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. 2019 Oct 4;47(22):e144. doi: 10.1093/nar/gkz851

Figure 2.

Figure 2.

Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled (A) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. (B) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. (C) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. (D) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.