Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-29
2001-09-25
Myers, Carla J. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C435S091500, C435S091510, C536S023100, C536S024330
Reexamination Certificate
active
06294337
ABSTRACT:
RELATED FIELDS
The present invention relates to a method for DNA sequencing utilizing the strand displacement amplification method. The present invention relates to a method of DNA sequencing using RNA polymerase in which amplification of a target DNA fragment and preparation of ribonucleotide fragment for DNA sequencing can be performed in parallel without variation of the temperature.
BACKGROUND TECHNOLOGY
Polymerase chain reaction (PCR) is an excellent method, and its field of application increases every year (Randall K. Saiki et al. (1988) Science 239, 487-491). In PCR, it is also possible to amplify DNA fragment starting with just of 1 molecule. A method in which an amplified product of PCR is sequenced without cloning (direct sequencing method) is also useful (Corinne Wong et al. (1988) Nature, 330, 384-386). This method requires neither preparation nor screening of a library, and it is a rapid method enabling to simultaneously obtain sequence information of multiple samples.
Moreover, the inventor introduced a completely novel DNA sequencing method which does not require to remove remaining unreacted primers and 2′deoxyribonucleoside5′triphosphate (2′ dNTPs), and which does not require denaturation so that the problem of quick regeneration of PCR products could be obviated [WO96/14434]. This method is a direct sequencing method using RNA polymerase such as T7 RNA polymerase and terminators for RNA transcription reaction (e.g. 3′deoxyribonucleoside5′triphosphate, 3′dNTPs).
The above-mentioned direct transcription sequencing method is performed as described below. RNA polymerase is reacted in a mixture of ribonucleoside5′triphosphates (NTPs) and deoxyribonucleotide(s) (3′ dNTP(s)) using DNA amplified by PCR method and the like as a template. In this reaction, ribonucleotides having the bases corresponding to the template DNA sequence are incorporated into a ribonucleotide sequence, termination occurs within corporation of 3′deoxyribonucleotide, and as a result a polynucleotide is synthesized. Resulted polyribonucleotides (nucleic acid transcription products) are separated, and the DNA sequence is determined by analyzing nucleic acid sequence of the separated fraction. Specifically, using florescence labeled 3′ dNTP derivatives as a terminator of nucleic acid transcription, nucleic acid sequence can be easily determined by analyzing the label which has been incorporated as a part of the terminator.
By this method, the nucleic acid sequence of PCR-amplified DNA products can be directly used for sequencing without having to remove primers and 2′deoxyribonucleoside5′triphosphates (2′ dNTPs). This is because 2′ dNTPs do not work as substrates for RNA polymerase. Furthermore, since no denaturation is required, the problem of quick regeneration of the PCR products can be avoided. Therefore the method is extremely powerful.
In the case that a large amount of nucleotide sequence such as the human genome is to be analyzed, a method much more rapid and easier than the existing methods is necessary in order to obtain results in a short time. The above-mentioned direct transcript sequencing method is a relatively rapid method compared to previous sequencing methods utilizing DNA polymerase, however an even more rapid and easier method is necessary. Therefore, it can be thought that a DNA amplification with polymerase chain reaction and a nucleic acid transcript reaction may take place in parallel in the same reaction solution using the above direct transcript sequencing method enabling sequencing rapidly and easily.
However, thepolymerase chain reaction requires an increase or decrease of the temperature of the reaction solution for an amplification of DNA fragments. Therefore, use of thermo-resistant DNA polymerase is required for the polymerase chain reaction. Thus RNA polymerase used for nucleic acid transcript reaction is also required to be thermo-resistant. The above combination method will be possibly performed if a thermo-resistant RNA polymerase having the thermo-resistance similar to that of DNA polymerase would be available. However, at present such thermo-resistant RNA polymerase is not known.
Therefore, an object of the present invention is to provide a method for sequencing DNA in which target DNA amplification and nucleic transcript generation can be operated simultaneously in parallel without use of thermo-resistant RNA polymerase.
SUMMARY OF THE INVENTION
The present invention relates to a method for sequencing a target DNA fragment in which along with amplification of the target DNA fragment, nucleic acid transcripts are generated using an RNA polymerase and the amplified target DNA fragments are used as templates in the presence of terminators for nucleic acid transcription reaction and the generated nucleic acid transcripts are analyzed, characterized in that the amplification of target DNA fragments and the generation of nucleic acid transcripts are carried out at a constant temperature (the first method).
The present invention also relates to a method for sequencing DNA comprising
a step of obtaining nucleic acid transcripts while DNA fragments comprising the target DNA fragment sequence are being amplified
by allowing
(g) a DNA polymerase and
(h) a RNA polymerase to work in the presence of
(a-1) a DNA fragment comprising the target DNA fragment sequence wherein the DNA fragment comprises a sequence accepting formation of a nick and on at least one strand, a promoter sequence for a RNA polymerase,
(b) a primer comprising a primer sequence for one strand of the target DNA fragment and a sequence accepting formation of a nick (hereinafter referred to primer G1)
(c) a primer comprising a primer sequence for the other strand of the target DNA fragment and a sequence accepting formation of a nick (hereinafter referred to primer G2),
provided that at least one of the primers G1 and G2 comprises the promoter sequence for the RNA polymerase,
(d) deoxyribonucleoside-5′-triphosphates comprising dATP, dGTP, dCTP and dTTP or derivatives thereof (hereinafter referred to dNTP derivatives),
(e) ribonucleoside-5′-triphosphates comprising ATP, GTP, CTP and UTP or derivatives thereof (hereinafter referred to NTP derivatives), and
(f) 3′-deoxyribonucleoside-5′-triphosphates comprising 3′dATP, 3′dGTP, 3′dCTP and 3′dUTP or derivatives thereof (hereinafter referred to 3′dNTP derivatives),
and by forming a nick at a site of the DNA fragment (a-1) accepting formation of a nick; and
a step of separating the resulting nucleic acid transcripts and reading the nucleic acid sequence from the separated fractions (the second method).
The present invention further relates to a method for sequencing a DNA comprising
a step in which primer B1 (a primer complementary to one strand of the target DNA fragment), primer B2 (a primer complementary to the other strand of the DNA fragment), primer G1, and primer G2 hybridize to the DNA fragment (provided that the primer B1 hybridizes to a site closer to 5′ end of one strand of the DNA fragment than the site recognized by primer G1, and the primer B2 hybridizes to a site closer to 5′ end of the other strand of the DNA fragment than the site recognized by primer G2), and
a step of obtaining nucleic acid transcripts while DNA fragments comprising the target DNA fragment sequence are being amplified, by allowing (g) a DNA polymerase and (h) a RNA polymerase to work on the target DNA (a-2) obtained from the hybridization in the presence of (b) primer G1, (c) primer G2, (d) dNTP derivatives, (e) NTP derivatives and (f) at least one kind of 3′ dNTP derivatives and by forming a nick at a site of the DNA fragment (a-2) accepting formation of a nick; and
a step of separating the resulting nucleic acid transcripts and reading the nucleic acid sequence from the separated fractions (The third method).
REFERENCES:
patent: 5270184 (1993-12-01), Walker et al.
patent: 6074824 (2000-06-01), Hayashizaki et
Burns Doane Swecker & Mathis L.L.P.
Myers Carla J.
Riken
Spiegler Alexander H.
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