Molecular Genetic Analysis and Biotechnology Benjamin A. Pierce GENETICS A Conceptual Approach FIFTH EDITION CHAPTER 19 Molecular Genetic Analysis and Biotechnology © 2014 W. H. Freeman and Company

Cutting and Joining DNA Fragments Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences Type II restriction enzyme: most useful enzyme By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria

Figure 19.2a Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

Cutting and Joining DNA Fragments Cohesive ends: fragments with short, single-stranded overhanging ends Blunt ends: even-length ends from both single strands

Cloning Genes Plasmid vectors Plasmids: circular DNA molecules from bacteria Insert foreign DNA into plasmid using restriction enzymes Linkers: synthetic DNA fragments containing restriction sites Transformation of host cells with plasmids Screening cells for recombinant plasmids Selectable markers are used to confirm whether the cells have been transformed or not

Cloning Genes Plasmid vectors Plasmids: circular DNA molecules from bacteria Insert foreign DNA into plasmid using restriction enzymes Linkers: synthetic DNA fragments containing restriction sites Transformation of host cells with plasmids Screening cells for recombinant plasmids Selectable markers are used to confirm whether the cells have been transformed or not

Figure 19. 6 The pUC19 plasmid is a typical cloning vector Figure 19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers—an ampicillin-resistance gene and a lacZ gene.

Figure 19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

Figure 19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

Figure 19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

Figure 19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

Cloning Genes Other gene vectors: Cosmids Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosome Ti plasmid

Figure 19.9 To ensure transcription and translation, a foreign gene may be inserted into an expression vector—in this example, an E. coli expression vector.

Figure 19. 10 The Ti plasmid can be used to transfer genes into plants Figure 19.10 The Ti plasmid can be used to transfer genes into plants. Flanking sequences TL and TR are required for the transfer of the DNA segment from bacteria to the plant cell. 15

Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

Figure 19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

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Knockout Mice A normal gene of the mouse has been fully disabled Knock-in mice: a mouse carries an inserted DNA sequence at specific locations

Silencing Genes with RNAi siRNAs Process called RNA interference (RNAi)