1.  Field of the Invention
The present invention relates to a PCR amplification reaction apparatus and a method for PCR amplification reaction using the apparatus.  Particularly, the present invention relates to a PCR amplification reaction apparatus that can be used for analyzing a genetic polymorphism, a single nucleotide genetic polymorphism (SNP) or the like, to a method for PCR amplification reaction using the apparatus, and to a method for detecting a genetic polymorphism, an SNP or the like to which the apparatus and/or the method are applied. 
2.  Related Background Art
Conventionally, PCR (polymerase chain reaction) has been used as typical means for amplifying a specific region of a nucleic acid chain.  PCR comprises adding a DNA polymerase enzyme and each nucleic acid as a substrate to a region to be amplified of a template nucleic acid chain, for example, a double-strand DNA using a corresponding DNA fragment called a PCR primer corresponding to the partial nucleotide sequences of both terminals of the region, and repeating a thermal cycle consisting of denaturing, annealing and extension to amplify only the DNA strand of a target region.  A one-cycle process consisting of, for example, denaturing at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and extension at 72° C. for one minute as the thermal cycle for PCR reaction is repeated 30 times in total.  Under these conditions, the time required for the thermal cycles as a whole is about 1.5 hours.  When amplifying a nucleic acid chain in which a region to be amplified has a larger length of nucleotides, the time interval for the extension step carrying out reaction of extending a DNA strand from the 3′-end of a primer is set longer.  Accordingly, it is fairly general that the total time required for the thermal cycles amounts to about 1.5 to 2.5 hours. 
In order to reduce as much as possible the time for such a thermal cycle process requiring a long period of time, several contrivances have been proposed.  For example, Light Cycler (Roche Diagnostics), an apparatus in which amplification reaction is completed within about 20 to 30 minutes has been commercially available in recent years.  However, the time reduction technique used in this commercially available apparatus is a technique of applying temperature controlled hot air to a capillary vessel simply in order to reduce the time spent for temperature change.  Specifically, the technique reduces the time spent for temperature change based on the finding that use of a capillary vessel can reduce the volume of the reaction vessel and a capillary vessel has a small heat capacity.  The temperature controlling technique itself is, in principle, the same as a conventional technique.  In addition, air used as a heating and cooling medium in this apparatus has a small density and a small thermal conductivity and is one of the least heat conductive media.  Taking these points into consideration, it cannot be said that air is not an optimal temperature controlling system. 
On the other hand, the greatest advantage of PCR is that the method can selectively amplify a nucleic acid chain having a specific nucleotide sequence in a sample containing various nucleic acid chains collected from the living body.  In analysis of genetic polymorphisms, SNPs or the like, only the nucleic acid chain which indicates a difference of nucleotide sequences to be analyzed must be selectively amplified in a sample containing various nucleic acid chains prior to the analysis.  In a process for preparing this DNA sample for analysis, PCR amplification has been widely used with the above-described selectivity against a nucleotide sequence. 
Conventionally, there has been no technique for analyzing gene polymorphisms, SNPs or the like that is specifically limited based on nucleotide sequences.  For analysis of various genetic polymorphisms, SNPs or the like, used is an oligonucleotide fragment for a detection probe which corresponds to a partial nucleotide sequence that is known to exhibit a difference based on technological accumulation in the past.  In a technology similar to a microarray technology, about 100 kinds of bead arrays of Luminex Corp. or electrode arrays of Nanogen, Inc. are used in order to achieve a hybridization reaction time remarkably shorter as compared with a conventional microarray technology. 
Other than the microarray technology using hybridization reaction with a detection probe, several analysis technologies have been known.  An RFLP (Restriction Fragment Length polymorphism) technology comprises effecting a restriction enzyme that can cleave a partial nucleotide sequence indicating a difference oh a selectively amplified double-strand DNA, and judging whether or not the corresponding partial nucleotide sequence exists based on the difference in electrophoresis patterns among a plurality of DNA fragments produced.  Direct nucleotide sequence determination comprises PCR amplifying using a Dye terminator for a DNA sequencer with a nucleic acid chain containing genetic polymorphisms, SNPs or the like as a template and directly determining the nucleotide sequence with a DNA sequencer to judge whether or not the partial nucleotide sequence indicating a difference exists. 
In the RFLP technology, a restriction enzyme that can cleave a partial nucleotide sequence indicating a difference between detectable genetic polymorphisms is required to be selected and used.  Accordingly, the technology has a methodologically limited range of application, for example, can be applied only to a case where a difference is indicated by a partial nucleotide sequence corresponding to the site of an available restriction enzyme.  On the other hand, there are no limitations, in principle, to the application range for the direct nucleotide sequence determination, because a DNA sequencer is actually used to determine the nucleotide sequence.  However, when a template contains a plurality of similar sequences, it is difficult to analyze a single gene, and a problem of ambiguity found in some HLA typings occurs.  In addition, the time required for pretreatment of a sample used for DNA sequencing, the running cost for a DNA sequencer apparatus, and the electrophoresis time of the technology are disadvantageous for working efficiency and economical efficiency.  In particular, the number of genetic polymorphisms that can be analyzed at the same time is limited according to the number of capillaries in a DNA sequencer apparatus.  On the other hand, when the number of capillaries is smaller than that of samples to be analyzed, the apparatus suffers from reduced operation efficiency.  Taking these limitations into consideration, it is hard to say that direct nucleotide sequence determination is not an optimal technology. 
On the other hand, the technology with a microarray can analyze a plurality of probes ranging from several thousand dots to ten thousand dots in one time, advantageously.  In contrast, a nucleic acid chain to be detected by each probe must be labeled such as fluorescently labeled.  Such a pretreatment of a sample requires several hours as in the case of direct nucleotide sequence determination, and such a pretreatment must be carried out for each objective probe, which leads to a complicated work.  Moreover, the probe hybridization reaction on a microarray is carried out as a solid phase reaction and requires several hours.  Furthermore, if the length of the DNA strand of a sample is too much larger than the length of probes involved in hybridization, crossreaction with other probes may be induced.  It is true that the technology with a bead array or electrode array is means for significantly improving the probe hybridization reaction time.  However, it is difficult to prepare a large-scale probe array like a conventional microarray, and only about 100 kinds of probe arrays are available.  For examples, regarding to HLA genetic polymorphisms, two hundred and several ten kinds of alleles have been already confirmed.  The technology with a bead array or electrode array cannot be adequately applied to analysis of such many kinds.