1. 11.1 Restriction and Modification Enzymes
Genetic engineering: using in vitro techniques to alter genetic material in the laboratory
Basic techniques include
Restriction enzymes
Gel electrophoresis
Nucleic acid hybridization
Nucleic acid probes
Molecular cloning
Cloning vectors
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2. 11.1 Restriction and Modification Enzymes
Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites
Widespread among prokaryotes
Rare in eukaryotes
Protect prokaryotes from hostile foreign DNA (e.g., viral genomes)
Essential for in vitro DNA manipulation
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3. 11.1 Restriction and Modification Enzymes
Three classes of restriction enzymes
Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation (Figure 11.1a)
Restriction enzymes recognize inverted repeat sequences (palindromes)
Typically 4–8 base pairs long; EcoRI recognizes a 6-base-pair sequence
Sticky ends or blunt ends
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4. Single-stranded “sticky” ends
Figure 11.1a
Figure 11.1 Restriction and modification of DNA.
Single-stranded “sticky” ends
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5. 11.1 Restriction and Modification Enzymes
Restriction enzymes protect cell from invasion from foreign DNA
Destroy foreign DNA
Must protect their own DNA from inadvertent destruction
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6. 11.1 Restriction and Modification Enzymes
Modification enzymes: protect cell’s DNA for restriction enzymes
Chemically modify nucleotides in restriction recognition sequence
Modification generally consists of methylation of DNA (Figure 11.1b)
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7. Figure 11.1b Figure 11.1 Restriction and modification of DNA.
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8. 11.1 Restriction and Modification Enzymes
Gel electrophoresis: separates DNA molecules based on size (Figure 11.2a)
Electrophoresis uses an electrical field to separate charged molecules
Gels are usually made of agarose, a polysaccharide
Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups
Gels can be stained with ethidium bromide and DNA can be visualized under UV light (Figure 11.2b)
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9. Figure 11.2a Figure 11.2 Agarose gel electrophoresis of DNA.
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10.  A B C D Size in base pairs Size in base pairs 5000 4000 3000 2000
Figure 11.2b  A B C D
Size in base pairs
Size in base pairs
5000
4000
3000
2000
1800
1000
Figure 11.2 Agarose gel electrophoresis of DNA.
500 
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11. 11.1 Restriction and Modification Enzymes
The same DNA that has been cut with different restriction enzymes will have different banding patterns on an agarose gel
Size of fragments can be determined by comparison to a standard
Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA (Figure 11.3)
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12. 11.2 Nucleic Acid Hybridization
Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix
Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe
Southern blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA
Northern blot: RNA is in the gel
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13. Figure 11.4 Figure 11.4 Southern blotting.
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14. 11.3 Essentials of Molecular Cloning
Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
Three main steps of gene cloning (Figure 11.5):
Isolation and fragmentation of source DNA
Insertion of DNA fragment into cloning vector
Introduction of cloned DNA into host organism
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15. Introduction of recombinant vector into a host
Figure 11.5
Foreign DNA
Cut with restriction enzyme
Add vector cut with same restriction enzyme
Sticky ends
Vector
Add DNA ligase to form recombinant molecules
Figure 11.5 Major steps in gene cloning.
Cloned DNA
Introduction of recombinant vector into a host
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16. 11.3 Essentials of Molecular Cloning
Isolation and fragmentation of source DNA
Source DNA can be genomic DNA, RNA, or PCR-amplified fragments
Genomic DNA must first be restriction digested
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17. 11.3 Essentials of Molecular Cloning
Insertion of DNA fragment into cloning vector
Most vectors are derived from plasmids or viruses
DNA is generally inserted in vitro
DNA ligase: enzyme that joins two DNA molecules
Works with sticky or blunt ends
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18. 11.3 Essentials of Molecular Cloning
Introduction of cloned DNA into host organism
Transformation is often used to get recombinant DNA into host
Some cells will contain desired cloned gene, while other cells will have other cloned genes
Gene library: mixture of cells containing a variety of genes
Shotgun cloning: gene libraries made by cloning random genome fragments
Animation: Recombinant DNA
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19. 11.3 Essentials of Molecular Cloning
Essential to detect the correct clone
Initial screen: antibiotic resistance, plaque formation
Often sufficient for cloning of PCR-generated DNA sequences
If working with a heterogeneous gene library you may need to look more closely
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20. Transformant colonies growing on agar surface
Figure 11.6
Transformant colonies growing on agar surface
Replica-plate onto membrane filter
Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form
Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash out unbound radioactivity
Figure 11.6 Finding the right clone.
Autoradiograph to detect radioactivity
X-ray film
Positive colonies
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21. 11.4 Molecular Methods for Mutagenesis
Synthetic DNA
Systems are available for de novo synthesis of DNA
Oligonucleotides of 100 bases can be made
Multiple oligonucleotides can be ligated together
Synthesized DNA is used for primers and probes, and in site-directed mutagenesis
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22. 11.4 Molecular Methods for Mutagenesis
Conventional mutagens produce mutations at random
Site-directed mutagenesis: performed in vitro and introduces mutations at a precise location (Figure 11.7)
Can be used to assess the activity of specific amino acids in a protein
Structural biologists have gained significant insight using this tool
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23. single-stranded vector Source
Figure 11.7
Clone into
single-stranded vector
Source
Single-stranded DNA from M13 phage
Add synthetic oligonucleotide with one base mismatch
Base-pairing with source gene
Extend single strand with DNA polymerase
Figure 11.7 Site-directed mutagenesis using synthetic DNA.
