recombinant dna technology recombinant dna technology is also known as gene cloning however, it...

Recombinant DNA Technology

Recombinant DNA technology is also known as gene cloning

However, it turned out to be safer than expected

- It also spread to industry faster and in more diverse ways than imagined

Figure 8.1 Overview of recombinant DNA technologyBacterial cell

Bacterialchromosome

Plasmid

Gene of interest

DNA containinggene of interest

Isolate plasmid.

Enzymatically cleaveDNA into fragments.

Isolate fragmentwith the gene ofinterest.

Insert gene into plasmid.

Insert plasmid and gene intobacterium.

Culture bacteria.

Harvest copies ofgene to insert intoplants or animals

Harvest proteinscoded by gene

Eliminateundesirablephenotypictraits

Produce vaccines,antibiotics,hormones, orenzymes

Createbeneficialcombinationof traits

DNA Cloning

Goal is to generate large amounts of pure DNA that can be manipulated and studied.

DNA is cloned by the following steps:

1. Isolate DNA from organism (e.g., extract DNA)

2. Cut DNA with restriction enzymes to a desired size.

3. Splice (or ligate) each piece of DNA into a cloning vector to create a recombinant DNA molecule.

Cloning vector = artificial DNA molecule capable of replicating in a host organism (e.g., bacteria).

4. Transform recombinant DNA (cloning vector + DNA fragment) into a host that will replicate and make copies.

5. E. coli is the most common host.

Creating Recombinant DNA Molecules

Manufacturing recombinant DNA requires restriction enzymes that cut donor and recipient DNA at the same sequence

These enzymes cut DNA at sites that are palindromic

The cutting action of many of these enzymes generate single-stranded extensions called “sticky ends”

The Tools of Recombinant DNA Technology

• Restriction Enzymes– Bacterial enzymes that cut DNA molecules only

at restriction sites– Categorized into two groups based on type of

cut• Cuts with sticky ends• Cuts with blunt ends

© 2012 Pearson Education Inc.

Figure 8.2 Actions of restriction enzymes-overview

Figure 19.3

MOLECULAR SCISSORS - TYPE II RESTRICTION ENDONUCLEASES

Hamilton Othanel Smith 1968

cohesive ends

SPECIFIC CUT SPECIFIC JOINING (LIGATION)

Ability to join two foreign pieces of DNA together

MOLECULAR BIOLOGY – Molecular biology techniques

BLUNT END

COH

ESIV

E EN

DS

Step 2-Cut DNA with restriction enzymes

Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond.

Cut occurs between the 3’ carbon of the first nucleotide and the phosphate of the next nucleotide.

Restriction fragment ends have 5’ phosphates & 3’ hydroxyls.

restriction enzyme

Step 2-Cut DNA with restriction enzymes (cont.)

Most restriction enzymes occur naturally in bacteria.

Protect bacteria against viruses (bacteriophages) by cutting up viral DNA.

Bacteria protects their DNA by modifying possible restriction sites (methylation).

More than 400 restriction enzymes have been isolated.

Names typically begin with 3 italicized letters.

Enzyme SourceEcoRI E. coli RY13HindIII Haemophilus influenzae RdBamHI Bacillus amyloliquefaciens H

Many restriction sites are palindromes of 4-, 6-, or 8-base pairs.

Short restriction site sequences occur more frequently in the genome than longer restriction site sequences.

Their frequency is a function of (1/4)n

Fig. 8.1, EcoRI

“6-base cutter”

Step 2-Cut DNA with restriction enzymes (cont.)

Some restriction enzymes produce blunt ends, whereas others produce sticky (overhanging staggered) ends.

Sticky ends are useful for DNA cloning because complementary sequences anneal and can be joined directly by DNA ligase without using ‘adapters’.

Fig. 8.2


Another “tool” used is a cloning vector

- Carries DNA from the cells of one species into the cells of another

Commonly used vectors include:

- Plasmids

- Bacteriophages

- Disabled retroviruses

RECOMBINANT DNA TECHNOLGYThe plasmid as DNA ‘vector’ (vehicle)

Possible to insert ‘interesting’ DNA’s into a plasmid using restriction

endonucleases

3

Fig. 8.3, Cut and ligate 2 DNAs with EcoRI ---> recombinant DNA

3 Why not clone whole genomes?

Each bacterial colony represents an amplified clone containing a recombinant plasmid harbouring a

distinct region of the genome

i.e. together they represent a ‘Genomic DNA Library’

Also possible to do this using cDNA copies of transcribed mRNAs resulting a ‘cDNA Gene Expression Library’

