Genomic sequencing method
First Claim
1. A method of sequencing DNA comprising the steps of:
- a) preparing from segments of a DNA strand to be sequenced, families of polynucleotides, each family including all polynucleotides, complementary to at least a portion of the DNA segment and at least a portion of the 3'"'"' flanking DNA segment of the DNA strand to be sequenced, of the formula;
space="preserve" listing-type="equation">K.sub.n '"'"' . . . K.sub.4 K.sub.3 K.sub.2 K.sub.1 X.sub.1 X.sub.2 X.sub.3 X.sub.4 . . . X.sub.n ranging in length from K1 X1 to Kn'"'"' -Xn'"'"' wherein K1 K2 K3 K4 . . . Kn'"'"' represents the nucleotides 5'"'"' to an internal reference point, the reference point defined as the dividing line between K1 and X1 ;
wherein X1 X2 X3 X4 . . . Xn represents the nucleotides 3'"'"' to the internal reference point;
wherein n and n'"'"' are integers and n+n'"'"', the number of nucleotides in a polynucleotide, is less than or equal to the number of nucleotides in a polynucleotide of length within the analyzable limit of the method for determining base composition and identity of the 3'"'"' terminal nucleotide of a polynucleotide; and
wherein each polynucleotide in the family conforms to the criterion that if the polynucleotide contains Xn it also contains Xn-1, Xn-2 . . . X1 ;
or the criteria that if the polynucleotide contains Kn'"'"' it also contains Kn'"'"'-1'"'"' Kn'"'"'-2 . . . K1 and if the polynucleotide contains Xn then it also contains Xn-1, Xn-2 . . . X1 ;
or the criteria that if any two polynucleotides have the same base composition, then they have different terminal bases and if any polynucleotide contains Xn, then it also contains Xn-1 Xn-2 . . . X1 ;
b) determining the base composition and the identity of the 3'"'"' terminal base of each polynucleotide of each family;
c) determining the base sequence of the longest polynucleotide in each family from the determined base composition and identity of the 3'"'"' terminal base of each polynucleotide in the family and the derived change in base composition and terminal base between polynucleotides in each family; and
d) determining the base sequence of the entire DNA strand to be sequenced based upon the overlapping sequences of the longest polynucleotides in each family.
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Abstract
A method of determining the nucleotide sequence of a DNA molecule of arbitrary length as a single procedure by sequencing portions of the molecule in a fashion such that the sequence of the 5'"'"' end of the succeeding contiguous portion is sequenced as the 3'"'"' end of its preceeding portion is sequenced, for all portions, where the order of contiguous portions is determined by the sequence of the DNA molecule. Sequencing of the individual portions is accomplished by generating a family of polynucleotides under conditions which determine that the elements are partial copies of the portion and are of random nucleotide length on the 3'"'"' and 5'"'"' ends about a dinucleotide which is an internal reference point; determining the base composition and terminal base identity of each element of the family and solving for the sequence by a method of analysis wherein the base composition and terminal base data of each element is used to solve for a single base of the sequence by assigning the base to either the 5'"'"' or 3'"'"' end of the partial sequence about the internal reference point as the entire sequence of the portion is built up from a dinucleotide.
