Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-09-17
2002-11-19
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C435S091200, C549S224000, C549S223000, C549S227000, C549S225000
Reexamination Certificate
active
06482938
ABSTRACT:
CONTINUING APPLICATION DATA
This application is a 371 of PCT/JP98/03093, filed Jul. 10, 1998.
1. Technical Field
The present invention relates to compounds having energy transfer function, and DNA sequence determination methods utilizing the compounds as a terminator, a primer or an initiator.
2. Background Art
There have been known methods for determining nucleotide sequences by preparing chain termination reaction products using a DNA polymerase or RNA polymerase, and subjecting them to separation and fractionation. Further, it has been tried to develop quicker nucleotide sequence determination methods in such ongoing projects as for elucidating gene sequences of higher animals. Some of such methods utilize a DNA fluorescence sequencer where sequencing reaction products obtained by using a fluorescence-labeled terminator are separated and fractionated by electrophoresis, the resulting DNA fragments are excited by a laser, and the emitted fluorescence is detected to determine nucleotide sequences.
It has been also known that quantum yield of fluorescent dyes can be increased based on the principle of energy transfer by using two fluorescent dyes (a donor dye and an acceptor dye). Recently, energy transfer primers have been developed which are composed of an oligonucleotide having covalently bonded two fluorescent dyes and used as a primer for sequencing reactions based on the principle of energy transfer [see, for example, Nature Medicine, 2, 246-249 (1996), WO95/21266, Japanese Patent Unexamined Publication (KOKAI) No. Hei 10-88124/1998 etc.].
In order to realize faster DNA sequencing, systems capable of analyzing multiple samples simultaneously have been developed. In particular, the capillary fluorescence sequencer has an ideal structure, in which loading of samples can be easily automated, and multiple capillaries can be easily used without crossing of lanes. For this fluorescence sequencer, two kinds of optical systems have been used in order to realize the use of larger number of capillaries.
One is the scanning method, and the other is the imaging method. For the both cases, it is essential for the use of larger number of capillaries that a highly sensitive detector, which can detect even a small amount of DNA, should be available. In the scanning method, in order to finish the sequencing within a certain period of time regardless of the number of capillaries, time assigned to each capillary should be shorter if the number of capillaries became larger. Therefore, a detector of higher sensitivity is required. In the imaging method, the range of the field covered by a detector is constant regardless of the number of capillaries. In order to use a larger number of capillaries, it is necessary to increase the number of picture elements of optics element, and decrease the diameter of each capillary. Therefore, it also requires a detector having higher sensitivity.
On the other hand, as discussed above, the technique for utilizing the fluorescence primer based on the principle of energy transfer for DNA sequencing has been developed in order to increase DNA detection sensitivity. However, because the technique utilizes the fluorescence primer, it requires complex operation procedure of which termination reaction should be performed for each of the four kinds of bases, A, G, C and T, and the mixed sequencing reaction products should be subjected to electrophoresis. In addition, because the sequencing based on the transcription reaction by a promoter-dependent RNA polymerase does not use a primer, the energy transfer primer cannot be used in it.
In order to realize faster DNA sequencing, it is necessary to use a highly sensitive detection system with high quantum yield such as energy transfer in a multiple capillary sequencer. On the contrary, however, the systems utilizing the energy transfer primers suffers from technical limitations such as complicated pretreatment (DNA polymerase system) and necessity of primer itself (RNA polymerase system).
Therefore, an object of the present invention is to provide a means which eliminates the drawbacks of the systems utilizing the energy transfer primers, i.e., which is adaptable to the transcription reaction systems utilizing RNA polymerases, and enables sequencing methods capable of high sensitivity detection utilizing the energy transfer and not requiring the complicated procedure consisting of mixing of the four kinds of reaction products for A, G, C and T separately obtained in the preliminary processes.
In particular, an object of the present invention is to provide a compound which can utilize the principle of energy transfer to afford high sensitivity, and a method for determining nucleotide sequences of DNA which utilizes such a compound as mentioned above as a terminator, and can detect labeled DNA fragments with high sensitivity based on the chain terminator method.
Meanwhile, the energy transfer primer disclosed in WO95/21266 is composed of two reporters causing the energy transfer which are connected with a part of oligonucleotide constituting the primer used as a linker. However, such a primer is practically disadvantageous, because a distinct primer must be synthesized for each of oligonucleotides used as primers having different sequences. On the other hand, the energy transfer primer of Japanese Patent Unexamined Publication (KOKAI) No. Hei 10-88124/1998 is composed of two reporters connected with an aliphatic or aromatic residue used as a linker. Therefore, it does not suffer from the problem observed in the primer of WO95/21266, but it may suffers from another problem that, when a longer linker is desired to realize a longer distance between the two reporters, it may be difficult to obtain a desired distance between the two reporters because of the bonding scheme of the linker even though the linker has a long chain.
Therefore, another object of the present invention is to provide a compound useful as the energy transfer primer which solves the above problem, i.e., which does not use a part of a primer sequence as the linker and enables easy control of the distance between the two reporters.
A further object of the present invention is to provide a method for determining nucleotide sequences of DNA which uses the above compound as the energy transfer primer.
In a method for determining nucleotide sequences using a terminator, an RNA polymerase such as T7 RNA polymerase is used for a reaction in a mixture of ribonucleoside 5′-triphosphates and 3′-deoxyribonucleotides. In this reaction, ribonucleotides and 3′-deoxyribonucleotides having a base corresponding to the sequence of the template are sequentially incorporated into a ribonucleotide sequence to synthesize a polyribonucleotide sequence. The resulting polyribonucleotides (nucleic acid transcription products) are then separated, and nucleic acid sequence is read from the resulting separated fractions to determine the nucleotide sequence of the DNA. For example, fluorescence-labeled 3′-dNTP derivatives are used as the terminators of the nucleic acid transcription for the nucleotide sequence determination.
However, terminators composed of 3′-dNTP having various kinds of labels may be difficult to be incorporated into a nucleic acid sequence, depending on the kind of the labels and the bonding scheme of the labels. In particular, when the chain length becomes longer, such a tendency becomes more serious. To deal with this problem, the nucleotide sequence may also be determined by using unlabeled compounds as the terminators and labeled initiators. Also in such a case, sensitivity of the label is important like in the labeled terminators mentioned above.
Therefore, a further object of the present invention is to provide a compound having energy transfer function which can be used as an initiator (transcription initiator) in a method for determining nucleotide sequences of DNA using an RNA polymerase without using a labeled terminator for the nucleotide sequence determination.
A still further object of the present invention is to
Hayashizaki Yoshihide
Tanaka Takumi
Burns Doane Swecker & Mathis L.L.P.
Riley Jezia
Wako Pure Chemical Industries Ltd.
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