摘要:
There are beforehand prepared a monomer having a reaction residue and a polynucleotide probe set comprising plural kinds of polynucleotide probes having a residue bonded to the reaction residue. The monomer is mixed with each kind of polynucleotide probes comprising any plural probes selected from the polynucleotide probe set. Each kind of the resultant mixtures is added to each of different small holes to make the mixture into gel matrix. Thus, a polynucleotide probe chip is produced. Sample DNA is forcibly migrated in the gels by electrophoresis. Laser light is projected onto the side face of the chip. The fluorescence emitted from the whole surface of the chip is collectively detected with a high-sensitive two-dimensional detector. Thus, the polynucleotide probe chip, holding various kinds of DNA probes, for detecting DNA can be provided. This chip has high hybridization-efficiency and makes high-sensitivity and high-speed DNA detection possible.
摘要:
Adenylation of a DNA fragment with a DNA polymerase occurs in the course of PCR, and thus two peaks are detected. To prevent the peak splitting, it is necessary to raise efficiency of adenylation a single peak to occur without changing reaction conditions. To this end, four types of PCR primers which, respectively, have an anchor sequence at 5′ terminus so that any of A, C, G or T is attached to at the 5′ terminus of the anchor sequence, and PCR is carried out by use of the respective primers to determine efficiencies of adenylation. Subsequently, an anchor sequence that is more likely to undergo adenylation is screened to decide an anchor sequence more likely undergo adenylation, followed by PCR by use of a primer having the decided anchor sequence.
摘要:
There are beforehand prepared a monomer having a reaction residue and a polynucleotide probe set comprising plural kinds of polynucleotide probes having a residue bonded to the reaction residue. The monomer is mixed with each kind of polynucleotide probes comprising any plural probes selected from the polynucleotide probe set. Each kind of the resultant mixtures is added to each of different small holes to make the mixture into gel matrix. Thus, a polynucleotide probe chip is produced. Sample DNA is forcibly migrated in the gels by electrophoresis. Laser light is projected onto the side face of the chip. The fluorescence emitted from the whole surface of the chip is collectively detected with a high-sensitive two-dimensional detector. Thus, the polynucleotide probe chip, holding various kinds of DNA probes, for detecting DNA can be provided. This chip has high hybridization-efficiency and makes high-sensitivity and high-speed DNA detection possible.
摘要:
The inventive method for assaying DNA fragments in mixture comprises step 1 of ligating different oligomers hybridizable to primers of the same melting temperature and the same length to individual groups of DNA fragments in a set of DNA fragments; step 2 of mixing together the groups of DNA fragments ligated with the oligomers; step 3 of simultaneous PCR of the groups of DNA fragments ligated with the oligomers in one receptacle by using the primers being complementary to the oligomers and corresponding to the individual groups; and step 4 of detecting PCR amplified DNA fragments; characterized in that the method enables the comparison of plural samples under no influence of PCR reproducibility.
摘要:
A fractionation method for DNA fragments according to the present invention comprises a first step of preparing a probe chip or a set of probe chips immobilizing independently a DNA probe having a first sequence part having a specific known sequence part together with a part of enzyme recognition sequence and a second sequence part composed of a combination of one to six bases adjacent to the first sequence part at 3' terminus, a second step of introducing a DNA oligomer composed of a part of enzyme recognition sequence and a sequence complementary to the known sequence part into the fragment termini of DNA fragments from restriction enzyme cleavage, and a third step of placing the probe chip or the set of probe chips in a solution containing the nucleotide fragments with the introduced DNA oligomer produced at the second step, for at least hybridization and the complementary strand extension of the DNA probe, whereby the DNA fragments are fractionated.
摘要:
A single molecule of single-stranded sample DNA (7) having a bead (5) at one end and a magnetic bead (6) at the other end is extended and fixed in the field of view of a fluorescent microscope by using a magnetic force (11) and a laser trap (3), and a primer (8) is bonded thereto, followed by elongation reaction (10) using polymerase. Only a single chemically modified nucleotide (9) labeled with at least one fluorophore which varies depending on the kind of the base is incorporated. Only the single fluorophore incorporated is measured as a fluorescence-microscopic image by evanescent irradiation (13) with exciting laser beams, and the kind of the base is determined from the kind of the fluorophore. The fluorophore labeling the nucleotide incorporated is released by evanescent irradiation (13) with ultraviolet laser beams (2), and the next nucleotide is incorporated. DNA sequencing is carried out by repeating the above procedure. The base sequence determination can be carried out by using the single DNA molecule, so that a DNA base sequence of hundreds kilos or more bases can be efficiently determined.
摘要:
A single molecule of single-stranded sample DNA (7) having a bead (5) at one end and a magnetic bead (6) at the other end is extended and fixed in the field of view of a fluorescent microscope by using a magnetic force (11) and a laser trap (3), and a primer (8) is bonded thereto, followed by elongation reaction (10) using polymerase. Only a single chemically modified nucleotide (9) labeled with at least one fluorophore which varies depending on the kind of the base is incorporated. Only the single fluorophore incorporated is measured as a fluorescence-microscopic image by evanescent irradiation (13) with exciting laser beams, and the kind of the base is determined from the kind of the fluorophore. The fluorophore labeling the nucleotide incorporated is released by evanescent irradiation (13) with ultraviolet laser beams (2), and the next nucleotide is incorporated. DNA sequencing is carried out by repeating the above procedure. The base sequence determination can be carried out by using the single DNA molecule, so that a DNA base sequence of hundreds kilos or more bases can be efficiently determined.
摘要:
A method of analysis or assay for nucleotides comprises: (1) a step of digesting DNA with a restriction enzyme; (2) a step of discriminating a difference in sequences of the DNA fragments obtained in step (1) above around the 3' termini thereof with a DNA probe and extending the DNA probe by a complementary strand synthesis to fractionate the DNA fragments into groups; and, (3) a step of measuring lengths of the DNA fragments which belong to said groups, or length of the DNA probe extended by said complementary strand extension reaction; wherein the thus measured lengths obtained for every sequence of the bases of the DNA fragments around the 3' termini thereof are employed as fingerprints.
摘要:
Focusing that abundant class genes abundantly present in a nucleic acid sample can comparatively easily be analyzed and readily be removed in a selective manner, a nucleic acid sample for expression analysis of rare expressed genes can be obtained by removing abundant genes therefrom, and analyses based upon the sample can be achieved.
摘要:
A cell sorting device using an inexpensive chip capable of being exchanged for each sample. The chip includes: a first flow path allowing buffer fluid containing cells to flow down; second and third flow paths which put the first flow path therebetween and allow buffer fluid not containing cells to flow down; a fourth flow path which allows the buffer fluid as a single flow path formed by joining the buffer fluids in the other three flow paths; a cell detecting region for detecting cells flowing with the buffer fluid down the fourth flow path; and a cell sorting region for sorting the cells according to a type of the cells detected. The first to fourth flow paths are cascaded, are supplied with the buffer fluid from reservoirs with the same fluid level, and have substantially the same width or cross-section area.