Bacteriophage Lambdavectors

phage linear DNA genome

Non-essential region that can be substituted by DNA to be cloned (approx 20Kb)

cos cos

Cosmids, phosmids, BACs and YACs to clone larger DNA fragments


• Vectors– Nucleic acid molecules that deliver a gene

into a cell

– Useful properties• Small enough to manipulate in a lab• Survive inside cells• Contain recognizable genetic marker• Ensure genetic expression of gene

– Include viral genomes, transposons, and plasmids


Figure 8.3 Producing a recombinant vectorAntibioticresistancegene

Restrictionsite

mRNA for humangrowth hormone (HGH)

Reversetranscription

Plasmid (vector)

cDNA for HGH

Restrictionenzyme

Restrictionenzyme

Sticky ends

Gene for humangrowth hormone

Ligase

Recombinant plasmid

Introduce recombinantplasmid into bacteria.

Recombinantplasmid

Bacterialchromosome

Inoculate bacteriaon media containingantibiotic.

Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.

Step 3-Splice (or ligate) DNA into some kind of cloning vector to create a recombinant DNA molecule

Six different types of cloning vectors:

1. Plasmid cloning vector

2. Phage cloning vector

3. Cosmid cloning vector

4. Shuttle vectors

5. Yeast artificial chromosome (YAC)

6. Bacterial artificial chromosome (BAC)

7. Fosmid cloning vector

Plasmid Cloning Vectors:

Bacterial plasmids, naturally occurring small ‘satellite’ chromosome, circular double-stranded extrachromosomal DNA elements capable of replicating autonomously.

Plasmid vectors engineered from bacterial plasmids for use in cloning.

Feeatures (e.g., E. coli plasmid vectors):

1. Origin sequence (ori) required for replication.

2. Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the anti-biotic resistance grow in colony).

3. Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction site.

4. Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ+ gene)

Fig. 8.4,

pUC19 Polylinker: restriction sites

Origin sequence Ampicillin

resistance gene

lacZ+

gene

Detailed map showing polylinker region in pUC57 (genecript.com)

Fig. 8.5

*Cut with same restriction enzyme

*DNA ligase

Some features of pUC19 plasmid vector:

1. High copy number in E. coli, ~100 copies/cell, provides high yield.

2. Selectable marker is ampR. Ampicillin in growth medium prevents growth of all other E. coli that do not contain plasmid.

3. Cluster of several different restriction sites called a polylinker occurs in the lacZ (-galactosidase) gene.

4. Cloned DNA disrupts reading frame and -galactosidase production.

5. Add X-gal to medium; turns blue in presence of -galactosidase.

6. Plaque growth: blue = no inserted DNA and white = inserted DNA.

7. Some % of digested vectors will reanneal with no insert. Remove 5’ phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques).

8. Plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane).

9. Good for <10kb; Cloned inserts >10 kb typically are unstable.

Phage cloning vectors:

1. Engineered version of bacteriophage (infects E. coli).

2. Central region of the chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted.

3. DNA is packaged in phage heads to form virus particles.

4. Phages with both ends of the chormosome and a 37-52 kb insert replicate by infecting E. coli.

5. Phages replicate using E. coli and the lytic cycle (see Fig. 3.13).

6. Produces large quantities of 37-52 kb cloned DNA.

7. Like plasmid vectors, large number of restriction sites available; phage cloning vectors useful for larger DNA fragments than pUC19 plasmid vectors.

Cosmid cloning vectors:

1. Features of both plasmid and phage cloning vectors.

2. Do not occur naturally; circular.

3. Origin (ori) sequence for E. coli.

4. Selectable marker, e.g. ampR.

5. Restriction sites.

6. Phage cos site permits packaging into phages and introduction to E. coli cells.

7. Useful for 37-52 kb.

Shuttle vectors:

1. Capable of replicating in two or more types of hosts..

2. Replicate autonomously, or integrate into the host genome and replicate when the host replicates.

3. Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells).

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Example:

*Insert firefly luciferase geneinto plasmid and transform Agrobacterium.

*Grow Agrobacterium in large quantities and infect tobacco plant.

Yeast Artificial Chromosomes (YACs):

Vectors that enable artificial chromosomes to be created and cloned into yeast.

Features:1. Yeast telomere at each end.

2. Yeast centromere sequence.

3. Selectable marker (amino acid dependence, etc.) on each arm.

4. Autonomously replicating sequence (ARS) for replication.

5. Restriction sites (for DNA ligation).

6. Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.

Bacterial Artificial Chromosomes (BACs):

Vectors that enable artificial chromosomes to be created and cloned into E. coli.

Features:

1. Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors.

2. Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects.

3. Like other vectors, BACs contain:

1. Origin (ori) sequence derived from an E. coli plasmid called the F factor.

2. Multiple cloning sites (restriction sites).

3. Selectable markers (antibiotic resistance).

Fosmid:

1. Based on the E. coli bacterial F-plasmid.

2. Can insert 40 kb fragment of DNA.

3. Low copy number in the host (e.g., 1 fosmid).

4. Fosmids offer higher stability than comparable high copy number cosmids. Contain other features similar to plasmids/cosmids such as origin sequence and polylinker.


Cut DNA from donor and plasmid vector with the same restriction enzyme

Mix to generate recombinant DNA molecule

When such a modified plasmid is introduced into a bacterium, it is mass produced as the bacterium divides

Figure 19.4

Several techniques are used to insert DNA into cells- Chemicals that open transient holes in plasma membrane- Liposomes that carry DNA into cells

- Electroporation: A brief jolt of electricity to open membrane

- Microinjection: Uses microscopic needles - Particle bombardment: a gun like device

shoots metal particles coated with foreign DNA

Techniques of Recombinant DNA Technology

• Inserting DNA into Cells– Goal of DNA technology is insertion of DNA into

cell – Natural methods

• Transformation• Transduction• Conjugation

– Artificial methods• Electroporation• Protoplast fusion• Injection: gene gun and microinjection


Electroporation:

Figure 8.9a Artificial methods of inserting DNA into cells: electroporation

Chromosome

Electroporation

Pores in wall and membrane

Competent cell

Electricalfield applied

DNA fromanother source

Cell synthesizesnew wall

Recombinant cell

Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion

Cell walls

Protoplast fusion

Polyethyleneglycol

Protoplasts

Enzymes removecell walls

Fused protoplasts

Recombinant cellNew wall

Cell synthesizesnew wall

Figure 8.9c Artificial methods of inserting DNA into cells: gene gun

Gene gun

Protoplasts

Nylonprojectile

Nylonprojectile

Blank .22caliber shell

DNA-coated beads

Vent

Target cell

Plate to stopnylon projectile

Figure 8.9d Artificial methods of inserting DNA into cells: microinjection

Microinjection

Target cell

Suction tubeto hold targetcell in place

Target cell’snucleus

Micropipettecontaining DNA

Selecting Recombinant Molecules

Three types of recipient cells can result from attempt to introduce a DNA molecule into a bacterial cell

1. Cells that lack plasmids

2. Cells with plasmids that do not contain foreign genes

3. Cells that contain plasmids with foreign genes

Selecting For Cells With Vectors

Vectors are commonly engineered to carry antibiotic resistance genes

Host bacteria without a plasmid die in the presence of the antibiotic

Bacteria harboring the vector survive

Growing cells on media with antibiotics ensures that all growing cells must carry the vector

Selecting For Cells With Inserted DNA

The site of insertion of the DNA of interest can be within a color-producing gene on the vector

Insertion of a DNA fragment will disrupt the vector gene

- And so the bacterial colony that grows will be colorless

Isolating Gene of Interest

Genomic library: Collections of recombinant DNA that contain pieces of the genome

DNA probe: Radioactively (or fluorescently) labeled gene fragments

cDNA library: Genomic library of protein encoding genes produced by extracting mRNA and using reverse transcriptase to make DNA


• Gene Libraries– A collection of bacterial or phage clones

• Each clone in library often contains one gene of an organism’s genome

– Library may contain all genes of a single chromosome

– Library may contain set of cDNA complementary to mRNA


Figure 8.4 Production of a gene library-overview

Genome

Isolate genomeor organism.

Generate fragments usingrestriction enzymes.

Insert each fragmentinto a vector.

Introduce vectorsinto cells.

Culture recombinant cells;descendants are clones.

Recombinant DNA Libraries (2 major types):

1. Genomic/chromosomal library, Collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome or chromosome.

2. Complementary DNA (cDNA) library, Collection of clones of DNA copies made from mRNA isolated from cells.

reverse transcriptase (RNA dependent DNA polymerase) Synthesizes DNA from an RNA template cDNA libraries reflect what is being expressed in cells.

# of clones required for a complete library can be calculated from the size of the genome and average size of overlapping fragments cut by restriction enzymes.

Library should contain many times more clones than the calculated minimum number of clones.

Genomic Library:

3 ways to cut the DNA for a genomic library:

1. Complete digestion (at all relevant restriction sites)

1. Choice of restriction enzyme determines size of fragments (e.g., 4-base cutter gives smaller fragments, 8-base cutter gives larger fragments).

2. Produces a large number of non-overlapping DNA clones.3. Genes containing two or more restriction sites may be cloned in two or more

pieces.

