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US 6,440,705 B1Granted
Method for analyzing polynucleotides
OwnerVincent P. Stanton, Jr., Jia Liu Wolfe +2inventors
InventorsVincent P. Stanton, Jr., Jia Liu Wolfe, Tomohiko Kawate, Gregory L. Verdine
Life of this patent
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Abstract
The present invention relates to methods for the analysis of polynucleotides including detection of variance in nucleotide sequence without the need for full sequence determination, full sequence determination of a polynucleotide, genotyping of DNA and labeling a polynucleotide fragment during the process of cleaving it into fragments.
Claims32
A method for cleaving a polynucleotide, comprising:
- a. replacing a natural A, C, G or T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;
- b. contacting the modified polynucleotide with a reagent or reagents that cleave(s) it at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments.
A method for cleaving a polynucleotide, comprising:
- a. replacing a first natural A, C, G or T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in the polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a once-modified polynucleotide;
- b. replacing a second, different natural A, C, G, T, or U nucleotide at greater than 90% of its points of occurrence in the once-modified polynucleotide with a second, different modified A, C, G, T or U nucleotide, respectively, to form a twice-modified polynucleotide;
- and,
- c. contacting the twice-modified polynucleotide with a reagent or reagents that cleaves it at each point where the first modified nucleotide is followed immediately in sequence by the second modified nucleotide.
1 more element
A method for detecting variance in nucleotide sequence among related polynucleotides, comprising:
- a. replacing three of four different natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of their points of occurrence in a polynucleotide with three stabilizing modified A, C, G, T or U nucleotides, respectively, to form a modified polynucleotide having one remaining natural nucleotide;
- b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the remaining natural nucleotide to give a set of fragments;
- c. determining a mass of each fragment of the set of fragments;
- and,
- d. comparing the mass of each fragment of the set of fragments with the mass of each fragment expected from cleavage of a related polynucleotide of known sequence, or
- e. repeating steps a-c with one or more related polynucleotides of unknown sequence and comparing the mass of each fragment of the set of fragments with the mass of each fragment obtained from cleavage of each related polynucleotide.
3 more elements
A method for detecting variance in nucleotide sequence among related polynucleotides, comprising:
- a. replacing two different natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T), nucleotides at greater than 90% of their points of occurrence in a polynucleotide with two modified A, C, G, T or U nucleotides, respectively, each having different cleaving characteristics from the others, to give a modified polynucleotide;
- b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the first modified nucleotide to give a first set of fragments;
- c. determining a mass of each fragment of the first set of fragments;
- d. cleaving the first set of fragments into a second set of fragments at greater than 90% of the points of occurrence in the first set of fragments of the second modified nucleotide:
- e. determining a mass of each fragment of the second set of fragments;
- and,
- f. comparing the mass of each fragment of the first set of fragments and the mass of each fragment of the second set of fragments with a mass of each fragment of first and second sets of fragments expected from cleavage of a related polynucleotide of known sequence, or
- g. repeating steps a-d with one or more related polynucleotides of unknown sequence and comparing the mass of each fragment of the first set of fragments and the mass of each fragment of the second set of fragments with a mass of each fragment obtained from first and second cleavages of each related polynucleotide.
5 more elements
A method for determining a nucleotide sequence of a polynucleotide, comprising:
- a. replacing a natural A, C, G, or T (or, if the polynucleotide is a ribonucleotide, U instead of T) nucleotide at a percentage of points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;
- b. cleaving the modified polynucleotide at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments;
- c. repeating steps a and b until each different natural A, C, G, T or U nucleotide in the polynucleotide has been replaced with a modified A, C, G, T or U nucleotide, respectively;
- d. determining a mass of each fragment obtained from each cleavage;
- and,
- e. constructing the nucleotide sequence of the polynucleotide from the masses of all the fragments, or,
- f. generating a sequence ladder from all the fragments obtained in step c;
- and,
- g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.
