Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-05-05
2000-06-13
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 435 915, 435 9151, 536 2433, 935 6, 935 8, 935 17, 935 77, 935 78, C12Q 168
Patent
active
060748249
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for determining nucleotide sequence of DNA. More precisely, the present invention relates to a method for determining DNA nucleotide sequence by a direct transcript sequencing method utilizing PCR technique.
BACKGROUND OF THE ART
Because of high performance of polymerase chain reaction (PCR), its applied fields have been expanded year by year (Randall K,. Saiki et al. (1988) Science 239, 487-491). Even a DNA fragment of one molecule can be amplified by the PCR. A direct sequencing method, which is one for sequencing a product amplified by the PCR, is also useful (Corinne Wong et al. (1988) Nature, 330, 384-386). This method enables to reveal information concerning sequence of numerous samples quickly and simultaneously without preparation of libraries and screening thereof.
The direct sequencing method, however, suffers from two serious problems.
First, primers and 2'-deoxyribonucleoside-5'-triphosphates (2'-dNTPs) which have not incorporated remain in reaction systems and interfere the sequencing reaction. Accordingly, the residual primers and 2'-dNTPs must be removed from the PCR products before sequencing in conventional methods. Various kinds of methods for purification of the PCR products are known and which include purification by electrophoresis, ethanol precipitation, filtration, HPLC and the like (see, for example, Dorit R. L. et al. (1991) Current Protocols in Molecular Biology, Vol .II, John Wiley and Sons, New York, 15.2.1-15.2.11). However, all of these methods are complicated.
Second problem is the quick renaturation of PCR products. When the PCR products are renatured into a double-stranded DNA, they are no longer a single-stranded template and interfere the annealing between primers and single-stranded templates. For minimizing the renaturation, there have been proposed, for example, quenching after the denaturation, biotilation of one primer and adsorption of PCR products onto a streptavidin coated surface, use of exonuclease, asymmetric PCR and the like (see, for example, Barbara Bachmann et al. (1990) Nucleic Acid Res. Vol. 18, 1309). However, most of these methods are time-consuming and complicated.
Therefore, an object of the present invention is to provide a completely novel method for determining DNA nucleotide sequence, which does not require to remove the primers and 2'-deoxyribonucleoside-5'-triphosphates (2'-dNTPs) unreacted and remained in PCR reaction systems and which also does not require denaturation so that the problem of quick renaturation of PCR products could be obviated.
SUMMARY OF THE INVENTION
The present invention relates to a method for determining nucleotide sequence of DNA product amplified by polymerase chain reaction, which does not require to remove primers and/or 2'-deoxyribonucleoside-5'-triphosphates and/or derivatives thereof, which comprises derivatives thereof and one or more of 3'-deoxyribonucleotide-5'-triphosphates comprising 3'-dATP, 3'-dGTP, 3'-dCTP, 3'-dUTP and derivatives thereof (3'-dNTP derivatives) in the presence of an RNA polymerase and a DNA product which has been amplified by polymerase chain reaction and contains promoter sequence for the RNA polymerase to obtain a nucleic acid transcription product,
The present invention solves the above-mentioned problems in the direct sequence methods and relates to a direct transcript sequencing method utilizing an RNA polymerase such as T7 RNA polymerase and a terminator of RNA transcription reaction (for example, 3'-deoxyribonucleoside-5'-phosphate (3'-dNTPs)).
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a photography of the electrophoresis gel showing the results of autoradiography by the direct transcript sequencing method obtained in Example 1.
FIG. 2 shows results of electropherogram by the fluorescence direct transcript sequencing method obtained in Example 2.
DISCLOSURE OF THE INVENTION
The method of the present invention is one for determining nucleotide sequence of DNA product amplified by polymerase chain reaction without remo
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Copy of European Search Report.
Hayashizaki Yoshihide
Sasaki Nobuya
Horlick Kenneth R.
Taylor Janell E.
The Institute of Physical and Chemical Research
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