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 Recombinant DNA Genetic engineering Small circular DNA that replicates within bacterial cells plasmid Recombinant DNA DNA with genes from two different sources ( often different species) Genetic engineering Direct manipulation of genes for practical purposes Restriction enzymes Enzymes that cut foreign DNA Restriction sites 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