How to Make Hybrid DNA and Grow It in Microbes

This patent describes the foundational method for cutting and pasting DNA from different sources to create new, functional DNA molecules, then inserting them into single-celled organisms like bacteria to make copies or produce new proteins.

What it covers

The patent details a method for creating "biologically functional DNA" (recombinant DNA) and getting a single-celled organism to replicate it. First, a circular piece of DNA, like a plasmid from a bacterium or a virus, is cut into a straight line (Claim 1a). This cut piece has special ends, called "termini," that are designed to stick to other DNA. Next, a "foreign" gene (a piece of DNA from a different source) is cut with matching ends and then "combined" with the first piece, essentially gluing them together (Claim 1b). This new, combined DNA is then put into a "unicellular organism," such as E. coli bacteria (Claim 2, 5). The patent explains how to select the organisms that successfully took up the new DNA by giving the DNA a "phenotypical trait," like resistance to a growth-inhibiting substance (Claim 4). These modified organisms then grow and make copies of the new DNA, or even produce proteins from the foreign gene (Claim 12). For example, one could insert the human insulin gene into a bacterial plasmid, transform bacteria with it, and then grow those bacteria to produce human insulin.

What it doesn't cover

• —Does not cover methods for creating recombinant DNA inside a living organism, as it specifies "prepared in vitro" (Claim 1).
• —Does not cover inserting DNA into multicellular organisms or complex eukaryotic cells, as it specifies "unicellular organisms" (Claim 1).
• —Does not cover methods that do not involve cleaving and ligating DNA segments with "ligatable termini" (Claim 1b).
• —Does not cover methods of selection that do not rely on a "phenotypical trait" imparted by the new DNA (Claim 1).
• —Does not cover methods where the inserted gene is from the same organism and would naturally exchange genetic information (Claim 11).

The clever bit

The novelty lay in the precise, controlled method of cutting DNA from different sources using "restriction enzymes" to create "cohesive ends" (Claim 8), then joining these pieces together with "enzymatic ligation" (Claim 7), and finally introducing this new hybrid DNA into a living cell to replicate and express the foreign gene. The key was making the foreign DNA functional within a new host and having a way to identify the successful modifications.

Why it matters

This patent, often referred to as the "Cohen-Boyer patent," is a landmark in biotechnology. It describes the fundamental techniques for recombinant DNA technology, which allowed scientists to combine genetic material from different species. This breakthrough enabled the industrial production of vital proteins like human insulin, growth hormone, and various enzymes, transforming the pharmaceutical and agricultural industries. It laid the groundwork for gene therapy and many modern biological research tools.

Real-world examples

• 1.Industrial production of human insulin
• 2.Production of human growth hormone
• 3.Manufacturing of enzymes for detergents or food processing
• 4.Creation of genetically modified microorganisms for bioremediation
• 5.Development of vaccines using recombinant proteins