title: In situ overlap and sequence synthesis during DNA assembly.
abstract: Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30-255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.
authors.affiliation: EMBL-CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) , Dr. Aiguader 88, 08003 Barcelona, Spain.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Paetzold
authors.forename: Carlo
authors.initials: C
authors.lastname: Carolis
authors.forename: Tony
authors.initials: T
authors.lastname: Ferrar
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.forename: Maria
authors.initials: M
authors.lastname: Lluch-Senar
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D004247:DNA