11.1 restriction and modification enzymes genetic engineering: using in vitro techniques to alter...
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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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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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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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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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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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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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Figure 11.2b
Size in basepairs
A B C D
50004000
3000
2000
1800
1000
500
Size in basepairs
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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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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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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):1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism
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Figure 11.5
Foreign DNA
Stickyends
Vector
ClonedDNA
Introduction of recombinantvector into a host
Cut with restrictionenzyme
Add vector cutwith same restriction enzyme
Add DNA ligase toform recombinantmolecules
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11.3 Essentials of Molecular Cloning
1. 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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11.3 Essentials of Molecular Cloning
2. 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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11.3 Essentials of Molecular Cloning
3. 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
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Animation: Recombinant DNAAnimation: Recombinant DNA
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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Figure 11.6 Transformant coloniesgrowing on agar surface
X-ray film
Positivecolonies
Replica-plate ontomembrane filter
Partially lyse cells; addspecific antibody; add agentto detect bound antibody inradiolabeled form
Lyse bacteria and denatureDNA; add RNA or DNAprobe (radioactive); washout unbound radioactivity
Autoradiographto detectradioactivity
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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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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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Figure 11.7
Single-strandedDNA from M13phage
Base-pairingwith sourcegene
Source
Clone andselect mutant
Clone intosingle-strandedvector
Add syntheticoligonucleotidewith one basemismatch
Extend singlestrand withDNA polymerase
Transformationand selection
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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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Figure 11.8Gene X EcoRI cut sites ()
Kanamycin cassette
BamHIcut site
Linearized plasmid
Chromosome
Sites of recombination
Gene X knockout
Cut with EcoRIand ligate
Cut with BamHI andtransform into cellwith wild-type gene X
Recombination and selectionfor kanamycin-resistant cells
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Figure 11.10
Target gene
Reporter gene
Gene fusion
Promoter
Promoter
Promoter
Coding sequence
Coding sequence
Cut and ligate
Reporter is expressed undercontrol of target gene promoter
Reporterenzyme
Substrate
Colored product
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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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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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Figure 11.11
Ampicillinresistance
Polylinker
Origin ofDNA replication
pUC192686 base pairs
lacI
lacZ
Order of restrictionenzyme cut sites inpolylinkerApoI - EcoRIBanII - SacIAcc651 - KpnIAvaI - BsoBI - SmaI - XmaIBamHIXbaIAccI - HincII - SalIBspMI - BfuAISbfIPstISphIHindIII
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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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Figure 11.12
AmpR
lacZ
VectorForeign DNA
Opened vector
Recyclized vector without insert Vector plus foreignDNA insert
Transformants blue(-galactosidaseactive)
Transformants white(-galactosidaseinactive)
Digestion with restriction enzyme
Join withDNA ligase
Transform into Escherichiacoli and select on ampicillinplates containing Xgal
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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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Figure 11.13
Well-developed geneticsMany strains availableBest known bacterium
Easily transformedNonpathogenicNaturally secretes proteinsEndospore formation simplifies culture
Well-developed geneticsNonpathogenicCan process mRNA and proteinsEasy to grow
Potentially pathogenicPeriplasm traps proteins
Genetically unstableGenetics less developed than in E. coli
Plasmids unstableWill not replicate most bacterial plasmids
Advantages Disadvantages
Escherichia coli Bacillus subtilis Saccharomycescerevisiae
Bacteria Eukaryote
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11.5 Gene Fusions and Reporter Genes
• 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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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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Figure 11.16
AmpicillinresistanceOrigin of
DNA replication
Polylinker(cloningsite)
S/D
T1
T2
trc promoterlacO
lacI
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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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Figure 11.17
Induce lacpromoter withIPTG
T7 RNApolymerase
Geneproduct
Chromosome
Cloned gene
T7promoter
pET plasmid
Gene forT7 RNA
polymerase
lacoperator
lacpromoter
lacl
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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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