Process for sequencing nucleic acids
First Claim
1. Method for sequencing a nucleic acid comprising:
- (a) combining(i) an oligonucleotide primer,(ii) a nucleic acid to be sequenced,(iii) four deoxyribonucleoside triphosphates,(iv) a polymerase, and(v) at least three dideoxyribonucleoside triphosphates in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced;
(b) labelling the nucleic acid fragments formed;
(c) separating the nucleic acid fragments by gel electrophoresis; and
(d) determining nucleic acid sequence by determination of position of incorporated dideoxynucleoside triphosphates in said labelled nucleic acid fragments, wherein said dideoxynucleoside triphosphates are differentiated from each other by intensity of the label in the nucleic acid sequence.
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Accused Products
Abstract
For the sequencing of nucleic acids by enzymatic extension of an oligonucleotide primer in the presence of a polymerase, the four deoxyribonucleoside triphosphates, one of the four dideoxynucleoside triphosphates and the nucleic acid to be sequenced as template in each of four different preparations, labelling of the nucleic acid fragments which form and which are dependent on the dideoxynucleoside triphosphate used, separation by gel electrophoresis of the preparations containing the fragments and detection of the sequence via the label, at least two of the dideoxynucleoside triphosphates are used in different amounts together in one preparation and the fragments are differentiated by the intensity of the labelling signal of the bands in the gel or in another separation procedure.
45 Citations
25 Claims
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1. Method for sequencing a nucleic acid comprising:
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(a) combining (i) an oligonucleotide primer, (ii) a nucleic acid to be sequenced, (iii) four deoxyribonucleoside triphosphates, (iv) a polymerase, and (v) at least three dideoxyribonucleoside triphosphates in different amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced; (b) labelling the nucleic acid fragments formed; (c) separating the nucleic acid fragments by gel electrophoresis; and (d) determining nucleic acid sequence by determination of position of incorporated dideoxynucleoside triphosphates in said labelled nucleic acid fragments, wherein said dideoxynucleoside triphosphates are differentiated from each other by intensity of the label in the nucleic acid sequence. - 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)
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Specification