Conventionally, capillary sequencing by the dideoxy method has been often used in order to determine base sequence of nucleic acid (DNA, RNA, etc.) or a fragment thereof (oligonucleotide, nucleotide, etc.). In this method, four normal DNA synthesis systems are prepared, to which chain terminating nucleotide (terminator) of a low concentration is added to cause a reaction therewith. As the terminator, only one type out of four types of dideoxynucleotides (ddATP, ddGTP, ddCTP, and ddTTP) is used. DNA polymerase continues to synthesize DNA while incorporating deoxyribonucleotide corresponding to a template sequence. However, the reaction sometimes stops upon incorporation of the corresponding terminator. As a result, DNA fragments of various lengths corresponding to a base of the terminator are obtained. For example, in a system where ddATP is added as the terminator, a 3′ end base of a resulting DNA fragment is adenine. This allow a sequence to be properly determined only with the four reaction systems without requiring simple DNA synthesis to be performed first. In this method, although a high accuracy is obtained, throughput such as the number of analyses that can be obtained in one performance of analysis is limited.
Currently, a significant improvement has been made in analysis throughput of sequencing. DNA sequencers capable of analyzing DNA or a fragment thereof having a large-scale base sequence of mega or giga order have been implemented (Illumina Inc. and Roche).
In this method, the extracted nucleic acid or a fragment thereof, which is a target of base sequence determination, is required to be fragmentated into base sequence of a substantially uniform size having a predetermined number of bases (e.g. approximately several tens to 1000 b). Furthermore, it is a prerequisite that the fragmentated pieces having a large number of bases are accommodated in a reaction vessel together with various reagents, a primer, DNA polymerase, nucleotide, and reaction buffer or the like with the nucleic acid functioning as a template DNA for performing amplification under controlled temperature.
However, when performing analysis of such a large number of base sequences, the target nucleic acid has to be amplified with a high accuracy. In particular, there may be cases where an erroneous conclusion is led by amplification where nucleic acid other than the target nucleic acid is mixed, or repeating processing many times from the beginning to the amplification after performing the amplification processing. In order to analyze base sequence of target nucleic acid, uniform fragmentation of nucleic acid or a fragment thereof into a predetermined number of base sequences is required for successful analysis of large-scale base sequences.
Therefore, to see whether the target nucleic acid or a fragment thereof having been extracted, fragmentated, and further amplified meets the treatment purpose, a portion is extracted manually from the nucleic acid or fragment thereof having been extracted and fragmentated and labeled with fluorescence or the like to monitor a size of molecular weight by electrophoresis. This is time to consuming as well as posing a risk of cross contamination. Moreover, performing the electrophoresis requires a high degree of technique, thus presently requiring a researcher or technician specialized in nucleic acid. Therefore, a doctor or the like not specialized in this field cannot easily use the technique in clinical application.
The above prevents generalization of genetic analysis or expansion of clinical application in hospitals using a large-scale sequencer. Therefore, it is important to provide accurate pretreatment where cross contamination is prevented and labor of a user is reduced in clinical use while allowing for the amplification process suitable for a large-scale sequencer and performing reliable amplification of nucleic acid with a high accuracy. Furthermore, full automation is required where extraction of nucleic acid to amplification and further to measurement is consistently automated. It is also important to downsize a device and to provide a high-accuracy device at a low cost.