1. 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

2. 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

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

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

6. 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

7. 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

8. 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.

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

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

11. 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.]

12. 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.]

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

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

15. Figure 19. 10 The Ti plasmid can be used to transfer genes into plants
Figure 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

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

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

20. 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

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