2. Partial digestion

1. Limiting the amount and time the enzyme is active results in a population of overlapping fragments.

2. Fragments can be size selected by agarose electrophoresis.3. Fragments have sticky ends and can be cloned directly.

3. Mechanical shearing

1. Produces longer DNA fragments.2. Ends are not uniform, requires enzymatic modification before fragments can

be inserted into a cloning vector.

cDNA Library:

1. cDNA is derived from mature mRNA, does not include introns.

2. cDNA may contain less information than the coding region.

3. cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue and with time).

4. mRNA degrades quickly after cell death, and typically requires immediate isolation (cryoprotectants can increase yield if immediate freezing is postponed by fieldwork).

5. Creating a cDNA library:

1. Isolate mRNA

2. Synthesize cDNA

3. Clone cDNA

Applications of Recombinant DNA

Recombinant DNA is used to:- Study the biochemical properties or genetic pathways of that protein- Mass-produce proteins (e.g., insulin)

Sometimes conventional methods are still the better choice because of economics

Textile industry can produce indigo dye in E. coli by genetically modifying genes of the glucose pathway and introducing genes from another bacterial species

Applications of Recombinant DNA Technology

• Pharmaceutical and Therapeutic Applications

– Protein synthesis• Creation of synthetic peptides for cloning

– Vaccines• Production of safer vaccines• Subunit vaccines• Genes of pathogens introduced into common fruits and

vegetables• Injecting humans with plasmid carrying gene from

pathogen– Humans synthesize pathogen’s proteins




– Genetic screening• DNA microarrays used to screen individuals for

inherited disease caused by mutations• Can also identify pathogen’s DNA in blood or

tissues

– DNA fingerprinting• Identifying individuals or organisms by their unique

DNA sequence




– Gene therapy• Missing or defective genes replaced with normal

copies• Some patients’ immune systems react negatively

– Medical diagnosis• Patient specimens can be examined for presence of

gene sequences unique to certain pathogens

– Xenotransplants• Animal cells, tissues, or organs introduced into

human body© 2012 Pearson Education Inc.

Table 19.3

Transgenic Animals

An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal

Transgenic sheep, cows, and goats have all expressed human genes in their milk, - Clotting factors- Clot busters- Collagen- Antibodies

Transgenic Animals

Finally, an organism must be regenerated from the altered cell

If the trait is dominant, the transgenic animal must express it in the appropriate tissue at the right time in development

If the trait is recessive, crosses between heterozygotes may be necessary to yield homozygotes that express the trait

Animal Models

Transgenic animals are far more useful as models of human diseases- Example: Inserting the mutant human beta globin gene that causes sickle-cell anemia into mice

Drug candidates can be tested on these animal models before testing on humans- Will be abandoned if they cause significant side effects

Animal Models

Transgenic animal models have limitations- Researchers cannot control where a transgene inserts, and how many copies do so- The level of gene expression necessary for a phenotype may differ in the model and humans- Animal models may not mimic the human condition exactly because of differences in development or symptoms

Bioremediation

Transgenic organisms can provide processes as well as products

Bioremediation: The use of bacteria or plants to detoxify environmental pollutants

Examples - Nickel-contaminated soils

- Mercury-tainted soils- Trinitrotoluene (TNT) in land mines


• Environmental Studies– Most microorganisms have never been grown

in a laboratory– Scientists know them only by their DNA

fingerprints• Allowed identification of over 500 species of

bacteria from human mouths• Determined that methane-producing archaea are a

problem in rice agriculture



• Agricultural Applications– Production of transgenic organisms

• Recombinant plants and animals altered by addition of genes from other organisms



• Agricultural Applications– Herbicide tolerance

• Gene from Salmonella conveys resistance to glyphosate (Roundup™)

– Farmers can kill weeds without killing crops

– Salt tolerance• Scientists have inserted gene for salt tolerance into

tomato and canola plants• Transgenic plants survive, produce fruit, and

remove salt from soil



• Agricultural Applications– Freeze resistance

• Crops sprayed with genetically modified bacteria can tolerate mild freezes

– Pest resistance• Bt toxin

– Naturally occurring toxin harmful only to insects – Organic farmers used to reduce insect damage to

crops

• Gene for Bt toxin inserted into various crop plants• Genes for Phytophthora resistance inserted into

potato crops© 2012 Pearson Education Inc.


• Agricultural Applications– Improvements in nutritional value and yield

• Tomatoes allowed to ripen on vine and shelf life increased

– Gene for enzyme that breaks down pectin suppressed

• BGH allows cattle to gain weight more rapidly– Produce meat with lower fat content and produce

10% more milk

• Gene for β-carotene (vitamin A precursor) inserted into rice

• Scientists considering transplanting genes coding for entire metabolic pathways


recombinant dna technology recombinant dna technology is also known as gene cloning however, it...

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