EP0223618A2
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EP
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Automatable process for nucleotide sequencing
Assignee: New York Medical College
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Filing: 1986-07-16
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Granted: —
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Expires: 2006-07-16
Abstract
Abstract This invention relates to a method for determining the nucleotide sequence of DNA and RNA molecules. The method is automatable and avoids the use of radioactive labels and gel electrophoresis. The method is also adaptable for introducing site-specific mutations in DNA and RNA molecules.
Inventors
- 👤 Robert Jean Melamede
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 10:25
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6858; C12Q1/6869
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
123 words
A method for determining the base sequence of nucleotides comprising: (a) adding an activated nucleoside 5'-triphosphate precursor having a known nitrogenous base to a reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer so that the primer is at least one nucleotide residue shorter than the template at the 3'-end of the primer under reaction conditions which allow incorporation of the activated nucleoside 5'-triphosphate precursor onto the primer at the 3'-end of the primer provided the nitrogenous base of the activated nucleoside 5'-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template; and (b) detecting whether or not the nucleoside 5'-triphosphate precursor was incorporated into the primer strand.
Claim 2
Independent Claim
123 words
A method for determining the base sequence of nucleotides comprising: (a) adding an activated nucleoside 5'-triphosphate precursor having a known nitrogenous base to a reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer so that the primer is at least one nucleotide residue shorter than the template at the 3'-end of the primer under reaction conditions which allow incorporation of the activated nucleoside 5'-triphosphate precursor onto the primer at the 3'-end of the primer provided the nitrogenous base of the activated nucleoside 5'-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template; and (b) detecting whether or not the nucleoside 5'-triphosphate precursor was incorporated into the primer strand.
Claim 3
Dependent Claim
46 words
The method according to claim 1 in which the detection of incorporation of the activated precursor is accomplished by: (a) separating unincorporated nucleoside 5'-triphosphate precursors from the reaction mixture; and (b) detecting the presence or absence of the unincorporated nucleoside 5'-triphosphate precursors in the separated component.
Claim 4
Dependent Claim
24 words
The method according to claim 2 in which the presence or absence of the separated unincorporated nucleoside 5'-triphosphate precursor is detected by absorption spectroscopy.
Claim 5
Dependent Claim
16 words
The method according to claim 3 in which the absorption wavelength is in the ultraviolet range.
Claim 6
Dependent Claim
24 words
The method according to claim 2 in which the presence or absence of the separated unincorporated nucleoside 5'-triphosphate precursor is detected by fluorescence spectroscopy.
Claim 7
Dependent Claim
43 words
The method according to claim 1 in which the detection of incorporation of the precursor is accomplished by: (a) separating unincorporated nucleoside 5'-triphosphate precursors from the reaction mixture; and (b) detecting the presence or absence of the separated unincorporated nucleoside 5'-triphosphate precursor electrochemically.
Claim 8
Dependent Claim
32 words
The method according to claim 2 in which the activated nucleoside 5'-triphosphate precursor is radiolabeled and the presence or absence of separated unincorporated radiolabeled nucleoside 5'-triphosphate precursors is detected by radioactive counting.
Claim 9
Dependent Claim
32 words
The method according to claim 1 or 2 in which the template comprises DNA, the nucleotide polymerase comprises a DNA-directed DNA polymerase, and the activated nucleoside 5'-triphosphate precursors comprise deoxyribonucleoside 5'-triphosphate precursors.
Claim 10
Dependent Claim
32 words
The method according to claim 1 or 2 in which the template comprises DNA, the nucleotide polymerase comprises a DNA-directed RNA polymerase, and the activated nucleoside 5'-triphosphate precursors comprise ribonucleoside 5'-triphosphate precursors.
Claim 11
Dependent Claim
32 words
The method according to claim 1 or 2 in which the template comprises RNA, the nucleotide polymerase comprises an RNA-directed DNA polymerase and the activated nucleoside 5'-triphosphate precursors comprise deoxyribonucleoside 5'-triphosphate precursors.
Claim 12
Independent Claim
254 words
A method for the site-specific mutagenesis of a nucleotide, comprising: (a) adding an activated nucleoside 5'-triphosphate precursor having a known nitrogenous base to a reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template having a known sequence hybridized with a primer that is at least one nucleotide residue shorter than the template at the 3'-end of the primer under reaction conditions which allow incorporation of the activated nucleoside 5'-triphosphate precursor onto the primer at the 3'-end of the primer provided the nitrogenous base of the activated nucleoside 5'-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template; and (b) detecting the incorporation of the 5'-triphosphate precursors by separating any unincorporated precursor from the reaction mixture and detecting the relative amount of unincorporated nucleotides in the separated component; (c) repeating steps 11(a) and 11(b) until the primer is elongated up to the nucleotide residue on the template preceding the residue which is to be mutagenized; (d) adding an analog of a nucleoside 5'-triphosphate precursor having a base comprising the desired mutation which will mis-pair with the template under conditions which allow incorporation of the analog into the primer strand; and (e) detecting the incorporation of the analog by separating any unincorporated analogs from the reaction mixture and detecting the relative amount of unincorporated analogs in the separated component; and (f) adding activated nucleoside 5'-triphosphate precursors comprising a mixture of all four bases to the reaction mixture so that the synthesis of the mutagenized nucleotide is completed.
Claim 13
Independent Claim
254 words
A method for the site-specific mutagenesis of a nucleotide, comprising: (a) adding an activated nucleoside 5'-triphosphate precursor having a known nitrogenous base to a reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template having a known sequence hybridized with a primer that is at least one nucleotide residue shorter than the template at the 3'-end of the primer under reaction conditions which allow incorporation of the activated nucleoside 5'-triphosphate precursor onto the primer at the 3'-end of the primer provided the nitrogenous base of the activated nucleoside 5'-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the template; and (b) detecting the incorporation of the 5'-triphosphate precursors by separating any unincorporated precursor from the reaction mixture and detecting the relative amount of unincorporated nucleotides in the separated component; (c) repeating steps 11(a) and 11(b) until the primer is elongated up to the nucleotide residue on the template preceding the residue which is to be mutagenized; (d) adding an analog of a nucleoside 5'-triphosphate precursor having a base comprising the desired mutation which will mis-pair with the template under conditions which allow incorporation of the analog into the primer strand; and (e) detecting the incorporation of the analog by separating any unincorporated analogs from the reaction mixture and detecting the relative amount of unincorporated analogs in the separated component; and (f) adding activated nucleoside 5'-triphosphate precursors comprising a mixture of all four bases to the reaction mixture so that the synthesis of the mutagenized nucleotide is completed.
Claim 14
Dependent Claim
18 words
The method according to claim 11 in which the analog of the nucleoside 5'-triphosphate comprises a nucleoside (1-thio)-triphosphate.
References Cited (Backward Citations)
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