lecture 9

Overview: DNA cloning
Introduction
When you hear the word “cloning,” you may think of the cloning of whole organisms, such as Dolly the sheep. However, all it means to clone something is to make a genetically exact copy of it. In a molecular biology lab, what’s most often cloned is a gene or other small piece of DNA.
Overview of DNA cloning
DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources.

Diagram showing the construction of a recombinant DNA molecule. A circular piece of plasmid DNA has overhangs on its ends that match those of a gene fragment. The plasmid and gene fragment are joined together to produce a gene-containing plasmid. This gene-containing plasmid is an example of recombinant DNA, or a DNA molecule assembled from DNA from multiple sources.
Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.
What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.
As you may know if you have friends or family members who are diabetic, the hormone insulin plays an important role in human health. In a healthy person’s body, insulin is produced in the pancreas. It travels through the bloodstream and binds to cells of the body, allowing them to take up the sugar glucose.
In a person with type I diabetes, the cells of the pancreas that produce insulin are damaged or destroyed. People with type I diabetes must regularly inject themselves with insulin from another source to prevent dangerously high levels of blood sugar (and to allow sugar to enter the cells of the body as fuel).