Fig. 2
Random access and sequential Gibson strategy for DNA storage. a Our sequential Gibson Assembly strategy enables random access and concatenation of any particular file from a pool of DNA oligonucleotides. We demonstrated up to 24-fragment assembly by performing two sequential assemblies of 6 and 4 fragments, respectively. b First, a given file is PCR-amplified using primers specific to its address (AD1 and AD2) and containing overlapping overhang sequences (Xn). For a three-fragment assembly, three separate PCR amplification reactions are carried out, each containing primer pairs with different overhangs based on a sequential assembly design (X1 and X2*, X2 and X3*, and X3 and X4*). Upon amplification, products are purified and combined by Gibson Assembly. The assembly product can consist of any combination of oligonucleotides in a given file pool separated by ordered overhangs with a consistent directionality (e.g., X1-AD1-Payload1-AD2-X2-AD1-Payload2-AD2-X3-AD1-Payload3-AD2-X4). This product is then amplified by using primers specific to ends of the assembly (X1 and X4*) and can be used as the starting material for a second assembly. c Gel electrophoresis size distribution corresponding to a PCR amplification of a 6-fragment Gibson Assembly. The expected band size was 1110 bp. d Gel electrophoresis size distribution corresponding to a PCR amplification of a 24-fragment second Gibson Assembly. The expected band size was 4590 bp. The correct fragment was gel extracted and used for nanopore sequencing
