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Figure 20.0 DNA sequencers
DNA Technology
DNA Cloning
• A molecular biologist studying a particular
gene faces a challenge. Characteristics of a
naturally occurring DNA molecule include:
• Very long molecule
• Carries many genes
• Genes occupy only a small portion of the
chromosome
• A specific gene probably only makes up 1/1000,000
of the DNA chromosomal molecule
• Subtle differences distinguish the gene from the
surrounding material
To work with a specific gene, scientists need
methods for preparing well-defined, gene-sized
pieces of DNA in multiple identical copies.
They need techniques for GENE CLONING!
Most techniques for gene cloning share several
general features – the use of bacteria and
plasmids.
Vocabulary Review
• plasmid
Small circular DNA that replicates
within bacterial cells
• Recombinant DNA
• Genetic
engineering
• Restriction
enzymes
• Restriction sites
DNA with genes from two different
sources ( often different species)
Direct manipulation of genes for
practical purposes
Enzymes that cut foreign DNA
Specific DNA sequence recognized by
a restriction enzyme
Figure 20.1 An overview of how bacterial plasmids are used to clone genes
Using a restriction
enzyme and DNA ligase
to make recombinant
DNA
Genes can be Cloned in Recombinant DNA
Vectors
• The plasmid is the vector. A vector is a means of
transportation. It will carry the foreign DNA and
will enter another cell and replicate. Bacterial
plasmids are widely used as cloning vectors.
• Bacteria are most commonly the host cell.
• Ease of isolation
• Ease of reintroduction
• Replicate quickly
Procedure for Cloning a Eukaryotic Gene in a
Bacterial Plasmid
• Isolation of vector and gene-source DNA
• Insertion of DNA into vector
• Introduce cloning vector into cells
• Cloning of cells with foreign DNA
• Identification of cell clones carrying the gene of
interest
Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 1)
Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 2)
Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)
Figure 20.4 Using a nucleic acid probe to identify a cloned gene
If the cells containing a
desired gene translate
the gene into protein,
then it is possible to
identify them by
screening for the
protein. This if often
done by using
antibodies that bind to
the protein.
Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
Figure 20.6 Genomic libraries
Figure 20.7 The polymerase chain reaction (PCR)
Figure 20.8 Gel electrophoresis of macromolecules