Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-23
2003-09-30
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C424S070170, C536S023100, C536S024100
Reexamination Certificate
active
06627399
ABSTRACT:
TECHNICAL FIELD
The present invention relates to polyamine compounds which accelerate the transcription reaction of RNA polymerase (hereinafter referred to as RNAP). Further, the present invention relates to a DNA sequencing method using RNAP with the polyamine compounds and an initiator of nucleic acid transcription reaction by RNAP.
BACKGROUND TECHNOLOGY
A DNA sequencing method is one of the most important means in molecular biological field. One of the most useful nucleic acid sequencing method at present is a direct transcription sequencing method (W096/14434) using RNAP such as T7 RNAP and a terminator of RNA transcription reaction (for example, 3′-deoxyribonucleotide-5′-triphosphate, 3′dNTPs). This method is an outstanding method utilizing the RNAP transcription reaction for sequencing nucleic acid sequences of DNA products amplified by polymerase chain reaction without removing primers and 2′-deoxyribonucleoside-5-triphosphates (2′ dNTP's). Recently, it has been suggested that the RNAP transcripton can be improved by the addition of a compound.
Under the circumstances, it is hoped that a compound with RNAP promoting effect will be developed. It has been reported that as a transcription from DNA to mRNA proceeds, a concentration of natural polyamines increases in in vivo system (C. W. Tabor H. Tabor. Ann. Rev. Biochem. 53, 749-790(1984) and J. Marton and D. R. Morris, “Inhibition of Polyamine Metabolism”, (P. P. McCann, A. E. Pegg, and A. Sjoerdsma, eds)). This report suggests that a natural polyamine participates in RNAP transcription process.”
M. Flugier, C. Florentz, M. W. Hosseini, J. M. Lehn and R. Giege, Nucleic Acids Research, 22(14), 2784-2790(1994) disclosed that the transcription activity of T7 RNAP is promoted by linear and cyclic synthetic polyamines. However, it is pointed out that the evaluation method of a transcription promoting ability in the report of M. Flugier et al has some problems. In fact, by using an evaluation method used by the present inventor, it has been found that polyamines described in the report have only extremely low accelerating effects. Therefore, the investigation of products having higher accelerating activity for RNAP transcription activity and an improvement of a sequencing method is highly desired.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel synthetic polyamine compounds with higher and outstanding accelerating activity for RNAP transcription activity than synthetic polyamines described in the state of the art, and to provide a method of DNA sequencing which can determine longer DNA sequences at a time by using the novel polyamine compounds which accelerate RNAP transcription activity.
The present invention relates to a RNA polymerase transcription accelerator comprising a compound represented by the general formula (I) below or a salt thereof.
wherein,
n represents an integer from 1 to 8,
R
1
represents a hydrogen atom or p-toluenesulfonyl group,
R
2
represents an ethyl group or a group represented by the general formula (II),
R
3
represents a hydrogen atom or p-toluenesulfonyl group,
R
4
represents an ethyl group or a group represented by the general formula (II).
wherein,
m represents 1 or 2,
R
5
represents a hydrogen atom,
R
6
represents a hydrogen atom or an ethyl group.
Compounds 5, 10, 15a-g, and 18-23 described in the following
Examples are represented by the above general formula (I). The relation between compounds 5, 10, 15a-g, and18-23, and the general formula (I) are shown in Table 1 below. In the table, No
a)
represents numbers of compounds in schemes below.
TABLE 1
Correlation between compounds 5, 10, 15a-g, and 18-23 and the
general formula (I)
No
a)
R
1
R
2
R
3
R
4
R
5
R
6
N
m
5
H
formula (II)
H
formula (II)
H
Et
2
1
10
H
Et
H
formula (II)
H
Et
1
2
15
H
formula (II)
H
formula (II)
H
ET
a: 2
2
b: 3
c: 4
d: 5
e: 6
f: 7
g: 8
18
H
Et
H
formula (II)
H
H
1
2
19
H
formula (II)
H
formula (II)
H
H
6
2
20
Ts
Et
Ts
Et
—
—
6
—
21
H
Et
H
Et
—
—
6
—
22
Ts
Et
Ts
Et
—
—
3
—
23
H
Et
H
Et
—
—
3
—
H represents a hydrogen atom, and Et represents an ethyl group in the table.
Salts may be either inorganic or organic acid salts. The inorganic acid salts include hydrochlorides, bromates and the like. The organic acid salts include acetates, citrates and the like. Among these, bromates are preferred, but not limited thereto.
A method of DNA Sequencing Using RNA Polymerase Transcription Accelerator
The present invention relates to a method of DNA sequencing wherein nucleic acid transcripts are produced by using an RNA polymerase and a DNA fragment as a template, the resulted nucleic acid transcripts are separated, the nucleic acid sequence is determined from the separated fractions, characterized in that said nucleic acid transcription reaction is carried out in the presence of at least one compound selected from the group consisting of compounds represented by the above-mentioned general formula (I).
The method of sequencing DNA of the present invention is characterized in that the nucleic acid transcription reaction is carried out in the presence of at least one of RNA polymerase transcription accelerators of the present invention.
When about 0.5-5 mmol of at least one of these compounds to 1 unit of an RNA polymerase is coexisted in the nucleic acid transcription reaction using RNA polymerase, sequencing of longer strands can be possible even if the amount of RNA polymerase used is not increased. Further, the amount of RNA polymerase or a template used in the nucleic acid transcription reaction can be reduced by the use of the compound of the present invention when the lengths of the DNAs to be subjected are not to be elongated.
A method of DNA sequencing is described below. Methods of DNA sequencing in which nucleic acid transcripts are obtained by using RNA polymerase and a DNA fragment as a template, the resulted nucleic acid transcripts are separated, and the nucleic acid sequence is determined from the separated fractions are already publicly known. Moreover, a method for enzymatically synthesizing nucleic acid transcription products by RNA polymerase using a DNA fragment comprising a promoter sequence for the RNA polymerase, a method for separating nucleic acid transcription products, and a method for determination of nucleic acid sequence from separated fractions are also publicly known. Therefore, for these purposes, any known methods, conditions, and equipments can be suitably used in this invention.
There is no limitation to the DNA fragment used as a template except that it comprises a promoter sequence for RNA polymerase. For example, the DNA fragment comprising a promoter sequence can be a DNA product amplified by polymerase chain reaction. Further, a nucleic acid transcription generation reaction in the method of the present invention can be carried out without removal of a primer and/or 2′-deoxyribonucleoside-5′-triphosphate and/or derivatives thereof used in the polymerase chain reaction from the amplified DNA product. The polymerase chain reaction used for the above DNA amplification can be a method which is widely used as PCR method. Further, the DNA fragment comprising a promoter sequence may be a DNA fragment which has been cloned using an adequate host after ligating the promoter sequence and a DNA fragment to be amplified. That is, in the present, invention, there is no limitation to a DNA sequence to be amplified, a primer, and conditions for the amplification and the like.
The polymerase chain reaction for the amplification of the DNA fragment comprising a promoter sequence can be performed, for example, in a 20 &mgr;l volume of solution containing 10-50 ng of genomic DNA or 1 pg of cloned DNA, 10 &mgr;M of each primer, and 200 &mgr;M of each 2′-deoxyribonucleoside-5′-triphosphate (dATP, dGTP, dCTP, dTTP) using a DNA polymerase such as a Tag polymerase.
Provided that, either one of primers for the polymerase chain reaction, or an insert DNA amplified is required to have a promoter sequence for RN
Hayashizaki Yoshihide
Iwata Masaaki
Benzion Gary
Riken
Tung Joyce
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