Abstract: Methods are provided for assembly of a target polynucleotide sequence comprising at least a first double stranded polynucleotide (DSP) and at least second DSP, and optionally further DSPs. The method comprises an assembly reaction comprising steps including providing a first single stranded polynucleotide (SSP) comprising the polynucleotide sequence of one strand of the first DSP, and a second SSP comprising the polynucleotide sequence of one strand of the second DSP and converting the SSPs to double stranded form via a primer and polymerase-mediated extension reaction. The DSPs comprise polynucleotide sequences that are complementary to other polynucleotide sequences within the assembly reaction such that the ordering and directionality of each of the first and second, and further DSPs is determined by unique overhang pairing. Nucleic acid libraries and methods of making such libraries are also provided.

Abstract: Methods are provided for assembly of a target polynucleotide sequence comprising at least a first double stranded polynucleotide (DSP) and at least second DSP, and optionally further DSPs. The method comprises an assembly reaction comprising steps including providing a first single stranded polynucleotide (SSP) comprising the polynucleotide sequence of one strand of the first DSP, and a second SSP comprising the polynucleotide sequence of one strand of the second DSP and converting the SSPs to double stranded form via a primer and polymerase-mediated extension reaction. The DSPs comprise polynucleotide sequences that are complementary to other polynucleotide sequences within the assembly reaction such that the ordering and directionality of each of the first and second, and further DSPs is determined by unique overhang pairing. Nucleic acid libraries and methods of making such libraries are also provided.