Figure 1.
A diagram of the Hot Fusion process for single fragment cloning.
Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
Figure 2.
A diagram of the Hot Fusion process for multi-fragment assembly.
Red, green, purple and blue boxes on the vector and PCR products indicate overlapping sequences (25–30 bp).
Table 1.
Examples of the oligo primers used in this study.
Figure 3.
Single fragment cloning by Hot Fusion.
(A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ. The linearized vector was directly used for Hot Fusion. (B) Amplified PCR products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb DNA ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct (Table 2).
Table 2.
PCR screening of colonies from nine Hot Fusion transformation plates.
Figure 4.
Creation of seamless constructs by Hot Fusion.
(A) A schematic diagram for the creation of a seamless construct. The vector was linearized by SphI digestion (leaving CATG 3′ overhangs), heat-inactivated and directly used for cloning. The PCR products contain the overlapping sequences at each end (excluding the CATG overhang). The ribosome binding site is underlined. (B) Examples of the transformation plates. Blue colonies contain the parental vector and white colonies contain potential positive clones. The negative control is shown as plate a (unpurified digested vector without added PCR product). Plates, b and c are two examples of transformation plates containing potential positive clones. (C) PCR screening results of 8 colonies for each construct (each plate). Eight PCR products of each construct were loaded every other lane on agarose gels by multi channel pipette.
Figure 5.
PCR screening of multi-fragment assembled clones from Table 3.
Eight clones (colonies) from each construct were grown overnight in the 96-well plate containing LB + Spectinomycin (50 ug/ml). One µl of the over-night grown cell culture was used for PCR screening with base vector primers (18 bp) located immediately upstream of the promoter and downstream of the terminator. PCR products for each construct were loaded every other lane on a 1.0% agarose gel (containing ethidium bromide) with a multi channel pipette. GeneRuler 1 kb Plus DNA Ladder was used as the DNA marker.
Table 3.
One step assembly of promoter, GOI and terminator by Hot Fusion reaction.
Figure 6.
Simultaneous cloning of seven fragments by Hot Fusion in order to eliminate methylation sites.
(A) Distribution of Agrobacterium methylation sites (ME) on T-DNA of a vector encoding e35s driving the GUS gene. (B) Alignment of two clones (HF902-3 and HF902-6) eliminating ten Agrobacterium methylation sites on T-DNA. Four fragments (gB1, gB2, gB5 and gB7) were synthesized to readily mutate the multiple Agrobacterium methylation sites within the regions and three fragments (PCR3, PCR4 and PCR6) were amplified from the template with oligo primers containing the mutated base pairs. All seven fragments plus the vector were combined in one Hot Fusion reaction.
Figure 7.
Creation of RNAi constructs by Hot Fusion.
(A) Design of RNAi elements for Hot Fusion. Anti-sense (AS) and sense (S) fragments of 150 bp (a), 300 bp (b) and 600 bp (c) overlapped with the vector and spacer as indicated. (B) Restriction digestion of plasmids containing RNAi elements. Three to five clones were analyzed for each construct (a, b and c). Each clone was digested by AscI and PmeI flanking the insert. The a, b and c contained the 150 bp, 300 bp and 600 bp of both anti-sense and sense fragments, respectively. M represents GeneRuler 1 kb Plus DNA Ladder.
Figure 8.
Comparison study of Hot Fusion and Gibson Assembly (NEB kit).
(A) Cloning strategy for single and multi-fragment assembly. A full lacZ gene and three pieces of the lacZ gene with overlapping sequences were amplified, and cloned into a base vector digested with SfaAI (unpurified). (B) Transformation efficiency of lacZ assembly by Hot Fusion and Gibson Assembly (from NEB). The top panels are the transformation plates from Hot Fusion. The bottom panels are the transformation plates from Gibson Assembly. The same amount of base vector was used in all reactions and the same amount of insert was used in the same treatment. Blue colonies contain the lacZ gene or all three pieces of the lacZ gene correctly cloned or assembled together (in frame). White colonies contain the parent vector. (C) Cloning efficiency of Hot Fusion and Gibson Assembly. Colonies were counted from each transformation plate.
Figure 9.
Comparison study of Hot Fusion under different cycling conditions.
A full lacZ gene with overlapping sequences to a vector was amplified and used for testing. The same amount of linearized base vector and lacZ insert were used in each reaction. The vector and insert were mixed and incubated at 37°C for 0, 1, 5 or 10 minutes, respectively, and then switched to 68°C for 5 or 10 minutes, respectively. The control used the regular Hot Fusion condition (1-hour at 50°C) as described in the Materials and Methods. Blue and red bars indicate numbers of clones containing either the lacZ gene or empty vector, respectively.