Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

12 - Gene Cloning

Gene cloning involves removing a gene from the genome of ay organism and then placing that gene into a the genome of a bacterial cell.  The bacterial cell is then responsible for copying and maintaining this foreign gene.  In some cases, the bacterial cell can then transcribe the foreign gene to make a messenger RNA (mRNA) molecule, and the mRNA can then be translated to make a protein product.  Many important human proteins, including insulin (for diabetes patients) and factor VIII (for type A hemophilia patients), have been produced successfully by bacterial cells via this gene cloning technique.

Gene Cloning (overview)

In the early 1970s, scientists isolated the DNA from two different organisms and covalently linked them together in a test tube.  This new DNA molecule, which contained DNA from two sources, is a recombinant DNA molecule.  This experiment demonstrated the first use of recombinant DNA techniques, methods to manipulate DNA molecules outside of a living organism.  Since then, many advances have made recombinant DNA techniques common practice among biologists. 

Our discussion will focus on a process called gene cloning (figure 12.1). Gene cloning involves isolating a particular gene of interest and inserting that gene into another DNA molecule, called a vector.  A common vector is a small circular plasmid DNA molecule isolated from bacteria (see below).  This recombinant DNA molecule, composed partly of the gene of interest and partly the microbe DNA molecule, is then introduced into a host cell (typically a bacterial cell).  The host cell maintains and copies this recombinant DNA molecule, so the gene of interest (i.e., a cloned gene) can be studied in more detail.  Once gene cloning is complete, the cloned gene can be used in:

7.3_Gene_Cloning_Overview.jpg
Figure 12.1 Gene cloning overview - Image by Khan Academy and modified by SL

Key Questions

  • What is a recombinant DNA molecule?
  • What is meant by the term gene cloning?
  • Why is it useful to clone a gene?

Vectors

Gene cloning involves removing a gene of interest from an organism and inserting that gene into a vector DNA molecule (figure 12.2).  The resulting recombinant DNA molecule is then introduced into a host for maintenance.  The host can be a bacterium such as Escherichia coli, the yeast Saccharomyces cerevisiae, or in some cases, a virus.

From this point on, let us assume that we are interested in studying the β-globin gene isolated from the human genome.  The overall goal of our gene cloning experiment is to insert the human β-globin gene into a vector DNA molecule.  In our scenario, the vector will function to:

What types of molecules can function as vectors?

7.4_Bacterial_Vector_1.png
Figure 12.2 The bacterial plasmid vector pBR322.  Important DNA sequences within the plasmid include the origin of replication (ori), the selectable marker genes (amp, tet) and gene cloning sites (EcoRI, HindIII, etc.).

Key Questions

  • What is a vector?
  • Identify two types of molecules that can serve as vectors.
  • Describe the functions of three types of DNA sequences that allow plasmids to serve as vectors.
  • What is a host cell?

Restriction Enzymes

How do we take the β-globin gene from the chromosome of a human cell and insert it into a vector DNA molecule?

An important tool that is used in the gene cloning process is a restriction enzyme.  Restriction enzymes are endonucleases that recognize a specific DNA sequence (a restriction enzyme site) and cleaves the phosphodiester bonds within both strands of the DNA molecule at the recognition enzyme site.  The restriction enzyme site is typically a palindrome DNA sequence.  As an example, suppose that the restriction enzyme EcoRI cuts the DNA sequence 5’-GAATTC-3’.  The complementary strand is 3’-CTTAAG-5’, which is identical to the original restriction enzyme sequence but in the reverse orientation (i.e., a palindrome).  EcoRI cuts both DNA strands within this palindromic site. 

Restriction enzymes are made by many bacteria to protect the cell from foreign DNA, particularly bacteriophage DNA injected into a bacterium during an infection.  Several hundred restriction enzymes have been isolated from bacteria and are available for purchase.

Key Questions

  • What is a restriction enzyme and a restriction enzyme site?
  • In terms of DNA sequences, what is meant by the term palindrome?

Producing Recombinant DNA Molecules

How can we use the restriction enzyme EcoRI to clone the β-globin gene into a plasmid vector?

Suppose that the restriction enzyme EcoRI recognizes the restriction enzyme site shown below in both the β-globin gene and in the plasmid DNA molecule (see figure 12.3).

