Fig 1.
A) Co-transformation cloning [2, 3]. The two homology regions are shown in red and blue. B) Ligation-independent cloning (LIC) [6] using complementary single-stranded ends of defined length. C) Sequence- and ligation-independent cloning (SLIC) [7, 8] using single-stranded ends of undefined length. After insert-to-plasmid annealing, single-stranded regions of excessive length result in single-stranded gaps (small arrows) in the circularized plasmids. D) Gibson assembly [9]. After insert-to-plasmid annealing at the overhangs of undefined length, the 3’-ends serve as primers for extension by DNA polymerase, which fills in the single-stranded gaps. Finally, the nicks are ligated.
Fig 2.
Critical factors for co-transformation cloning.
A and B) E. coli cells were co-transformed with the lacZ insert PCR fragment and either PCR-linearized plasmid or restriction-enzyme digested/linearized plasmid. A) Data points represent the percentage of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. C and D) E. coli cells were co-transformed with the lacZ insert PCR fragment and PCR-linearized plasmid. The same experiment was also performed in the presence of a 10-fold molar excess over insert of each of two 15 nt long oligonucleotides comprising the sequence of the primer homology overhangs. All experiments were performed with and without a heat-denaturation / slow cooling step. C) Data points represent the percentage of positive (blue) colonies averaged over three experiments ±SD. D) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD.
Fig 3.
Producing two single-stranded ends by incomplete PCR.
If primer extension in late PCR cycles is incomplete in a subset of PCR products, double-stranded DNA molecules with a single-stranded end may form. To produce double-stranded DNA molecules with two single-stranded ends, the PCR mixtures have to be denatured after the final polymerase extension step to allow new strand combinations upon renaturation. The concept shown here for the plasmid also applies to the insert. By the same mechanism, double-stranded DNA molecules containing two single-stranded ends could potentially form during PCR cycling if critical reagents are used up prior to the last PCR cycle(s), so that no DNA synthesis takes place in the last cycle(s).
Fig 4.
Single-stranded gaps in plasmids reduce cloning efficiency.
A) Schematic overview of the concept of recombinant circle PCR (RCPCR)[28]. The insert and the plasmid are each amplified in two separate PCR reactions. One of the resulting insert and plasmid PCR products contain only homology region 1 (red). The other insert and plasmid PCR products contain only homology region 2 (blue). The four PCR products are mixed, heated and allowed to cool slowly, allowing new strand combinations. This results in the formation of double-stranded DNA molecules that can circularize through insert-to-plasmid annealing at single-stranded terminal complementary regions. B) A further downstream primer annealing site (green arrow) was chosen for PCR 1b. The resulting PCR 1 products lacking homology region 1 (red) were therefore shortened (green cross), and in combination with insert 2 and the two plasmid PCR products, after heating and slow cooling, formed a circular plasmid containing a single-stranded gap in addition to the nicks. The length of the gap depended on the (green) primer annealing site and for our experiments measured 5, 10, 20 or 40 nucleotides, respectively. C) Chemically competent E. coli cells were transformed with the plasmids described in (A) and (B). Plasmids with no gaps, or with 5, 10, 20 or 40 nucleotides long single-stranded gaps were tested. Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. D) Percentage of positive (blue) colonies. Data points represent the percentage of positive colonies averaged over three experiments ±SD.
Fig 5.
“Dissection” of the Gibson assembly into its component reactions.
In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E. coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.