times to form one of the spots.
Accordingly, it is possible to improve the yield of the DNA chip. In this process, it is preferable that the sample solution is supplied by an ink-jet system.
When the ink-jet system is used, a large number of liquid droplets in a required amount can be supplied onto the base plate at a high speed (to 100 kHz) in a manner to make no contact with the base plate. The supply source is continuously supplied via a pouring port and a cavity connected to a discharge port for discharging the liquid droplets. Therefore, unlike the conventional pin system, it is unnecessary to move the pin to a supply source (sample well) for the sample solution to immerse the pin tip in the sample solution every time the spot is formed. Thus, it is possible to form the spots on a large number of base plates for a short period of time.
It is preferable that the sample solution is obtained by diluting a sample containing a DNA fragment to give a predetermined concentration. In this process, it is preferable that the sample solution is obtained by diluting the sample containing the DNA fragment with water or an aqueous solution containing sodium chloride or an aqueous solution containing a polymer. It is preferable that the sample solution is diluted to give a concentration of such a degree that final desired base pairs per one spot are satisfied, by performing the supply a plurality of times to form one of the spots.
The following advantage is obtained by diluting the concentration of the sample. That is, it is possible to relatively decrease the amount of the expensive DNA fragment in the sample solution adhered or remained in the supply flow passage path at the stage at which the supply of the sample solution onto the base plate comes to an end. The following effect is also obtained. That is, it is possible to avoid the occurrence of any defect which would be otherwise caused such that the solution is dried, thickened, and solidified due to the concentrated sample solution, and the discharge port is clogged to cause defective discharge. A greater advantage is obtained that when the sample solution is supplied onto the base plate, then the sample solution becomes not hemispherical but flat. In this case, almost all of the sample solution supplied to the base plate is immobilized on the base plate. Therefore, most of the sample solution is not washed away during the washing step to be performed thereafter. Thus, it is possible to improve the efficiency of the use of the sample solution.
Further, it is possible to realize a uniform spot diameter of the sample solution formed on the base plate, by changing the degree of the dilution depending on the type of the DNA fragment contained in the sample solution so that the viscosity and the surface tension of the sample solution are varied.
Further, it is preferable that the sample solution is diluted with the aqueous solution containing the polymer. Accordingly, the shape-retaining performance is increased for the spot shape after being supplied onto the base plate.
Hirota Toshikazu
Ohnishi Takao
Takahashi Nobuo
Takeuchi Yukihisa
Burr & Brown
NGK Insulators Ltd.
Riley Jezia
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