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1. (EP1711631) CARACTERISATION D'ACIDES NUCLEIQUES
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique
Claims

1. A method of sequencing and distinguishing between nucleic acid sequences on a single array, which sequences originate from different sources, which method comprises the steps of,

a) ligating target nucleic acid sequences comprising DNA polynucleotides from a first source to double-stranded nucleic acid anchoring molecules thereby producing tagged target nucleic acid sequences, wherein said double-stranded nucleic acid anchoring molecules comprise a nucleic acid sequence tag characteristic of the target nucleic acid sequence source and a functionality capable of effecting immobilisation to said array, and wherein the 3' end of each strand of said DNA polynucleotide is ligated to the 5' end of one strand of said double-stranded anchoring molecule, which 5' end is not used for anchoring;

b) repeating step a) for target nucleic acid sequences from second and subsequent sources using second and subsequent double-stranded nucleic acid anchoring molecules having characteristic nucleic acid sequence tags for each of said sources;

c) immobilising tagged target nucleic acid sequences from different sources to said array via the functionality capable of effecting immobilisation, thereby producing immobilised molecules, each immobilised molecule comprising a target nucleic acid sequence and a nucleic acid sequence tag characteristic of the target nucleic acid sequence source wherein said nucleic acid sequences on said array are disposed at a density such that they are capable of individual resolution using optical microscopy; and

d) sequencing said immobilised individually resolvable molecules by addition of nucleotides to a 3' hydroxyl group on the double-stranded anchor whereupon said sequencing identifies a sequence of each of the target nucleic acid molecules and the sequence of the tag to identify the corresponding source of the target nucleic acid molecule.


  2. A method according to claim 1 wherein said double-stranded nucleic acid anchoring molecule is a hairpin oligonucleotide and said DNA polynucleotide is ligated to the 5' end of said hairpin nucleotide.
  3. A method according to claim 1 or claim 2 wherein said double stranded anchoring molecule or hairpin comprises a 5' overhanging sequence and the nucleic acid sequence tag is located on said overhanging sequence.
  4. A method according to claim 2 wherein said hairpin oligonucleotide comprises a single stranded nucleic acid sequence which contains a region of internal self complementarity, said region being capable of forming a intramolecular duplex.
  5. A method according to claim 4 wherein said hairpin oligonucleotide comprises said nucleic acid sequence tag characteristic of said target nucleic acid sequence source positioned immediately adjacent the 5' end of said hairpin and the complement of said nucleic acid sequence tag positioned immediately adjacent the 3' end of said hairpin.
  6. A method according to claim 5 wherein said target nucleic acid is ligated to said hairpin oligonucleotide by removing any phosphate groups from 5' and 3' ends of said target nucleic acid whilst providing a single phosphate group at the 5' end of said hairpin oligonucleotide, and incubating said target nucleic acid and hairpin oligonucleotide in the presence of a ligation reagent, whereby a 3' end of the target nucleic acid is ligated to the 5' end of said hairpin oligonucleotide.
  7. A method according to any of claims 3 to 6 wherein said overhanging sequence is generated in a cleavage step comprising cleavage of the 3' strand of said double stranded anchoring molecule or hairpin at a cleavage position upstream (5') of or adjacent to the complement of the nucleic acid tag sequence, thereby removing the complement of said tag sequence on said 3' strand.
  8. A method according to claim 7 wherein cleavage is carried out by providing on said hairpin a recognition sequence for an endonuclease capable of cleaving the 3' strand of the anchoring molecule or hairpin at a cleavage site upstream of or adjacent to the complement of nucleic acid tag sequence to remove the complement of said tag sequence on said 3' strand and contacting with said endonuclease.
  9. A method according to claim 7 or 8 wherein said cleavage step to generate the overhanging sequence is carried out prior to said sequencing and sequencing comprises adding one or more nucleotides simultaneously or sequentially to the 3' hydroxyl group generated by cleavage of the 3' strand and determining the identity of one or more of the added nucleotides.
  10. A method according to claim 7 or claim 8 wherein sequencing of a portion of the target nucleic acid sequence is carried out prior to said cleavage step, said sequencing comprising adding one or more nucleotides simultaneously or sequentially to the 3' end of the anchor molecule or hairpin and determining the identity of one or more of the added nucleotides and sequencing of the nucleic acid sequence tag is carried out after said cleavage step said sequencing comprising adding one or more nucleotides simultaneously or sequentially to the 3' hydroxyl group generated by cleavage of the 3' strand and determining the identity of one or more of the added nucleotides.
  11. A method according to claim 1 wherein said immobilised molecules are present on said array at a density of 10 6-10 9 immobilised molecules per cm2.
  12. A method according to any one of the preceding claims wherein the nucleic acid sequence tag is from 1 to 10 nucleotides in length.
  13. A method according to claim 12 wherein the nucleic acid sequence tag is 4, 5 or 6 nucleotides in length.
  14. A method according to any of the preceding claims wherein said sequencing comprises at least one cycle of sequencing and is performed in the presence of a polymerase, said sequencing comprising addition of one or more nucleotides simultaneously or sequentially wherein each nucleotide comprises a characteristic label, and a blocking group that is capable of preventing uncontrolled polymerisation, wherein a cycle of sequencing comprises identifying any incorporated nucleotide incorporated by said polymerase and removing the blocking group and characteristic label from said nucleotide incorporated by said polymerase.