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Gene Technology
Chapter 11
The Basics
The process of manipulating genes for practical (useful) purposes is called genetic engineering.
This process may involve recombinant DNA, which is DNA from two or more different organisms.
Steps In A G.E. Experiment
Step 1: The DNA from the organism containing the gene of interest is cut by restriction enzymes which are bacterial enzymes that bind to specific short sequences of DNA and cut it between specific nucleotides.
Steps In A G.E. Experiment
The DNA from a vector is also cut. A vector is an agent that is used to carry the gene of interest into another cell.
Commonly used vectors include viruses, yeast, and plasmids, which are circular DNA molecules that replicate independently from the main chromosomes of bacteria.
Steps In A G.E. Experiment
Step 2: The DNA fragments from the organism containing the gene of interest are combined with the DNA fragments from the vector using ligase to bond the ends together. This produces recombinant DNA.
Steps In A G.E. Experiment
Step 3: In a process called cloning, many copies of the gene of interest are made each time the host cell reproduces.
Step 4: During screening, cells that have received the particular gene of interest are separated from those which did not.
Steps In A G.E. Experiment
Step 1: Cutting DNAStep 2: Making recombinant DNA
Step 3: CloningStep 4: Screening
Confirmation of a Cloned Gene
One method of identifying the presence of the gene of interest is to use a technique called a Southern blot.
Step 1: The DNA from each bacterial clone colony is isolated and cut into fragments by restriction enzymes.
Confirmation of a Cloned Gene
Step 2: The DNA fragments are separated by gel electrophoresis, which is a technique that uses an electric field within a gel to separate molecules by their size.
Gel Electrophoresis
Since DNA has a negative charge, it moves toward the + pole when the electrical field is applied. The smaller DNA fragments move faster and a pattern of bands develops in the gel.
Confirmation of a Cloned GeneStep 3: The DNA bands are then transferred (blotted) directly onto a piece of filter paper. The filter paper is then moistened with a probe solution. Probes are radioactive or fluorescent-labeled RNA or single-stranded DNA pieces that are complementary to the gene of interest.
Confirmation of a Cloned GeneStep 4: Only the fragments complementary to the probe will bind with the probe and form visible bands which confirm the presence of a cloned gene.
