How Does Molecular Cloning Work?

Introduction to Cloning

Molecular cloning is the process of making copies of a piece of genetic information for further analysis. This process is done by transferring a piece of DNA from an organism into a molecule that can reproduce copies of the transferred DNA. The molecule most often used to make copies of DNA in molecular cloning is a substance called a bacterial plasmid. Plasmids are independent forms of life that do not have to be in the host bacteria to generate DNA copies. Molecular cloning techniques are used frequently in the laboratory and have been in use since their discovery in the 1970s.

DNA Isolation and Transfer

Before the DNA being studied can be inserted into a plasmid it must first be isolated, separated form the rest of the DNA and broken into fragments. The first step in this process, the isolation of the section of DNA that will be replicated, is done using a variety of techniques, the most common of which is called polymerase chain reaction, or PCR. The PCR method not only isolates a piece of DNA, it also increases the quantity of DNA available. This amplified portion of DNA is then inserted into a plasmid (or other similar vector) and treated with specialized enzymes that prepare the DNA for replication. These enzymes break the DNA into linear fragments and process these fragments into DNA strands that contain only the DNA of interest. The processed plasmid is now ready for transfer.

Transfection

The prepared plasmid is next transferred into a host bacteria cell through a process called transfection. Transfection is most often done using a process called electroporation, although there are a number of techniques which can accomplish transfection. Electroporation works by increasing the host cells' permeability using an electrical field to create pores in the cell wall. The bacteria containing the processed plasmid are then transferred onto a growth plate where colonies of the cells are grown.

Identification and confirmation of success

The processes involved in creating a molecular clone are not at all efficient. Prior to transfer to the growth plate the bacteria are marked in a way so that bacteria containing the desired DNA can be identified visually with a color marker or so that only those bacteria with the desired DNA are able to grow. The colonies that develop are then tested to assure that the desired DNA is present. Once the DNA sequence is confirmed the bacteria can then be used for investigative studies.

Resources

• Human Genome Project cloning fact sheet
• Detailed description of molecular cloning lab techniques