Fig. 1From: AFEAP cloning: a precise and efficient method for large DNA sequence assemblyAFEAP cloning. a Schematic details show the flow chart of multi-fragment assembly with AFEAP cloning. The first PCR yields linear DNA fragments (step 1), and is followed by a second asymmetric (one primer) PCR (step 2) and subsequent annealing (step 3) that inserts overlapping overhangs at 5′ end of each DNA fragment. These double-stranded DNA fragments are then subsequently assembled “hand-in-hand” (step 4). The nicks in the annealed multi-part DNAs are sealed by DNA ligase to form transformable plasmid (step 4), followed by bacterial transformation to yield the desired plasmids, which is confirmed by DNA sequencing (step 4). b A typical AFEAP cloning showing the region of overhang, and the two set primers of two adjacent fragments. Fw: forward primer, Rv: reverse primer, OH: overhang region, ssDNA: single-stranded DNABack to article page