Assembly of genes from partially overlapping fragments using single-stranded DNA and sequence-specific synthetic oligodeoxynucleotides.
A simple procedure for the precise assembly of functional DNA sequences from overlapping fragments is described. The fragments to be joined are cloned in tandem in the proper relative orientation into a vector from which single-stranded DNA copies can be obtained. Single-stranded DNA is cut by a restriction enzyme at corresponding sites in the two overlap regions, which are made double-stranded by annealing an oligonucleotide of appropriate sequence to them. This results in the excision of the unwanted sequences between the two overlap regions. After removal of the restriction enzyme the DNA is reannealed using the same oligonucleotide, ligated to give closed circular molecules and used to transform competent cells. Clones with the desired structure appear in the progeny at high frequency. The method has the advantage that restriction enzymes with short recognition sequences, cutting frequently in the target DNA, can be used and hence the overlap region required can be quite short.
Clegg
J C
JC
Pathology Division, Centre for Applied Microbiology and Research, Salisbury, United Kingdom.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics*; D005807:Genes, Overlapping; D005813:Genes, Synthetic; D009841:Oligonucleotides / Q000235:genetics
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes