Table 1.
PCR condition for amplification of longer inserts.
Table 2.
PCR condition for amplifying DNA shorter than 80 bp.
Fig 1.
Comparison of enzymes with exonuclease activities for SLIC experiments.
(A) A pBluescript II SK+ vector was chosen as a PCR template for amplifying the indicated lengths of fragments. Both PCR products, depicted in blue and red, were combined and assembled into the intact pBluescript II SK+ vector using SLIC techniques. The black lines represent the 15 bp complementary sequences of each product. (B) A schematic diagram of phosphodiester and phosphorothioate (PT) bonds used in the preparation of primer synthesis. (C) Primers for amplifying the two different regions of pBluescript II SK+ in (A) were modified with a 5’ end phosphate (P-15 mer-15 mer), a 5’ end phosphate together with five PTs (P-15 mer-5PT-10 mer), five PTs only (15 mer-5PT-10 mer), or were not modified (15 mer-15 mer). The orange braces represent the complementary sequences. (D) Transformation efficiencies were calculated by counting the number of colonies in triplicate experiments. 1×108 competent cells were used for transforming the mixture of 50 ng of each purified PCR product after undergoing SLIC experiments. Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01, triple asterisks; P-value lower than 0.001. All experiments were repeated three times. (E) Representative plates of (D) are shown. CFU; colony forming unit, Ctl; control, Lambda Exo; lambda exonuclease, T5 exo; T5 exonuclease.
Fig 2.
Optimization of the T5-exonuclease-based SLIC method.
(A) Subcloning efficiency was increased by reducing the reaction time and the amount of T5 enzyme. The reaction was carried out at 30°C on a heat block. Double asterisks represent a P value less than 0.01, while triple asterisks indicate a P value less than 0.001, derived from three independent experiments. A linear DNA mixture without T5 exonuclease (-T5 Exo) was used as a negative control. (B) Representative plates of (B) are shown. CFU; colony forming unit.
Fig 3.
At least 5 PTs are required for the subcloning of small DNA fragments.
(A) A schematic illustration of the subcloning procedure of a 6xHis epitope into an XbaI site of the pCS2+ vector. To amplify a 48 bp-length PCR product encompassing a 6xHis epitope (red uppercase), the indicated primers, depicted with arrows, were annealed via complementary sequences before undergoing PCR. The asterisks indicate the position and number of PT modifications. (B) Electrophoresis of PCR samples on an 8% acrylamide gel. The 4 μl of crude PCR products (20% of PCR) without purification were treated with or without 0.1 U of T5 exonuclease in a total reaction volume of 10 μl for 30 min at 30°C. Note that an increased number of PT internucleotide linkages proportionally stabilized the PCR products. M stands for the 100 bp size marker. (C) The PCR product with primers incorporating 5 PTs showed the highest subcloning efficiency into the pCS2+ vector. The experiments were carried out three times. Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01, triple asterisks; P-value lower than 0.001. (D) Representative images of (C). (E) Five randomly selected colonies from the plates in (D) were validated by Sanger sequencing. Only the samples with 5PT showed 100% subcloning accuracy. CFU; colony forming unit.
Fig 4.
Comparison of DAPE and Gibson assembly in terms of cloning efficiency for small DNA fragments.
(A) A 48 bp PCR product was subjected to DAPE or Gibson assembly, using primers with or without 5’ PT modifications. A purified 50 pg of pCS2+ vector, linearized by Xba I digestion, was used for cloning with 1 μl of crude PCR samples. Gibson-/linear vector; enzyme free reaction with linearized vector only. Gibson+/linear vector; Gibson technology reaction with linearized vector only. Gibson+/no PT; Gibson technology reaction with linearized vector and PCR amplified insert without 5PT modification. Gibson+/5PT; Gibson technology reaction with linearized vector and 5PT modified PCR product. T5-/linear vector; T5 free reaction with linearized vector only. T5+/linear vector; T5 (0.1 U) reaction with linearized vector only. T5+/no PT; T5 (0.1 U) reaction with linearized vector and PCR amplified insert without 5PT modification. T5+/5PT; T5 (0.1 U) reaction with linearized vector and 5PT modified PCR product. (B) Graphical representation of the data shown in (A). Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01, triple asterisks; P-value lower than 0.001. All experiments were repeated three times. CFU; colony forming unit.
Fig 5.
(A) A schematic diagram of gRNA cloning into the px330-puro vector. After linearization with BbsI treatment, 50 ng of the linear vector was subjected to cloning procedures with 1 μl (5%) of 54 bp PCR products for DAPE or the same amount of 52 bp PCR products for Golden Gate cloning. While the linearized vector was purified using purification kits, the crude PCR products were directly used for cloning. (B) Comparison of Golden Gate cloning and DAPE. Although Golden Gate cloning (left) yielded more colonies than DAPE (right), the former also resulted in a higher number of non-positive clones. DAPE without PTs failed to form positive clones. A linear vector without gRNA was used as a negative control (labeled as "linear vector"). Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01. The triple asterisks represent P value lower than 0.001, derived from triple experiments. (C) Representative individual images of triplet (B). (D) Cloning accuracies of Golden Gate cloning and DAPE were validated by sequencing. Five randomly selected clones were sequenced to identify the orientation and sequences of the gRNA. Only DAPE with 5 PTs showed 100% accuracy of cloning. CFU; colony forming unit.
Fig 6.
Five PTs are required when the DNA fragments are smaller than 80 bp.
(A) A schematic diagram of the experimental procedure. Different sizes of DNA fragments encoding a portion of PCR template (EGFP) were amplified with the indicated primers depicted by arrows. The red and blue lines attached to the primers represent the 15 bp complementary sequences between the pCS2+ vector and the insert. The pCS2+ vector was linearized with XbaI before purification. 50 ng of linearized vector was used for subcloning with the same amount of DNA, except for a 50 bp and 80 bp inserts, of which 1 μl (5%) of crude PCR products was directly used for DAPE cloning. (B) Five PTs are obligatory for cloning when the DNA fragments are smaller than 50 bp, and are highly recommended when they are less than 80 bp. Double asterisks represent a P value less than 0.01, while triple asterisks indicate a P value less than 0.001, derived from three independent experiments. (C) Representative plates of triplet (B). (D) Five randomly selected clones from the plates in (C) were sequenced to evaluate the accuracy of DAPE. Except for clones obtained from samples without 5 PTs of 50 bp inserts, all others showed 100% accuracy in cloning. CFU; colony forming unit.
Fig 7.
5PT-incorporated primers may prevent secondary structure formation at both ends of PCR products after reaction with T5 exonuclease.
(A) A schematic diagram of the experimental condition. The linearized pCS2+ vector, digested with XbaI, was reacted with an insert encoding a kanamycin resistance gene and containing 30-bp leadzyme sequences at both ends, which are prone to forming secondary structures. (B) Leadzyme sequence and its expected secondary structure. (C) Representative plates showing subcloning results. Gibson+/linear vector; Gibson technology reaction with linearized vector only. Gibson+/no PT; Gibson technology reaction with linearized vector and PCR amplified insert without 5PT modification. Gibson+/5PT; Gibson technology reaction with linearized vector and 5PT modified PCR product. T5-/linear vector; T5 free reaction with linearized vector only. T5+/linear vector; T5 (0.1 U) reaction with linearized vector only. T5+/no PT; T5 (0.1 U) reaction with linearized vector and PCR amplified insert without 5PT modification. T5+/5PT; T5 (0.1 U) reaction with linearized vector and 5PT modified PCR product. (D) Graphical and statistical representation of the data shown in (C). Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01, triple asterisks; P-value lower than 0.001. All experiments were repeated three times. (E) Colony PCR analysis of the emerging clones on the plate, performed on a 1% agarose gel. Eight randomly selected colonies were subjected to PCR using primers that amplify the expected DNA size (~1 kb). (F) Graphical representation of the date shown in (E). CFU; colony forming unit.
Fig 8.
Multiple DNA fragments assembly with DAPE.
(A) A schematic illustration of the DNA assembly. The identically colored ends of DNA fragments represent 15 bp complementary sequences. Asterisks on the primers represent 5PT linkages. The pCS2+ vector was linearized with XbaI, and then 50 ng of the vector was used for the DNA assembly experiment. The 54 bp length of 6xHis and the 57 bp length of Flag epitope were amplified with 5 PTs primers. The full-length EGFP was amplified with non-PT primers. While 50 ng of EGFP PCR product was used for DNA assembly, 1 μg (5%) of each crude small DNA epitope was added to the reaction tube for DAPE. (B) Comparison of DAPE with conventional SLIC using T5 exonuclease. The single asterisk represents P-values lower than 0.05, derived from triple DNA assembly experiments. (C) Representative images of (B) from among the triple experiments. (D) Electrophoresis of PCR products on a 2% agarose gel. M represents 100 bp size marker. The small epitopes amplified by PCR migrated slower compared to the primer dimers. (E) Ten randomly selected clones from the plates in (C) were validated by sequencing to analyze the accuracy of DNA assembly. DAPE is superior in both accuracy and efficiency compared to conventional SLIC when using small DNA fragments in multiple DNA assembly. CFU; colony forming unit.