Method for determining polynucleotide sequence variations
First Claim
1. A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, comprising:
- a) providing a sample of the first polynucleotide;
b) selecting a region of the first polynucleotide potentially containing the variation;
c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which include the selected region;
d) selecting a region of the first polynucleotide sequence lying within the templates for analysis;
e) producing a family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including, i) a primer pair, ii) dATP, dCTP, dGTP and either dTTP or dUTP or both dTTP and dUTP, and iii) two non-Watson-Crick-pairing dideoxyterminators;
where the primer pair flank the selected region of the template strands;
where each of the primer pair is labeled;
where at least a portion of one of the dATP, dCTP, dGTP and either dTTP or dUTP or both dTTP and dUTP is labeled;
where each of the two non-Watson-Crick-pairing dideoxyterminators is labeled;
where each of the labels on the primer pair and labels on the two non-Watson-Crick-pairing dideoxyterminators are all distinguishable from each other;
where each of the family of labeled, linear polynucleotide fragments from both strands of the templates are terminated by one of the two labeled non-Watson-Crick-pairing dideoxyterminators at the 3′
end of the fragment; and
where the labeled, linear polynucleotide fragments from both strands of the templates include at least one fragment terminating at each possible base, represented by either of the two non-Watson-Crick-pairing dideoxyterminators of that portion of the selected region of both template strands flanked by one of the labeled primer pair; and
f) determining the location and identity of the bases in the selected region of the first polynucleotide by detecting the labels present in the fragments.
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Abstract
A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, comprising a) providing a sample of the first polynucleotide; b) selecting a region of the first polynucleotide potentially containing the variation; c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which include the selected region; d) selecting a region of the first polynucleotide sequence lying within the templates for analysis; e) producing a family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction.
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Citations
62 Claims
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1. A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, comprising:
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a) providing a sample of the first polynucleotide;
b) selecting a region of the first polynucleotide potentially containing the variation;
c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which include the selected region;
d) selecting a region of the first polynucleotide sequence lying within the templates for analysis;
e) producing a family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including, i) a primer pair, ii) dATP, dCTP, dGTP and either dTTP or dUTP or both dTTP and dUTP, and iii) two non-Watson-Crick-pairing dideoxyterminators;
where the primer pair flank the selected region of the template strands;
where each of the primer pair is labeled;
where at least a portion of one of the dATP, dCTP, dGTP and either dTTP or dUTP or both dTTP and dUTP is labeled;
where each of the two non-Watson-Crick-pairing dideoxyterminators is labeled;
where each of the labels on the primer pair and labels on the two non-Watson-Crick-pairing dideoxyterminators are all distinguishable from each other;
where each of the family of labeled, linear polynucleotide fragments from both strands of the templates are terminated by one of the two labeled non-Watson-Crick-pairing dideoxyterminators at the 3′
end of the fragment; and
where the labeled, linear polynucleotide fragments from both strands of the templates include at least one fragment terminating at each possible base, represented by either of the two non-Watson-Crick-pairing dideoxyterminators of that portion of the selected region of both template strands flanked by one of the labeled primer pair; and
f) determining the location and identity of the bases in the selected region of the first polynucleotide by detecting the labels present in the fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A method of determining the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, comprising:
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a) providing a sample of the first polynucleotide;
b) selecting a region of the first polynucleotide potentially containing the variation;
c) subjecting the selected region to a template producing amplification reaction to produce a first plurality of double stranded polynucleotide templates which include the selected region;
d) selecting a region of the first polynucleotide sequence lying within the templates for analysis;
e) producing a family of labeled, linear polynucleotide fragments from both strands of the templates simultaneously by a fragment producing reaction including, i) a primer pair, ii) dATP, dCTP, dGTP and either dTTP or dUTP or both dTTP and dUTP, and iii) two non-Watson-Crick-pairing dideoxyterminators;
where the primer pair flank the selected region of the template strands;
where each of the family of labeled, linear polynucleotide fragments from both strands of the templates are terminated by one of the two non-Watson-Crick-pairing dideoxyterminators at the 3′
end of the fragment; and
where the first family of fragments include at least one fragment terminating at each possible base, represented by either the first terminator or the second terminator of that portion of the selected region of both template strands flanked by a primer; and
where the labeled, linear polynucleotide fragments from both strands of the templates include at least one fragment terminating at each possible base, represented by either of the two non-Watson-Crick-pairing dideoxyterminators of that portion of the selected region of both template strands flanked by one of the primer pair; and
f) determining the location and identity of the bases in the selected region. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
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Specification