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Fig. 1 | BMC Biotechnology

Fig. 1

From: Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

Fig. 1

Schematic diagram representing the two different approaches used for gene synthesis: a. Polymerase chain assembly using DNA template (PCA-DT), and b. Polymerase chain assembly DNA template-free (PCA-DTF). Each oligonucleotide is represented as an arrow; black arrows correspond to internal (inner) oligonucleotides while external (outer) oligonucleotides are denoted as orange arrows. Each strand of the desired gene is dissected into two or more oligonucleotides that are amplified (A) or overlap extended (b) by a DNA polymerase. A1-Two long oligonucleotides were designed with a 20 bp overlap region with the cloning vector; A2-Two sets of 40-mer oligonucleotides containing the 5’-end and 3’-end sequences of the desired gene were assembled by PCR; A3 – Successive oligonucleotides with 40 nt in length were designed with a 20 nt overlaps regions between adjacent oligonucleotides to construct the full-length of cloning vector containing the desired gene. The PCR products from A1, A2 and A3 strategies were inserted in E. coli cells through homologous recombination. Strategy B corresponds to a single-step PCR assembly step that does not require template DNA. B1 – Gapless 40-mer oligonucleotides containing 20 nt overlap regions between primers were used to assemble the synthetic gene; B2 – 40-mer oligonucleotides with 15 bp overlaps between forward and reverse primers, with gaps of 10 nt were mixed to produce the gene of interest; B3 – The synthetic gene was synthesized by mixing a 60-mer overlapping oligonucleotides containing gaps of 20 nt. After overlap extension by DNA polymerase, gene fragments from B1, B2 and B3 were cloned into a vector using a ligation-independent cloning method. All outer primers contain a vector complementary region at the 5’-end (highlighted in orange rectangles) or a 16 bp complementary sequence (highlighted in blue rectangles) to facilitate the cloning reaction

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