Nucleic acid adaptors and uses thereof

摘要

Provided herein are compositions, systems, methods, and kits for joining together the ends of one or more polynucleotides using at least one pair of blocking oligonucleotide adaptors. Blocking oligonucleotide adaptors can be used to reduce the formation of adaptor dimers or trimers (or higher-order concatemers) which can improve the yield of desirable polynucleotide-adaptor products in any recombinant nucleic acid workflow. Blocking oligonucleotide adaptors can comprise a double-stranded oligonucleotide adaptor (duplex) having an overhang cohesive portion that anneals with a blocking oligonucleotide which can be a separate single-stranded oligonucleotide. A blocking oligonucleotide, when annealed to an overhang portion, can prevent undesirable hybridization of the overhang portion to another nucleic acid, such as the overhang portion from another blocking oligonucleotide adaptor or a polynucleotide of interest.

主权项

1. A method for circularizing a polynucleotide, comprising:
a) providing (i) a linear target polynucleotide having a first end and a second end, (ii) a first double-stranded nucleic acid adaptor with a target joining end and a first overhang end wherein the first overhang end comprises a first overhang nucleic acid sequence, (iii) and a second double-stranded nucleic acid adaptor with a target-joining end and a second overhang end wherein the second overhang end comprises a second overhang nucleic acid sequence, wherein the first and second overhang nucleic acid sequences being at least partially complementary to each other, wherein the first overhang is hybridized to a first single-stranded blocking oligonucleotide over at least a portion of its length and the second overhang is hybridized to a second single-stranded blocking oligonucleotide over at least a portion of its length, and wherein the target-joining end of the first double-stranded nucleic acid adaptor is joined to the first end of the linear target polynucleotide and the target joining end of the second double-stranded nucleic acid adaptor is joined to the second end of the linear target polynucleotide; b) removing the first and the second single-stranded blocking oligonucleotides from the first and the second overhangs; and c) hybridizing the first overhang having a first nucleic acid sequence with the second overhang having a second nucleic acid sequence, thereby circularizing the polynucleotide, wherein the first and second overhangs are not enzymatically ligated together.