7.5_Cut_Site_and_Sticky_Ends_1.jpg
Figure 12.3 Cut Site and Sticky Ends - Image from Khan Academy and modified by SL

EcoRI cleaves the cloning site within the plasmid and the β-globin molecules to produce complementary single-stranded regions called sticky ends.  Thus, when mixed, the sticky ends of the β-globin DNA can form hydrogen bonds with the sticky ends from the vector DNA. 

DNA ligase then forms the final covalent bonds in each DNA strand, linking the insert (β-globin) to the vector (plasmid).

Key Questions

  • What are sticky ends?
  • What is the function of DNA ligase in a gene cloning experiment?

A Gene Cloning Experiment

Recall that in a gene cloning experiment, the goal is to insert a gene of interest (i.e., the β-globin gene) into a vector DNA molecule.  The approach used to clone the β-globin gene is outlined below and is shown in figure 12.4:

  1. The plasmid and chromosomal DNA molecule containing the β-globin gene are digested with the same restriction enzyme. A restriction enzyme is chosen to cut the chromosomal DNA into many small pieces.  One of these chromosome pieces contains the β-globin gene.  The same restriction enzyme is used to cut the plasmid DNA within the cloning site.  The DNA fragment containing the β-globin gene and the plasmid DNA molecule have complementary sticky ends.  For the purposes of this hypothetical experiment, let us assume that the plasmid DNA molecule contains the ampR gene as a selectable marker.
     
  2. The cleaved plasmid and β-globin gene fragment are mixed. Three different events can occur:

  1. DNA ligase is added. DNA ligase catalyzes the covalent linkage of the β-globin gene fragment to the plasmid DNA molecule.  Ligation produces a population of circular DNA molecules, called recombinant DNA molecules, in which an insert is contained within the plasmid.  Some of these recombinant DNA molecules contain the β-globin gene insert.

  1. Transformation of a host cell. Now that recombinant DNA molecules have been created, the recombinant DNA molecules are introduced into an E. coli host cell for maintenance.  When E. coli cells are treated with calcium, the bacteria become competent to take up DNA from the environment.  When the recombinant DNA molecule is added to these competent bacterial cells, and the bacteria are shocked by a brief heat treatment, the recombinant DNA molecule is taken into the cytoplasm of the E. coli cell via transformation
     
  2. Host bacteria are grown on a medium that contains ampicillin. Growth produces colonies of bacterial cells, with each cell containing many copies of the recombinant DNA molecules.  Two scenarios are possible after transformation: 

  1. Identify colonies that contain an insert by the blue-white screening method. Many cloning experiments are designed so that the gene cloning site in the plasmid is within a DNA sequence called lacZ.  The lacZ gene produces the enzyme β-galactosidase.  Cloning the insert disrupts the lacZ gene, allowing researchers to distinguish colonies that contain an insert versus colonies that do not contain an insert.

How do we determine if β-galactosidase is produced?  This is done by plating the bacterial cells on an agar plate that not only includes ampicillin but also includes a compound called IPTG, which activates the lacZ gene to produce the β-galactosidase protein, and X-Gal, which is a substrate for β-galactosidase.

  1. Identify colonies that contain the β-globin insert. All the white colonies contain an insert; however, at this point, we cannot distinguish the white colonies that contain the β-globin insert from the white colonies that have other DNA fragments as inserts. 

Several techniques can be used to identify which bacterial cells contain a recombinant vector with the β-globin gene.  For example, vectors isolated from white colonies can be cut with the same restriction enzyme used at the beginning of the cloning experiment to release the insert.  The digested DNA can then be analyzed by agarose gel electrophoresis.  The presence of a DNA fragment of the appropriate size for the β-globin gene indicates that the chosen colony likely contains the β-globin insert.  Alternatively, the polymerase chain reaction (PCR), using primers specific for β-globin can be used to amplify the β-globin insert.  If a PCR product is produced using these β-globin primers, then the β-globin gene was cloned successfully.  Finally, determining the nucleotide sequence of the cloned insert by DNA sequencing will determine if the recombinant molecule contains the β-globin gene.

7.6_Gene_Cloning_Overview.jpg
Figure 12.4 Gene cloning overview --- This image is used from OpenStax (access for free at https://books.byui.edu/-vuzA

Key Questions

  • Describe the seven steps involved in cloning the β-globin gene into a plasmid.
  • How are untransformed host cells eliminated in the cloning experiment?
  • How can blue-white screening be used to identify recombinant DNA molecules that contain an insert?
  • How can you identify the recombinant DNA molecule that contains the β-globin insert?

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