Rapid assembly of multiple arbitrary length DNA fragments

摘要

Aspects herein relate to composition, and related methods, for isolating and assembling DNA molecules without intermediate cloning steps.

主权项

1. A method of preparing a double-stranded (ds)DNA molecule, comprising:
providing a circular DNA vector comprising a vector backbone VB and a polynucleotide P-X-S, wherein P is a prefix nucleic acid sequence and S is a suffix nucleic acid sequence, each comprising one or more enzyme recognition sites, and X is a DNA part of interest; linearizing the circular DNA vector with one or more enzyme that cleaves one or more enzyme recognition sites in the suffix to produce a linear double-stranded (ds) DNA having a 5′ overhang at each end of the dsDNA; ligating to the 5′ overhang of the suffix a first oligonucleotide having a length of at least 20 nucleotides; removing the vector backbone from the linear dsDNA with one or more enzyme that cleaves one or more enzyme recognition sites in the prefix, producing a 5′ overhang; and ligating to the 5′ overhang of the prefix a second oligonucleotide having a length of at least 20 nucleotides, thereby preparing a dsDNA molecule.