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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
Cutting and Joining DNA
Fragments
• Cohesive ends: fragments with short, singlestranded 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
Cloning Genes
• Other gene vectors:
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Cosmids
Bacterial Artificial Chromosomes (BACs)
Yeast Artificial Chromosome
Ti plasmid
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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)