PMC

Agarose gel showing a restriction analysis of potential ORF1-containing plasmids. Plasmids from 32 independent colonies were digested using SpeI enzyme and run on an agarose gel. An aliquot of a molecular weight marker was loaded in the outermost lanes and in the middle lane. An ∼3.9-kb band (black arrow), which represents the full-length ORF1, was seen in 29 out of the 32 digestions (∼90%).

Primer design in USER fusion. The blue lines encompass the desired DNA fusion product, in which X and Y are any pair of complementary deoxynucleotides. Only the sequence near a junction site is shown. The only requirement for USER fusion is the presence of a T located ∼8 residues downstream from an A (both residues shaded in black, as well as their complementary residues) in the proximity of the junction site. The sequences of the reverse primer (that will amplify the upstream fragment) and the forward primer (that will amplify the downstream fragment) are shown. In both primers, the selected T is replaced by a U (shaded in red).

Strategies for USER fusion. The two colored strands in the middle of each figure represent the desired DNA fusion product. The fragments to be joined together are presented in red (left) and in blue (right). In cases (C), (D), (E) and (F), a linker (gray) is desired at the junction site. The arrows represent the primers designed for USER fusion, and their color code indicates the origin of their sequence. (A) The selected A and T residues surround the junction site. (B) Both residues are chosen at one side of the junction site. (C) The A and T residues lie in the linker itself, and the linker sequence is partially included in each primer. (D) Both residues are chosen at one side of the linker, and the entire linker sequence is included in the primer that amplifies the sequence at the other side. (E) The residues are located at each side of the linker, and the entire linker sequence is included in both primers. (F) The residues are located at each side of the linker, but the linker sequence is not included in any of the primers. Two complementary oligonucleotides that carry the linker sequence are used as an intermediate DNA fragment to be fused.

(A) Outline of ORF1. The coding regions of genes X1, X2, and X3 are linked together by two different ∼60-bp linkers, L1 and L2. ORF1 has a length of ∼3.9 kb. (B) Assembly of ORF1. X1, X2 and X3 sequences were amplified using primers that contained a single U (instead of a T) near their 5′ end. The L1 sequence was included as two separate fragments in primers X1-rev and X2-fwd, and the L2 sequence was similarly included in primers X2-rev and X3-fwd so that both ∼60-bp linkers were constructed after the fusion procedure. The PCR products were mixed with a pre-digested USER-compatible vector and treated with the deoxyuridine-excising USER™ enzyme mix. This generated 3′ overhangs that were designed so that the internal ones complemented each other (indicated by arrows), while the outermost ones complemented the overhangs of the pre-digested vector. This design enabled the formation of a stable circular hybridization product that was transformed directly into E. coli without prior ligation.