Insertion of synthetic copies of human globin genes into bacterial plasmids

Abstract

Double stranded human globin cDNA was synthesized by use of viral reverse transcriptase from globin mRNA of cord blood of premature infants requiring exchange transfusions. The cDNA was introduced into plasmids and the recombinant DNA plasmids used to transform E. coli X1776. A number of transformants were obtained. Plasmid DNA from selected colonies was isolated and characterized for the type of globin cDNA it contained by three types of procedures: 1) hybridization to previously characterized 3H-labeled alpha,beta and gamma cDNA; 2) analysis of the size and nature of fragments produced by digestion of the plasma DNA by different restriction endonucleases; and 3) by rapid DNA sequence analysis of selected DNA fragments produced by restriction endonuclease digestion. Analysis by these techniques of plasmid DNA from different colonies has definitively identified the presence of human alpha, beta or gamma cDNA sequences in different plasmids.