Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-09
2001-06-05
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024330, C536S025300
Reexamination Certificate
active
06242193
ABSTRACT:
TECHNICAL FIELD
This invention relates to an apparatus for analysis of DNA, RNA and the like. In particular, it relates to an apparatus effective in determination of the base sequences of DNA and RNA or analysis of restriction fragments and specific fragments.
BACKGROUND ART
Techniques for analysis of DNA, RNA and the like have become important in the medical and biological fields including gene analysis and genetic diagnosis. Both the determination of the base sequences of DNA or RNA and the analysis of restriction fragments or specific fragments are based on separation carried out on the basis of molecular weight by electrophoresis. In this case, a fragment or a group of fragments is previously labeled with a radioactive or fluorescent label, and a development pattern of separation on the basis of molecular weight is measured after or during the electrophoresis of the labeled fragment or fragments, whereby the fragment or fragments are analyzed. The need for, in particular, a DNA sequencing apparatus has recently increased in relation to genome analysis, so that the development of the apparatus is in progress. DNA sequencing using a fluorophore label is explained below. Dideoxy reaction according to the Sanger method is carried out before electrophoretic separation. An oligonucleotide having a length of 20 bases which is complementary to the known base sequence portion of a sample DNA to be analyzed is synthesized and then labeled with a fluorophore. This oligonucleotide is complementarily bonded as a primer to about 10
−12
mol of the sample DNA, and the elongation reaction of a complementary strand is carried out with polymerase. In this case, as substrates, there are added four deoxynucleotide triphosphates, i.e., deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), as well as dideoxyadenosine triphosphate (ddATP). When ddATP is incorporated by the elongation of the complementary strand, the complementary strand is not further elongated, so that fragments of various lengths terminated by adenine (A) are prepared. The same reaction as above is independently carried out except for adding each of dideoxycytidine triphosphate (ddCTP), dideoxyguanosine triphosphate (ddGTP) and dideoxythymidine triphosphate (ddTTP) in place of ddATP. The primers used in these reactions, respectively, have the same base sequence but are labeled with four kinds of fluorophores, respectively, which can be distinguished from one another by fluorescence separation into spectral components.
The 4 kinds of the reaction products thus obtained are mixed to prepare fragments which are complementary to the sample DNA, have a length up to about 1000 bases, differ in length by steps of 1 base, and have any of the 4 kinds of the fluorophore labels, depending on the kind of the terminal base. For each of the fluorophore labels, the amount of fragments having each length (number of bases) is about 10
−15
mol. Then, the samples prepared are separated by electrophoresis with a resolving power of 1 base. In the electrophoresis, there is widely used a slab gel obtained by polymerizing acrylamide between two glass plates about 0.3 mm apart from each other. When the samples are placed at the upper end of the slab gel and an electric field is applied to the upper and lower ends of the slab gel, the samples migrate toward the lower end while being separated. When a position about 30 cm below the upper end is irradiated with laser beams while carrying out the electrophoresis, the fluorophore-labeled fragments separated pass the laser irradiation position to be subjected to excitation, in the order of increasing length. By measuring the emitted fluorescence while separating it into spectral components with a plurality of filters, the kinds of terminal bases of all the fragments can be determined in the order of increasing fragment length, from the change with time of the fluorescence intensity of the four kinds of the fluorophores. Since the thus determined order of the bases is complementary to that of the sample DNA, the base sequence of the sample DNA can be determined.
There have been proposed several novel DNA sequencing methods using no electrophoresis. In a first prior art, in carrying out the elongation reaction of a complementary strand by using polymerase and a sample DNA as template, the 4 kinds of the substrates are added one by one, and the amount of the substrates incorporated into the complementary strand is determined at each stage by utilizing light absorption or fluorescence, to determine the base sequence of the sample DNA (JP-A-4-505251). In a second prior art, the elongation reaction of a complementary strand is carried out by using polymerase, a sample DNA as template and the 4 kinds of the substrates labeled with different labels, respectively, after which bases are released one by one from the 3′-end of the thus synthesized complementary strand with exonuclease, and the labels of the released bases, respectively, are measured in turn to determine the base sequence of the sample DNA (Journal of Biomolecular Structure & Dynamics 7, 301-309 (1989)). In a third prior art, the base sequence of a sample DNA is determined by repeating a cycle consisting of a step of carrying out DNA polymerase reaction by using four dNTP derivatives (MdNTPs) which have a detectable label and can be incorporated into a template DNA as substrates for DNA polymerase to stop DNA strand elongation reaction, owing to the presence of their protecting group, a step of detecting the incorporated MdNTP, and a step of returning this MdNTP to its original state at which the elongation is possible. In this prior art, the DNA strand elongation is stopped every time the DNA strand is elongated by one base, and the enzyme and the substrates are removed from a system (a solution) containing the template, a primer and the MdNTPs, after which the MdNTP incorporated is detected, and the protecting group (and the label) of the MdNTP incorporated into the template are removed to return this MdNTP to its original state at which the DNA strand elongation is possible (Japanese Patent Application No. 2-57978). These proposals, however, set forth mere ideas at present and no practical application thereof has been reported.
DISCLOSURE OF THE INVENTION
In DNA sequencing methods which have been put to practical use, separation on the basis of molecular weight is carried out by electrophoresis. For DNA sequencing, the resolving power for the separation on the basis of molecular weight should be a length of one base. Usually, the resolving power of the electrophoresis decreases with an increase of the length (number of bases) of DNA fragments to be separated. That is, even if a DNA fragment having a length of 50 bases and that having a length of 51 bases can be separated from each other on the basis of the length difference of one base, a DNA fragment having a length of 500 bases and that having a length of 501 bases cannot always be separated from each other on the basis of the length difference of one base. The length (number of bases) at separation limit which indicates the limit of the length (number of bases) within which a resolving power of one base can be attained, is determined by the electrophoresis conditions, i.e., the composition of a medium for separation, the length of electrophoresis lanes and the intensity of electric field. Various optimizations have been carried out for increasing the length (number of bases) at separation limit, but there is no report that the limit exceeds 1000 bases (Electrophoresis 13, 495-499 (1992) and Electrophoresis 13, 616-619 (1992)). There has been theoretically explained the fact that the length (number of bases) at separation limit is at most 1000 bases (Electrophoresis 13, 574-582 (1992)). That is, when electrophoresis is employed, the length of a base sequence which can be determined from one kind of sample DNA is at most 1000 bases. On the other hand, in large-scale base sequence determinatio
Anazawa Takashi
Kambara Hideki
Okano Kazunori
Uematsu Chihiro
Hitachi , Ltd.
Mattingly Stanger & Malur, P.C.
Riley Jezia
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