Transformation and selection
Clone and select mutant
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24. 11.4 Molecular Methods for Mutagenesis
Cassette mutagenesis and knockout mutations
DNA fragment can be cut, excised, and replaced by a synthetic DNA fragment (DNA cassettes or cartridges)
The process is known as cassette mutagenesis
Gene disruption is when cassettes are inserted into the middle of the gene (Figure 11.8)
Gene disruption causes knockout mutations
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25. Gene X knockout Figure 11.8 EcoRI cut sites () Gene X
Kanamycin cassette
Cut with EcoRI and ligate
BamHI cut site
Cut with BamHI and transform into cell with wild-type gene X
Linearized plasmid
Sites of recombination
Figure 11.8 Gene disruption by cassette mutagenesis.
Chromosome
Recombination and selection for kanamycin-resistant cells
Gene X knockout
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26. Figure 11.9 Figure 11.9 Green fluorescent protein (GFP).
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27. Target gene Reporter gene Gene fusion Promoter Coding sequence
Figure 11.10
Target gene
Promoter
Coding sequence
Reporter gene
Promoter
Coding sequence
Cut and ligate
Gene fusion
Promoter
Reporter is expressed under control of target gene promoter
Figure Construction and use of gene fusions.
Reporter enzyme
Substrate
Colored product
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28. 11.6 Plasmids as Cloning Vectors
Plasmids are natural vectors and have useful properties as cloning vectors
Small size; easy to isolate DNA
Independent origin of replication
Multiple copy number; get multiple copies of cloned gene per cell
Presence of selectable markers
Vector transfer carried out by chemical transformation or electroporation
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29. 11.6 Plasmids as Cloning Vectors
pUC19 is a common cloning vector (Figure 11.11)
Modified ColE1 plasmid
Contains ampicillin resistance and lacZ genes
Contains polylinker (multiple cloning site) within lacZ gene
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30. Origin of DNA replication
Figure 11.11
Order of restriction enzyme cut sites in polylinker
Ampicillin resistance
lacZ
ApoI - EcoRI BanII - SacI Acc651 - KpnI AvaI - BsoBI SmaI - XmaI BamHI XbaI AccI - HincII - SalI BspMI - BfuAI SbfI PstI SphI HindIII
Polylinker
pUC base pairs
Figure Cloning vector plasmid pUC19.
lacI
Origin of DNA replication
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31. 11.6 Plasmids as Cloning Vectors
Blue/white screening
Blue colonies do not have vector with foreign DNA inserted
White colonies have foreign DNA inserted
Insertional inactivation: lacZ gene is inactivated by insertion of foreign DNA (Figure 11.12)
Inactivated lacZ cannot process Xgal; blue color does not develop
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32. Vector plus foreign DNA insert
Figure 11.12
lacZ
AmpR
Foreign DNA
Vector
Digestion with restriction enzyme
Join with DNA ligase
Opened vector
Figure Cloning into the plasmid vector pUC19.
Recyclized vector without insert
Vector plus foreign DNA insert
Transform into Escherichia coli and select on ampicillin plates containing Xgal
Transformants blue (-galactosidase active)
Transformants white (-galactosidase inactive)
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33. 11.7 Hosts for Cloning Vectors
Ideal hosts should be
Capable of rapid growth in inexpensive medium
Nonpathogenic
Capable of incorporating DNA
Genetically stable in culture
Equipped with appropriate enzymes to allow replication of the vector
Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae
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34. Bacteria Eukaryote Escherichia coli Bacillus subtilis
Figure 11.13
Bacteria
Eukaryote
Escherichia coli
Bacillus subtilis
Saccharomyces cerevisiae
Well-developed genetics Many strains available Best known bacterium
Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture
Well-developed genetics Nonpathogenic Can process mRNA and proteins Easy to grow
Figure Hosts for molecular cloning.
Potentially pathogenic Periplasm traps proteins
Genetically unstable Genetics less developed than in E. coli
Plasmids unstable Will not replicate most bacterial plasmids
Advantages
Disadvantages
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35. 11.5 Gene Fusions and Reporter Genes
Encode proteins that are easy to detect and assay (Figure 11.9)
Examples: lacZ, luciferase, GFP genes
Gene fusions
Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions (Figure 11.10)
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36. 11.8 Shuttle Vectors and Expression Vectors
Expression vectors: allow experimenter to control the expression of cloned genes (Figure 11.16)
Based on transcriptional control
Allow for high levels of protein expression
Strong promoters
lac, trp, tac, trc, lambda PL
Effective transcription terminators are used to prevent expression of other genes on the plasmid
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37. Polylinker (cloning site)
Figure 11.16
trc promoter
lacO
S/D
lacI
Polylinker (cloning site) T1 T2
Figure Genetic map of the expression vector pSE420.
Ampicillin resistance
Origin of DNA replication
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38. 11.8 Shuttle Vectors and Expression Vectors
In T7 expression vectors, cloned genes are placed under control of the T7 promoter (Figure 11.17)
Gene for T7 RNA polymerase present and under control of easily regulated system (e.g., lac)
T7 RNA polymerase recognizes only T7 promoters
Transcribes only cloned genes
Shuts down host transcription
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39. Gene for T7 RNA polymerase
Figure 11.17
Induce lac promoter with IPTG
T7 RNA polymerase
Gene product
lac operator
Gene for T7 RNA polymerase
lac promoter
T7 promoter
Cloned gene
pET plasmid
Figure The T7 expression system.
Chromosome
lacl
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40. 11.8 Shuttle Vectors and Expression Vectors
mRNA produced must be efficiently translated and there are problems with this always happening
Bacterial ribosome binding sites are not present in eukaryotic genomes
Differences in codon usage between organisms
Eukaryotic genes containing introns will not be expressed properly in prokaryotes
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