Compositions and methods for capturing and amplifying target polynucleotides using modified capture primers

A method for capturing and amplifying first target polynucleotides on a surface of a substrate, the method comprising:

• providing a composition comprises:
• a plurality of capture primers coupled to the surface of the substrate, each of the capture primers comprising modified nucleic acids;
• and
• a plurality of orthogonal capture primers coupled to the surface of the substrate, each of the orthogonal capture primers comprising modified nucleic acids;
• providing a fluid comprises:
• first target polynucleotides, wherein each of the first target polynucleotides comprises a first adapter that is complementary to the capture primers and a second adapter that is complementary to the orthogonal capture primers;
• and
• second target polynucleotides that are complementary to the first target polynucleotides;
• contacting the composition with the fluid under a condition that inhibits hybridization of the second target polynucleotides to the first target polynucleotides
• forming first duplexes on the surface of the substrate by hybridizing the first adapter of some of the first target polynucleotides to the capture primers;
• forming second duplexes on the surface of the substrate by hybridizing the second adapter of some of the first target polynucleotides to the orthogonal capture primers;
• and then
• generating amplicons of the first target polynucleotides by amplifying the first target polynucleotides of the first duplexes and/or the second duplexes,
• wherein each of the first duplexes and each of the second duplexes has a melting temperature (Tm) that is greater than a Tm of third duplexes that are formed by hybridization of the second target polynucleotides to the first target polynucleotides.