6 more elements
A method for determining nucleotide sequence in a polynucleotide, comprising:
- a. replacing a natural A, C, G, T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of the points of its occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide;
- b. cleaving the modified polynucleotide at a percentage of points of occurrence of the modified A, C, G, T or U nucleotide to give a first set of fragments;
- c. repeating steps a and b until each different natural A, C, G,T or U nucleotide in the polynucleotide has been replaced with a modified A, C, G, T or U nucleotide, respectively, to form a modified polynucleotide and each modified polynucleotide has been cleaved to give a set of fragments;
- d. determining a mass of each fragment of each set of fragments from each cleavage reaction;
- and,
- e. constructing the nucleotide sequence of the polynucleotide from all the masses;
- or,
- f. generating a sequence ladder from all the fragments obtained in step c;
- and
- g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.
7 more elements
A method for determining a nucleotide sequence of a polynucleotide, comprising:
- a. replacing A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of each of their points of occurrence in a polynucleotide with four modified A, C, G, T or U nucleotides, respectively, each of which has a different cleaving characteristic from the others, to form a modified polynucleotide;
- b. separating the modified polynucleotide into four aliquots;
- c. cleaving the modified polynucleotide in each aliquot at greater than 90% of the points of occurrence of a different one of the modified nucleotides such that each aliquot contains a set of fragments resulting from cleavage at a different modified A, C, G, T or U nucleotide;
- d. determining a mass of each fragment of each set of fragments in each aliquot;
- and,
- e. constructing the nucleotide sequence of the polynucleotide from the masses of all the fragments;
- or,
- f. generating a sequence ladder from all the fragments obtained in step c;
- and,
- g. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.
7 more elements
A method for cleaving a polynucleotide during polymerization, comprising:
- providing two or three natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides, and one or two modified A, C, G, T or U nucleotides, four nucleotides in all;
- providing one or more polymerases, at least one of which produces or enhances cleavage during polymerization at points where the modified nucleotide is, or the modified nucleotides are, being incorporated into the polynucleotide;
- mixing the four nucleotides with the polymerase or polymerases under polymerizing conditions;
- and,
- running the polymerization with concurrent cleavage.
2 more elements
A method for cleaving a polynucleotide wherein fragments obtained from the cleavage are labeled, comprising:
- a. replacing a natural A, C, G, T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a modified A, C, G, T or U nucleotide to form a modified polynucleotide;
- b. contacting the modified polynucleotide with a labeled phosphine and a chemical base that cleaves the modified polynucleotide at greater than 90% of the points of occurrence of the modified nucleotide to give a set of fragments in which the labeled phosphine has covalently bonded to the 3′ position of sugar moieties of the modified nucleotides.
A method for determining a nucleotide sequence of a polynucleotide, comprising:
- a. replacing natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at greater than 90% of each of their points of occurrence in a polynucleotide with four modified A, C, G. T or U nucleotides, respectively, each of which has a different cleavage characteristic from the others, to form a modified polynucleotide;
- b. separating the modified polynucleotide into four aliquots;
- c. cleaving the modified polynucleotide in each aliquot at a percentage of points of occurrence of a different modified A, C, G, T or U nucleotide such that each aliquot comprises a set of fragments from cleavage at a different modified nucleotide;
- d. creating a sequence ladder from all the fragments;
- and,
- e. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.
3 more elements
A method for determining a nucleotide sequence of a polynucleotide, comprising:
- a. replacing natural A, C, G and T (or, if the polynucleotide is a ribonucleotide, U in place of T) nucleotides at a percentage of points of occurrence in a polynucleotide with four modified A, C, G, T or U nucleotides, respectively, each of which has a different cleavage characteristic from the others to form a modified polynucleotide;
- b. separating the modified polynucleotide into four aliquots;
- c. cleaving the modified polynucleotide in each aliquot at greater than 90% of the points of occurrence of a different one of the modified nucleotides such that each aliquot comprises a set of fragments from cleavage at a different modified A, C, G, T or U nucleotide;
- d. creating a sequence ladder from all the fragments;
- and,
- e. analyzing the sequence ladder to obtain the nucleotide sequence of the polynucleotide.
3 more elements
Dependencies are inferred from the claim text.