11.1 restriction and modification enzymes genetic engineering: using in vitro techniques to alter...

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

© 2012 Pearson Education, Inc.


• 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



• 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


Figure 11.1a

Single-stranded“sticky” ends



• Restriction enzymes protect cell from invasion from foreign DNA– Destroy foreign DNA

– Must protect their own DNA from inadvertent destruction



• 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)


Figure 11.1b



• 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)


Figure 11.2a


Figure 11.2b

Size in basepairs

A B C D

50004000

3000

2000

1800

1000

500

Size in basepairs



• 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)


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


Figure 11.4


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


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



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



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



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


Animation: Recombinant DNAAnimation: Recombinant DNA


• 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


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


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



• 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


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



• 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


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


Figure 11.9


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


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



• 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


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



• 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


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


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


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


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)


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


Figure 11.16

AmpicillinresistanceOrigin of

DNA replication

Polylinker(cloningsite)

S/D

T1

T2

trc promoterlacO

lacI



• 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


Figure 11.17

Induce lacpromoter withIPTG

T7 RNApolymerase

Geneproduct

Chromosome

Cloned gene

T7promoter

pET plasmid

Gene forT7 RNA

polymerase

lacoperator

lacpromoter

lacl



• 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


11.1 restriction and modification enzymes genetic engineering: using in vitro techniques to alter...

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