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Patexia Research

Patent 07695904 - Reducing non-target nucleic acid dependent amplifications: amplifying repetitive nucleic acid sequences > Claims

  • 1. A method of amplifying repetitive units in a repetitive region of a human target nucleic acid comprising:na) contacting a target nucleic acid comprising substantially complementary first and second strands with a first and a second primer, wherein said first primer hybridizes to at least one repetitive unit of said first strand and said second primer hybridizes to at least one repetitive unit of said second strand, wherein said hybridized primers are capable of primer extension when hybridized to their respective strands, and wherein at least one nucleotide of said first primer produces an internal base pair mismatch between said first primer and a nucleotide of said repetitive unit when said first primer is hybridized to at least one repetitive unit of said first strand, wherein said first primer also produces a mismatch with the 3′ terminal nucleotide of said second primer when first and second primers hybridize to each other, wherein at least one nucleotide of said second primer produces an internal base pair mismatch between said second primer and a nucleotide of said repetitive unit when said second primer is hybridized to at least one repetitive unit of said second strand, wherein said second primer also produces a mismatch with the 3′ terminal nucleotide of said first primer when first and second primers hybridize to each other; andb) amplifying the target nucleic acid by the polymerase chain reaction.
    • 4. A method according to claim 1, or 3, wherein said first and second primers further comprise 5′ terminal sequences which do not hybridize to said repetitive units.
    • 7. A method according to claim 1, or 3, wherein said repetitive unit comprises hexanucleotide repeats.
    • 8. A method according to claim 1, 2 or 3, wherein said repetitive unit comprises pentanucleotide repeats.
    • 9. A method according to claim 1, 2, or 3, wherein said repetitive unit comprise tetranucleotide repeats.
    • 10. A method according to claim 1, 2 or 3 wherein said repetitive units comprise telomere repetitive units.
      • 11. A method according to claim 10, wherein said first primer comprises SEQ ID NO: 1 and said second primer comprises SEQ ID NO: 2.
      • 12. A method according to claim 10, wherein said first primer comprises SEQ ID NO: 8 and said second primer comprises SEQ ID NO: 9.
        • 14. A method according to claim 12 wherein said measuring is by real time quantitative PCR.
        • 15. A method according to claim 12, further comprisingna) amplifying a target nucleic acid of known copy number (S); andb) determining the T/S ratio to determine repetitive unit copy number.
    • 13. A method of determining the number of repetitive units in a repetitive region of a target nucleic acid according to claim 1, 2 or 3, further comprising measuring the amount of amplified product (T).
  • 2. A method of amplifying repetitive units in a repetitive region of a human target nucleic acid comprising:na) contacting a target nucleic acid comprising substantially complementary first and second strands with a first and second primer, wherein said first primer hybridizes to more than one repetitive unit of said first strand and said second primer hybridizes to more than one repetitive unit of said second strand, wherein said hybridized primers are capable of primer extension when hybridized to their respective strands, and wherein nucleotides of said first primer produce internal base pair mismatches with nucleotides at the identical nucleotide positions of each repetitive unit of said first strand, wherein said first primer also produces a mismatch with the 3′ terminal nucleotide of said second primer when first and second primers hybridize to each other; andb) amplifying the target nucleic acid by the polymerase chain reaction.
    • 3. A method according to claim 2, wherein nucleotides residues of said second primer produce internal base pair mismatches with nucleotides at the identical nucleotide positions of each repetitive unit of said second strand.
      • 5. A method according to claim 3, wherein at least two of the mismatches in the complex formed by hybridization of said first and said second primer are on adjacent nucleotide positions of said repetitive unit.
    • 6. A method according to claim 2, wherein said mismatches are on non-adjacent nucleotide positions of said repetitive unit.
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