Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides.
The lamda phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.
Lu
Lin-Yu
LY
University of Hong Kong, Pokfulam, Hong Kong, SAR, China.
Huen
Michael S Y
MS
Tai
Andrew Chi-Pang
AC
Liu
De-Pei
DP
Cheah
Kathryn S E
KS
Huang
Jian-Dong
JD
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D005838:Genotype; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D017384:Sequence Deletion
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D009841:Oligonucleotides; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli