Adaptors, and synthesis and cloning of proinsulin genes

#This invention is concerned with the synthesis of exact DNA genetic sequences and the joining of the synthetic gene to replicable cloning vehicles. Of particular concern is the total synthesis of the human-like proinsulin gene, the insertion of the gene into cloning vehicles, and the transferring of the hybrid DNA molecules into host cells, the transformed cells thus having the ability to produce the specified human-like proinsulin protein. In this application, several adaptors for trimming DNA have been described, and they can trim any DNA down to a desired length.

#In recent years, methods have been developed (see "Molecular Cloning of Recombinant DNA", eds., W. A. Scott and R. Werner, Academic Press Inc., 1977),

#(1) for the in vitro joining by DNA ligase of a DNA segment to be cloned (scheme 1, structure I) to a cloning vehicle (DNA capable of independent replication, structure II),

#The principle behind this type of retrieving (trimming) adaptor is based on the knowledge that certain restriction endonucleases such as HphI and MboII cleave DNA eight nucleotides away from their recognition sequence (R. J. Roberts, Methods in Enzymol. 68, 27, 1980). In formula 3, the five nucleotides with a line drawn above constitute the recognition sequence, where N stands for any nucleotide. ##STR5##

#We have designed and synthesized adaptors for trimming DNA as symmetric adaptors (non-symmetric adaptors based on formula 3 can also be used); ##STR6##

#This type of adaptor for trimming DNA can be blunt-end ligated to any DNA from which 8 nucleotides at the end can be removed. MboII adaptor is used as an example to remove eight nucleotides N N N N N N N N from the end of a cloned insulin A-chain gene. ##STR7## the dXTP is chosen so that it is identical to the one adjacent to N at the 3' end of the cloned A-chain gene, e.g. if the nucleotide "X" is C then dCTP is used.