Methods and compositions for forming ligation products

A method of conducting a targeted rolling circle amplification, comprising:

• (a) providing a circular polynucleotide, wherein the circular polynucleotide is formed by:
• (i) forming a first polynucleotide complex by mixing a plurality of cell-free DNA polynucleotides and a single-stranded adaptor with a capture probe, wherein a first segment of the capture probe hybridizes with a target polynucleotide sequence from the plurality of cell-free DNA polynucleotides via sequence complementarity and a second segment of the capture probe hybridizes with the single-stranded adaptor via sequence complementarity, and 3′ end of single-stranded adaptor and 5′ end of the target polynucleotide sequence from the plurality of cell-free DNA polynucleotides of the first polynucleotide complex are located adjacent from each other on the capture probe and 5′ end of the target polynucleotide sequence from the plurality of cell-free polynucleotides of the first polynucleotide complex lacks a sequence complementarity to the capture probe and forms a flap structure;
• (ii) contacting the first polynucleotide complex with a reaction mixture comprising a flap endonuclease under conditions such that the flap structure located on 5′ end of the target polynucleotide sequence from the plurality of cell-free polynucleotides of the first polynucleotide complex is cleaved and producing a second polynucleotide complex comprising a cleaved target polynucleotide sequence, the single-stranded adaptor and the capture probe, wherein the flap endonuclease acts as a structure specific endonuclease for cleaving the flap structure located on 5′ end of the target polynucleotide sequence of the first polynucleotide complex;
• (iii) ligating the cleaved target polynucleotide sequence to the single-stranded adaptor of the second polynucleotide complex, thereby forming a ligation product;
• and
• (iv) producing the circular polynucleotide by circularizing the ligation product;
• (b) generate a plurality of amplification products comprising concatemers of the ligation product by subjecting an amplification reaction mixture to multiple cycles of a rolling circle amplification, wherein the amplification reaction mixture comprises (i) a polymerase having strand displacement activity, (ii) the circular polynucleotide obtained from step (a), and (iii) at least a first primer that hybridizes to a sequence of at least a segment of the single-stranded adaptor.