Method of using mobile priming sites for DNA sequencing
AssigneeW. R. Grace & Co.-Conn.
InventorJack T. Leonard
Life of this patent
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Abstract
Doubled-stranded oligonucleotide molecules for use in providing enchaned sequencing of DNA, comprising: a first stand having 1) a priming site; 2) a labeling region contiguous with the priming site, wherein the labeling region consists essentially of at least six identical nucleotides; and 3) a cohesive end contiguous with the labeling region, having a nucleotide sequence complementary to a nucleotide sequence generated by the action of a restriction enzyme; and a second strand annealed to the labeling region of the first stand, but not overlapping with the cohesive end. A method of DNA gene sequencing, involving the use of mobile priming sites for DNA polymerases in DNA sequencing, is also disclosed.
Claims39
A double-stranded oligonucleotide molecule for use in providing enhanced sequencing of DNA, comprising:
- a first strand having 1) a priming site;
- 2) a labeling region contiguous with said priming site;
- and 3) a cohesive end contiguous with said labeling region, said cohesive end having a nucleotide sequence complementary to a nucleotide sequence generated by the action of a restriction enzyme;
- and
- a second strand annealed to said labeling region of said first strand, but not overlapping with said cohesive end.
A kit for sequencing selected restriction fragments comprising a library of prinkers, each of said prinkers comprising a double-stranded oligonucleotide portion having a first strand having 1) a priming site;
- 2) a labeling region contiguous with said priming site, said labeling region consisting essentially of at least six identical nucleotides;
- and 3) a cohesive end contiguous with said labeling region, said cohesive end having a nucleotide sequence selected from the group consisting of 5'-AATT, 5'-CG, ACGT-3', 5'-AGCT, AGCT-3', 5'-TA, 5'-CATG, CATG-3', 5'-CCGG, 5'-CGCG, 5'-CTAG, 5'-GATC, AT-3', CG-3', 5'-GCGC, GCGC-3', 5'-GGCC, GC-3', GGCC-3', 5'-GTAC, GTAC-3', 5'-TATA, 5'-AT, 5'-TCGA, 5'-TGCA, TGCA-3', and 5'-TTAA;
- and a second strand annealed to said labeling region of said first strand, but not overlapping with said cohesive end.
A method of sequencing DNA, comprising the steps of
- a identifying on a DNA molecule a segment containing genetic information of interest;
- b. selecting a restriction enzyme that will excise said segment and leave at least one end of said segment with a sticky end;
- c. making a restriction digest by treating said DNA molecule with said restriction enzyme, excising said segment and leaving at least one end of said segment with a sticky end;
- d. selecting a prinker comprising a double-stranded oligonucleotide molecule for use in providing enhanced sequencing of DNA, comprising:
- a first strand having 1) a priming site;
- 2) a labeling region contiguous 30 with said priming site;
- and 3) a cohesive end contiguous with said
- labeling region, said cohesive end having a nucleotide sequence complementary to said sticky end of said segment;
- and
- a second strand annealed to said labeling region of said first strand, but not overlapping with said cohesive end;
- e. ligating said prinker to said segment to form an activated DNA segment;
- f. isolating said activated DNA segments from said restriction digest;
- g. directly sequencing some or all of said activated DNA segments.
A method of sequencing DNA, comprising
- a) subjecting DNA to the action of more than one restriction enzyme so as to prepare a restriction digest, said action resulting in restriction fragments having at least one sticky end;
- b) forming activated DNA segments by ligating to said restriction fragments a double-stranded oligonucleotide molecule comprising a first strand having a priming site, a labeling region contiguous with said priming site, and a cohesive end contiguous with said labeling region, said cohesive end having a nucleotide sequence complementary to the nucleotide sequence of said sticky end, wherein the double-stranded oligonucleotide molecule has been tagged with a light-responsive dye having a photometric response unique to that particular double-stranded oligonucleotide molecule;
- c) separating said activated DNA segments, and
- d) directly sequencing some or all of said activated DNA segments.
Dependencies are inferred from the claim text.