Fig 1.
Double-stranded circular plasmid is linearized and amplified by PCR. Alternatively, the vector can be linearized by restriction digestion. The primers used for amplification of the insert must comprise segments that are complementary to the primers used for vector linearization. The start point of matching nucleotide segments are marked by yellow and red points. The PCR products (blue) are combined, treated with 3’-exonuclease and the hybridized mixture is used to transform competent E. coli cells.
Fig 2.
Modified QuikChange mutagenesis scheme using a 3’-exonuclease to generate single-stranded 5’ overhangs.
Plasmid DNA is amplified by PCR using a high-fidelity DNA polymerase such as Q5 (New England Biolabs). Forward and reverse primers overlap partially [20]. The red and yellow circles (triangles) mark the 5’ (3’) ends of the primers. The green crosses mark mismatches between the mutation primers and the template DNA. The present work demonstrates the advantage of using an attenuated 3’-exonuclease to generate single-stranded overhangs. Filling in of single-stranded DNA and re-ligation of the circular plasmid is achieved by endogenous E. coli enzymes following transformation.
Fig 3.
Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb linearized vector.
DNA was digested at 37 °C with the T4P mutants E114A or Y320A at a final concentration of 0.3 μM. Experiments were performed in triplicate. Vector and insert were used in 1:1 molar ratio. (A) Digestion with E114A for the durations indicated. Column c shows the result of a negative control without T4P treatment, which indicates that the presence of the enzyme made no significant difference. (B) Digestion with Y320A for the durations indicated. (C) Result of digestion with the mutant Y320A for different lengths of insert–vector overlaps (see Table A in S1 File for the primers used to amplify the inserts) and two different sizes of insert (1.2 kb N388 and 2.7 kb wild-type T4P, respectively).
Fig 4.
DNA digestion by the E2 enzyme and wild-type T4P.
The substrate was a 98-bp double-stranded DNA fragment (Fig A in S1 File) amplified by PCR from pETMCSI with the primers 98bp-f and v-r (Table A in S1 File) and purified by a PCR clean-up kit. The digestion product was purified by phenol extraction, precipitated with ethanol, dried and analysed on a 12% continuous denaturing PAGE run in TBE buffer (89 mM Tris-borate, pH 8.3, 2 mM EDTA) containing 7 M urea. Lane M: DNA markers (number of nucleotides indicated at the side). See Text B in S1 File for more details. (A) Digestion of a 157 nM solution of 98-bp DNA with 1.6 μM E2 at 37 °C. Aliquots were taken at 15 minute intervals as indicated. (B) Digestion of 98-bp DNA (157 nM solution) with wild-type T4P (New England Biolabs) at 16 °C for 30 minutes, using the enzyme at different concentrations. Reactions were terminated by adding 1 μL 10% SDS. Lanes 1–4: T4P used in 100-, 30-, 10-, and 3-fold dilution relative to the amount (5 units or 0.6 μg) used for the reaction in lane 5, which corresponded to a concentration of wild-type T4P of 0.3 μM.
Fig 5.
Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb vector.
The plasmid (pETMCSI) [21] was linearised by PCR using as template (A) undigested and (B) NdeI-EcoRI double-digested plasmid. Digestions with E2 and DpnI were performed at 37 °C for the durations indicated and all experiments were performed in triplicate. (C) Negative control conducted with vector or insert only, following 60 minutes of E2/DpnI digestion. V1: linearized vector. V2: vector double-digested with NdeI and EcoRI prior to linearization by PCR. I: insert.
Table 1.
Cloning yields obtained with the standard cloning protocola.
Table 2.
Comparison of the cloning efficiency of RQ-SLIC compared to three established methods based on wild-type T4Pa.