Amplification and characterization of a beta-globin gene synthesized in vitro
Abstract
Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.
Citations
Rapid purification of covalently closed circular DNAs of bacterial plasmids and animal tumor viruses
Related Concepts
Related Feeds
Cell eTOC
Cell is a scientific journal publishing research across a broad range of disciplines within the life sciences field. Discover the latest research from Cell here.
CZI Human Cell Atlas Seed Network
The aim of the Human Cell Atlas (HCA) is to build reference maps of all human cells in order to enhance our understanding of health and disease. The Seed Networks for the HCA project aims to bring together collaborators with different areas of expertise in order to facilitate the development of the HCA. Find the latest research from members of the HCA Seed Networks here.