Recombinant DNA and Cloning


Recombinant DNA technology involves taking naturally occurring DNA molecules and recombining them in vitro, creating new molecules. The tools used in recombinant DNA technology are naturally occurring and were discovered in the process of deciphering how living cells replicate and modify their DNA. Enzymes involved in these processes were cloned, and in some cases altered, to manipulate DNA in vitro.

Another tool used frequently in biotechnology is cloning, but not in the 'Dolly' sense. When we talk about cloning we are referring to making multiple identical copies of a portion of a genome. For example, in the sequencing of the human genome the first step was to break the total physical genome into manageable pieces and then insert the pieces into vectors, in order to make multiple copies that could then be sequenced. Each copy of the original piece of the genome would be called a clone because it is identical to the DNA that was originally taken from a human cell.

Cloning is also important for the study of gene regulation and function. In this approach a piece of genomic DNA that includes the gene of interest is cloned. Once cloned the gene can be studied in many ways. For example, the gene can be mutated and put back into a cell to see what happens. It the gene produces a protein product; we can produce large quantities of the product for in vitro studies. If the whole gene, including non-coding regulatory regions, is cloned, we can study how its expression is regulated.

Recombinant DNA technology is also used extensively to produce products in the pharmaceutical industry; for example, human insulin is produced by cloning and expressing the human insulin gene in bacteria. Recombinant DNA technology is also used in the agriculture industry to improve crop yields and food quality and to reduce production costs.

General DNA cloning steps

The basic steps involved in producing recombinant DNA are outlined in Figure 1.

  1. Plan the cloning strategy. This includes selecting a method for obtaining the DNA fragment to be cloned, selecting a vector into which the DNA fragment will be incorporated, and selecting the bacterial host that will be used to produce the recombinant DNA.
  2. Isolate the vector and the DNA fragment to be cloned. For the vector this may involve growing an appropriate bacterial strain carrying the vector, and isolating the vector DNA. The method used to obtain the DNA for cloning depends on where it currently resides (in a plasmid, in genomic DNA, in mRNA etc.).
  3. Prepare the vector and the target DNA for ligation. The DNA that will be cloned needs to be an appropriate size for ligation (size range depends on the vector being used) and the vector needs to be opened up (made linear) so that the insert can be ligated to it. This could involve using restriction enzymes or mechanical methods to breakup the target DNA. Vectors are usually opened up using restriction enzymes.
  4. Ligate the digested vector with the DNA fragment(s) of interest.
  5. Screen the new plasmids to find the desired recombinant plasmid(s). This involves transforming host bacteria with the ligation mixture and using selection methods to isolate single bacteria carrying the newly created plasmids. Plasmids are then re-isolated and checked for correct structure.
Figure 1. Cloning Steps

More information


Educational Sites


Several molecular biology supply companies are good sources for general molecular biology background.
Here are a few of my favorites

  • Molecular Cloning: a laboratory manual. 2001. Joseph Sambrook, David W. Russell. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. (Available in the lab & on reserve in the library.)
  • Current Protocols in Molecular Biology. 1994. Edited by Frederick M. Ausubel. Current Protocols, N. Y. (In the reference section of the library.)
  • Search the library catalog for other useful books.
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