摘要:
A DNA fragment preparation method for DNA analysis comprising, i) preparing a plurality of DNA fragments from a sample DNA, and ii) amplifying a specific DNA fragment by PCR, using a pair of primers which hybridize with terminus sequences of the DNA fragments, and a specific primer which hybridizes specifically with a base sequence of the specific DNA fragment at a position between a priming site of one of primer the pair of primers and a priming site of another primer of the pair of primers. The specific primer hybridizes specifically with a base sequence at a middle position of the specific DNA fragment. Products of PCR are separated, by electrophoresis, and signals from DNA fragments originated in a known genes and signals from DNA fragments originated in a unknown genes are displayed separately on a display.
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:
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 present invention comprises a method for analysis of a nucleic acid which comprises:(1) a step of digesting a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments;(2) a step of ligating a plurality of oligonucleotides to the double-stranded DNA fragments respectively at the both ends thereof;(3) a step of dispensing a solution containing the double-stranded DNA fragments into a plurality of tubes;(4) a step of adding DNA primers comprising combinations of DNA primers selected from each set of a plurality of DNA primer sets comprising a plurality of labeled primers having a base sequence complementary to the base sequence of oligonucleotide and a part or all of the base sequence contiguous to the base sequence complementary to the base sequence of the oligonucleotide and recognized by the restriction enzymes and a selective base sequence of 1 to 4 bases at the 3'-end thereof, to the respective tubes corresponding to the combinations and performing a complementary strand synthesis reaction of the region of the double-stranded DNA fragments between the base sequences recognized by the two restriction enzymes; and,(5) a step of subjecting the products obtained by the complementary strand synthesis reaction to electrophoresis to produce a large number of DNA fragments from the long double stranded DNA sample digested with restriction enzymes and obtain fingerprinting patterns therefrom which enables to inspect the long double-stranded DNA sample.
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:
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:
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:
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.