TABLE 1.
Accuracy of DSB rejoining
Plasmida | Ade+ colonies
|
Ade− colonies
|
Fraction precise joiningd | |||||
---|---|---|---|---|---|---|---|---|
Fraction of total coloniesb | Sequencing resultsc
|
Fraction of total coloniesb | Sequencing resultsc
|
|||||
Precise | Imprecise | Precise | Imprecise | Other | ||||
pTW423-34 | 0.59 ± 0.07 | 38/40 | 2/40 | 0.41 ± 0.07 | 1/20 | 5/20 | 14/20 | 0.81 ± 0.07 |
pTW423-313 | 0.84 ± 0.04 | 20/20 | 0/20 | 0.16 ± 0.04 | 15/20 | 0/20 | 5/20 | 1.00 ± 0.04 |
pTW423-313/4 | 0.06 ± 0.04 | 11/15 | 4/15 | 0.94 ± 0.04 | 0/24 | 4/24 | 20/24 | 0.20 ± 0.03 |
pTW423-313/9 | 0 | 1.00 | 0/24 | 3/24 | 21/24 | 0 |
See Fig. 7 (also see Fig. S1 in the supplemental material) for plasmid configurations.
For each plasmid, the mean fraction of Ade+ and Ade− colonies ± 1 standard deviation was calculated from four transformations.
Joints were amplified, sequenced, and categorized as “precise,” “imprecise,” or “other.” “Precise” indicates either wild-type ADE2 sequence or, in the case of Ade− colonies, joints that showed correct overhang alignment but had mutations outside the overhangs introduced during oligonucleotide synthesis. In the case of pTW423-313/4 and pTW423-313/9, precise alignment refers to pairing of the available 4-base microhomology. “Imprecise” indicates sequences in which oligonucleotide ligation could be verified but overhangs were misaligned or recessed microhomologies were utilized. “Other” indicates parent plasmids, sequences in which one oligonucleotide pair was missing, or joints that could not be amplified or sequenced due to deletion of primer sites. The data are shown as the number of colonies with each sequencing result relative to the total number of colonies sequenced.
The fraction of precise rejoining was calculated from the combined data from Ade+ and Ade− colonies, but excluding the “other” category. This category was excluded, because it could not be determined that the plasmids responsible for these events had been successfully ligated to both oligonucleotide pairs. The number thus reflects the fraction (not frequency) of precise joints among all recovered transformants known to result from plasmids bearing both oligonucleotide pairs.