Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2000-03-16
2002-08-06
Marschel, Ardin H. (Department: 1631)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S050000, C435S006120, C435S007100
Reexamination Certificate
active
06428749
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an advanced thermal gradient DNA chip (ATGC), a substrate for ATGC, a method of manufacturing of ATGC, a method and an apparatus for biochemical reaction and a storage medium.
DESCRIPTION OF THE RELATED ART
As a method for determining a base sequence of a nucleic acid, the method for detecting hybridization between a single stranded polynucleotide of interest and a single stranded oligonucleotide probe previously designed, by using the polynucleotide detection chip with the single stranded oligonucleotide probes immobilized on its different areas depending on the type of sequences,are known. Examples of the polynucleotide detection chips include polynucleotide detection chips for diagnosis, where DNAs complementary to specific mutated sequences of interest are arranged (Science, Vol. 270, 467-470, 1995) and those for SBH (Sequencing By Hybridization) method, in which the oligonucleotide probes capable of hybridizing with all the possible base sequences existing in a sample are provided on the chips, for determining the base sequences of the subjects of measurement (J. DNA Sequencing and Mapping, Vol.1, 375-388, 1991).
The thermal stability of hybridization between oligonucleotide probes and the single stranded polynucleotide varies depending on the types of base sequences. The reason for this is as described in the following. The bonding between adenine (A) and thymine (T) or adenine (A) and uracil (U) is of double hydrogen bond per base pair, while the bonding between guanine (G) and cytosine (C) is of triple hydrogen bond per base pair (see FIG.
11
), resulting in some differences in bonding strength between these two types of bondings. Since the G—C bond is greater in strength than the A—T bond (see FIG.
12
A), the thermal stability of the former is higher. Therefore, comparing the thermal stability of hybridization of sequences with equal base length, the thermal stability of hybridization involved by only A—T or A—U bond is lowest, while that involved by only G—C bond is highest. In general, the thermal stability of hybridization is represented by the temperature (melting temperature, hereinafter referred to as Tm) at which both bonding and dissociation exist at rate of 50% respectively (FIG.
12
B).
Taking an example of the oligoucleotide DNA probe of octamer, the Tm of the duplex DNA which consist of the A-T bondings, is 15.2° C. (a value calculated by the % GC method (Breslauer K. J., et. al., “Predicting DNA Duplex stability from the base sequence”, Proc., Natl. Acad. Sci., USA83, 3746-3750), while the Tm of the duplex DNA which consist of the G—C bondings, is 56.2° C., giving a difference of 41.0° C. (FIG.
12
C).
As indicated above, when the value of Tm of the hybridization for each probe varies largely, it is necessary to carry out hybridization assay at Tm of each probe, respectively. When a temperature is higher than Tm, a single stranded polynucleotide is hard to bond effectively with a probe. On the other hand, when a temperature is lower than Tm, the background noise resulting from the mismatch bonding increases, leading to the decline of measuring resolution. Thus, in a case where different kinds of probes are immobilized on the polynucleotide detection chip, when the probes are hybridized with the single stranded polynucleotide sample while keeping the temperature constant on the chip, this gives rise to problems such as differences in the amount of the formation of hybridization and differences in mismatching probability occurring due to the difference in thermal stability among individual probes.
Conventionally, in order to resolve the above-described problems, an attempt has been made such as adjusting the salt concentration in solvent or varying the density or the base length of the probes to be immobilized on the detection chip for each probe, while keeping the temperature equal for hybridization for all the probes on the detection chip. Such an attempt, however, is not sufficient for fully eliminating the effect of the difference in Tm.
As an example of the means for resolving this problem, there is Laid Open Japanese Patent No. H11-127900 disclosing a method wherein conductive heating track is provided around each analytical electrode or a method wherein each analytical electrode is heated by means of laser. However, the Laid Open Japanese Patent No. H11-127900 discloses a method characterized by only heating the analytical electrode and no means controlling, for example, the temperature of the analytical electrode to a constant level, are disclosed.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a biochemical reaction detection chip and its substrate capable of controlling the temperature for biochemical reaction including hybridization of the oligonucleotide probe with polynucleotide.
Another object of the present invention is to provide a apparatus and a method for enabling the biochemical reactions in a plurality of reaction systems to progress simultaneously at temperatures controlled for individual reaction systems and an associated data storage medium.
Further another object of the present invention is to provide a substrate of a biochemical reaction detection chip comprising a plurality of islands of a heat conductive material formed on a membrane, the islands being placed apart from each other and each island being provided with a temperature controller.
It is preferable for the membrane to be formed from a material having a high insulating ability, heat insulating ability and physical strength. The electric conductivity of 10
8
&OHgr;·m or more is sufficient for the membrane material, preferably, 10
10
&OHgr;·m or more. The heat conductivity of 10 w/mk or less is sufficient for the membrane material, preferably, 1 w/mk or less.
It is easier to control the temperature of each island by forming the membrane from a material having a high (electrical) insulating ability and a high heat insulating ability. The membrane may be formed, for example, from at least one of a group of materials such as silicon nitride, silicon oxide, aluminum oxide, Ta
2
O
5
, or may be a composite membrane of these materials. Among these, the composite membrane of SiN and SiO
2
is preferable. Since SiN has resistance to alkali, probes can be immobilized on SiN membrane by means of silane coupling in alkali solution. Further, the SiN membrane is capable of protecting the electronic circuit for temperature control provided thereunder from the solution such as sample solution.
The film thickness of 1-500 &mgr;m is sufficient, preferably, 5-20 &mgr;m.
It is preferable to make an indent for the area for fixing the probe of the membrane. Such indent is convenient for holding the sample solution on a chip when letting the biochemical reaction take place by bringing sample solution into contact with the probe.
Further, a resist membrane may be formed on the surface opposite to the islands. The resist membrane may be of a photosensitive polyimide resin or the like.
A plurality of islands of a heat conductor are formed on the membrane. “A plurality of islands” means at least 2 islands, preferably 10-1000 islands, although the number of the islands is not defined. A plurality of islands may be arranged either in line or 2-dimensionally, that is, in a first direction (row) and a second direction (column).
The islands are formed from a heat conductor. Examples of heat conductors include crystals of Si, metals such as Ag, Au, Cu and silicones such as polysilicone and amorphous silicone. The heat conductor constituting the islands is preferable to be electrically insulatable from the temperature controller. Silicone is preferable as a heat conductor to form the islands, since it is a good heat conductor and can be electrically insulated from the temperature controller. The insulation between the heat conductor and the temperature controller can be secured by forming a pn junction in the silicone.
The islands are spaced from each other. The spaces among the islands serve as a substitute for h
Kajiyama Tomoharu
Miyahara Yuji
Murakawa Katsuji
Hitachi , Ltd.
Marschel Ardin H.
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