127 Citations
29 Claims
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1. A method of sequencing DNA comprising the steps of:
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a) preparing from segments of a DNA strand to be sequenced, families of polynucleotides, each family including all polynucleotides, complementary to at least a portion of the DNA segment and at least a portion of the 3'"'"' flanking DNA segment of the DNA strand to be sequenced, of the formula;
space="preserve" listing-type="equation">K.sub.n '"'"' . . . K.sub.4 K.sub.3 K.sub.2 K.sub.1 X.sub.1 X.sub.2 X.sub.3 X.sub.4 . . . X.sub.nranging in length from K1 X1 to Kn'"'"' -Xn'"'"' wherein K1 K2 K3 K4 . . . Kn'"'"' represents the nucleotides 5'"'"' to an internal reference point, the reference point defined as the dividing line between K1 and X1 ;
wherein X1 X2 X3 X4 . . . Xn represents the nucleotides 3'"'"' to the internal reference point;
wherein n and n'"'"' are integers and n+n'"'"', the number of nucleotides in a polynucleotide, is less than or equal to the number of nucleotides in a polynucleotide of length within the analyzable limit of the method for determining base composition and identity of the 3'"'"' terminal nucleotide of a polynucleotide; and
wherein each polynucleotide in the family conforms to the criterion that if the polynucleotide contains Xn it also contains Xn-1, Xn-2 . . . X1 ;
or the criteria that if the polynucleotide contains Kn'"'"' it also contains Kn'"'"'-1'"'"' Kn'"'"'-2 . . . K1 and if the polynucleotide contains Xn then it also contains Xn-1, Xn-2 . . . X1 ;
or the criteria that if any two polynucleotides have the same base composition, then they have different terminal bases and if any polynucleotide contains Xn, then it also contains Xn-1 Xn-2 . . . X1 ;b) determining the base composition and the identity of the 3'"'"' terminal base of each polynucleotide of each family; c) determining the base sequence of the longest polynucleotide in each family from the determined base composition and identity of the 3'"'"' terminal base of each polynucleotide in the family and the derived change in base composition and terminal base between polynucleotides in each family; and d) determining the base sequence of the entire DNA strand to be sequenced based upon the overlapping sequences of the longest polynucleotides in each family. - View Dependent Claims (2, 3, 4)
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5. A method of sequencing DNA comprising the steps of:
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a) cleaving the DNA to be sequenced to produce fragments of about 20 to about 400 nucleotides in length; b) separating and isolating the DNA fragments according to size; c) separating and isolating the individual strands of each fragment; d) preparing RNA/DNA hybrid polynucleotides by; i) making an RNA transcript(s) of at least a portion of the fragment strand; ii) isolating the RNA transcript(s); iii) extending the RNA transcript(s) with deoxyribonucleotides, using the DNA to be sequenced as template for the extension, and terminating the extension randomly to produce a set of RNA/DNA polynucleotides ranging in length up to about 400 nucleotides; e) separating and isolating each of the RNA/DNA hybrid polynucleotides in each set; f) determining the base composition and the identity of the 3'"'"' terminal base of each RNA/DNA polynucleotide of each set; g) randomizing the 5'"'"' end of at least one RNA/DNA polynucleotide of length greater than one half the length of the longest RNA/DNA polynucleotide of each set and at least one of the smallest RNA/DNA hybrid polynucleotides in the set, to produce RNA/DNA polynucleotides having an RNA portion containing from one ribonucleotide to the number of ribonucleotides in the original transcript; h) separating and isolating the 5'"'"' randomized RNA/DNA polynucleotides; i) determining the base composition and terminal base of each 5'"'"' randomized RNA/DNA hybrid molecule; j) determining the base sequence of the longest RNA/DNA polynucleotide in each set from the determined base composition and identity of the 3'"'"' terminal base of the RNA/DNA hybrid polynucleotides; and k) determining the base sequence of the entire DNA to be sequenced from the overlapping sequences of the longest polynucleotides in each family. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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14. A method of sequencing DNA, comprising the steps of:
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a) isolating the DNA to be sequenced; b) preparing 3'"'"' randomly ended RNA transcripts of the DNA in multiple reaction mixtures, all transcripts initiating from the 3'"'"' end of the DNA to be sequenced, such that for any reaction n the succeeding reaction n+1 results in RNA transcripts which are on average longer than those in reaction n; c) isolating the RNA transcripts from each reaction; d) extending the transcripts with deoxyribonucleotides using the DNA to be sequenced as template and terminating the extension reaction randomly to produce a set of RNA/DNA hybrid polynucleotides; e) degrading the RNA portion of the polynucleotides; f) separating the DNA molecules according to size; g) determining the base composition and the identity of the 3'"'"' terminal base of the set of DNA molecules generated from the transcripts of each reaction; h) determining the sequence of the longest DNA molecule in each set from the determined base composition and identity of the 3'"'"' terminal base of the DNA molecules; and i) determining the sequence of the entire DNA to be sequenced from the region of overlap of the longest DNA molecule of each set. - View Dependent Claims (15, 16, 29)
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Specification