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Enhanced probe binding

  • US 10,294,516 B2
  • Filed: 01/16/2014
  • Issued: 05/21/2019
  • Est. Priority Date: 01/18/2013
  • Status: Active Grant
First Claim
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1. A method for producing a biomolecule analyte, the method comprising:

  • a) providing a single-stranded DNA or RNA template comprising (i) two or more secondary structures and (ii) a plurality of probe recognition sites comprising at least two different types of nucleotide recognition sites complementary to at least two different types of sequence-specific oligonucleotide probes, wherein each type of nucleotide recognition sites of the at least two different types of nucleotide recognition sites comprises multiple identical nucleotide recognition sites;

    b) hybridizing a first type of the at least two different types of sequence-specific oligonucleotide probes to the template such that multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes bind to the multiple identical nucleotide recognition sites of the first type of nucleotide recognition sites of the at least two different types of recognition sites and form a hybridized template comprising multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes;

    c) after the hybridizing step, conducting a base extension reaction from a 3′

    end of each of the multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes along the hybridized template comprising multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes;

    d) terminating the base-extension reaction after step c) is conducted for a time period such that (i) the multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes are securely hybridized to the template and (ii) other types of recognition sites of the at least two different types of recognition sites of the hybridized template are left unoccupied, thereby forming a base extension product comprising the hybridized template;

    e) after step d), applying heat or chemicals to the base extension product to denature the hybridized template such that at least a portion of said two or more secondary structures of the hybridized template is broken, thereby forming a denatured DNA or RNA comprising the multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes; and

    f) thereafter, performing steps b) to d) by hybridizing a second type of the at least two different types of sequence-specific oligonucleotide probes to the denatured DNA or RNA, thereby producing the biomolecule analyte, wherein the biomolecule analyte comprises DNA or RNA (i) comprising a reduced number of the secondary structures and (ii) comprising multiple probes of the first type of the at least two different types of sequence-specific oligonucleotide probes and multiple probes of the second type of the at least two different types of sequence-specific oligonucleotide probes, wherein the first type of the at least two different types of sequence-specific oligonucleotide probes and the second type of the at least two different types of sequence-specific oligonucleotide probes have different nucleotide sequences and are hybridized to different types of nucleotide recognition sites of the at least two different types of nucleotide recognition sites.

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