Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli.
Lambda-red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5'-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required.
Wong
Queenie N Y
QN
Department of Biochemistry, The University of Hong Kong Pokfulam, Hong Kong SAR, China.
Ng
Vivian C W
VC
Lin
Marie C M
MC
Kung
Hsiang-Fu
HF
Chan
Danny
D
Huang
Jian-Dong
JD
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D024121:Collagen Type X / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011995:Recombination, Genetic; D013940:Thymidylate Synthase / Q000235:genetics*
D024121:Collagen Type X; D004274:DNA, Recombinant; D013940:Thymidylate Synthase