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Fig. 9. Recombineering-mediated mutagenesis. (A) `Blind' mutagenesis.
To perform a site-specific change in a fragment within a target plasmid, a PCR
fragment or oligonucleotide that contains the desired mutation is transformed
into bacteria that contain recombineering functions and a target plasmid.
Recombination results in the incorporation of the desired mutation:
substitution, deletion or insertion. The bacteria are then screened by PCR for
the proper mutagenic event. (B) Mutagenesis using a positive/negative
selectable marker. First, in the positive-selection step, a PCR fragment
containing a positive (+)/negative (-) selectable marker is transformed into
bacteria that contain recombineering functions and the target plasmid.
Individual colonies containing the correct recombinant plasmid are then
selected. Second, during the counterselection step, a PCR fragment containing
the desired change, such as a tag, might replace the positive/negative
selectable marker. Counterselection or negative selection may result in the
selection of a correct recombinant plasmid that can be identified through PCR.
(C) RecA-assisted modification. A specialized plasmid that contains a
selectable marker (+), a counterselectable marker (-), RecA and a mutation
flanked by two homology boxes (A and B) is transformed into bacteria. During a
first recombination event, identified through selection, this plasmid can
integrate through homology box A (shown) or B (not shown), resulting in a
co-integrate. During a second recombination event, identified through
counterselection, this co-integrate can resolve to the original plasmid (not
shown) or the modified plasmid (shown).
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