Recombinant DNA Technology and Genetic Engineering

7/27/2019 Recombinant DNA Technology and Genetic Engineering

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Biotechnology

The use of microbiological and biochemical

techniques to solve problems and produce

product

Recombinant DNA techniques

Methods used to manipulate DNA to

intentionally genetically alter organisms

through genetic engineering

Often to give organisms more useful traits


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Familial Hypercholesterolemia

Gene encodes protein that serves as cells LDL

receptor

Two normal alleles for the gene keep blood level ofLDLs low

Two mutated alleles lead to abnormally high cholesterol

levels & heart disease

Woman with familial hypercholesterolemia

Part of her liver was removed

Virus used to insert normal gene for LDL receptor

into cultured liver cells


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Modified cells alive in womans liver

Blood levels of LDLs down 20 percent

No evidence of atherosclerosis

Cholesterol levels remain high

Remains to be seen whether procedure willprolong her life


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GMOs are organisms that havehad genetic material removedand/or inserted in order to changea particular trait or traits of the

organism. The process is called gene

splicing or genetic engineering

Organisms produced by

transplanting genetic materialsbetween different types oforganisms are called transgenicorganisms.


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Genes from bacteria are splicedinto corn and cotton to makethem less susceptible to insectdamage

Human growth hormoneimplanted into mice & otheranimals so that it can beharvested

ANDi (first transgenic monkey) isa rhesus monkey carrying GFPprotein, showing foreign genecan be inserted into primatechromosome

May lead to primate models of


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Humans have been changing the

genetics of other species for

thousands of yearsArtificial selection of plants and animals

(breeding)

Natural processes also at workMutation, crossing over


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Recombinant DNA technology:

techniques & tools used to analyze genes

Cut DNA up and recombine piecesAmplify modified pieces

Genetic Engineering:uses the above technology to isolate &

modify genes in organisms or even to insert

new genes


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rDNA technology

involves cloning DNA by cutting & pasting

DNA from different sources

Restriction enzymes & DNA ligases

are important enzymes for thisprocess


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Basic components of molecular

biologists toolkit

Restriction enzymes Gel electrophoresis

DNA probes

Primers


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Restriction enzymes areDNA-cutting enzymes that arefound in bacteria

They are also called

endonucleases (cut withinDNA

sequences)

Microbiologists from 1960s

discovered that some bacteriaare protected from destructionby viruses because they cutviral DNA, restricting viral

replication


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Molecular scissors that cut DNA at a

specific nucleotide sequence

Over 200 different restriction enzymes

are known, each isolated from

bacteria and able to cut DNA in aunique manner


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In 1970, Hamilton Smith isolated HindIII(1st restriction enzyme well characterizedand used for DNA cloning), which comesfrom Haemophilus influenzae.

In 1970, Hamilton Smith isolated HindIII(1st restriction enzyme well characterizedand used for DNA cloning), which comesfrom Haemophilus influenzae.

They cut DNA by cleaving phosphodiesterbonds (in sugar-phosphate backbone) that

join adjacent nucleotides


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Restriction enzymesshow specificity forcertain substrates (DNAin this case)

They recognize, bind to,and cut DNA at specificsites called restrictionsites (recognition site)

Usually a 4-base pair or

6-base pair cutter Restriction sites are

palindromes


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Some cut DNA to

create fragments with

overhanging single-

stranded ends (stickyends or cohesive

ends), while others

create fragments with

non-overhanging ends(blunt ends)

Enzymes that create stickyends are favored for cloningexperiments since the DNA

fragments can be easily joinedtogether

DNA from any source can bedigested (as long as it has thespecific restriction site)


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Want to study or isolate a particular gene

Need to get many copies (amplification)of the gene so it can be studied

adequately Most organisms only have one or two

copies of any gene per cell, so we need a

way to amplify copies of that gene Dothat via cloning into a vector

This allows scientists to make additional

copies of the gene using bacteria


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Plasmids can be used as vectors(pieces of DNA

that can accept, carry, and replicate other pieces

of DNA)


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1. Size (must be small enough toseparate easily)

2. Origin of replication (ori) - DNA

sequence at which replication is initiated 3. Multiple cloning site (MCS)

a stretch of DNA with recognition sequences

for common restriction enzymes(Engineered into plasmid so that digestiondoes not result in loss of DNA fragment)


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4. Selectable marker genes -allow for selection andidentification of transformedbacteria

Most common selectablemarkers are antibioticresistance. Lac z gene widely used (gene

of interest inserted within lac zgene)

Plated on X-gal (substratesimilar to lactose but turns bluewhen cleaved by -gal); so,recombinant bacteria turn blue& nonrecombinant are white


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5. RNA polymerase promoter

sequences - place where RNA

polymerase binds to begintranscription

6. DNA sequencing primer sequences

- known sequence that allowssequencing of cloned DNA fragments

that have been inserted into the

plasmid


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One primary limitation of bacterialplasmids as vectors is the size of DNAfragments (usually cannot exceed 6-7kb:

6000-7000 base pairs).Bacteriophage vectors

Expression vectors

Bacterial artificial chromosomes (BACs)Yeast artificial chromosomes (YACs)

Tumor-inducing (Ti) vectors


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Cohen discovered that plasmid DNA entersa bacterial cell (transformation) treated withcalcium chloride, chilled on ice, then brieflyheated

A more recent transformation technique iselectroporation (brief pulse of high-voltageelectricity to create tiny holes in bacterialcell wall allowing DNA to enter)

Cells that have been treated fortransformation (so they are more receptiveto take up DNA) are called competentcells


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Sometimes, biolistics are used inorder to have foreign DNA entera cell

DNA is blasted into the cell using

tiny bullets composed oftungsten or gold particles withDNA attached

Done with a gene gun (aka

bioblaster) Can be used on bacteria, yeasts,& mammalian cell lines


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Southern Blot


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Genetically engineered organisms

have variety of usesProtein production

DNA production

Researching gene function andregulation


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Produce commercially important proteins Pharmaceutical proteins

Human insulin

Vaccines

Hepatitis B vaccine

Commercially valuable proteins

Chymosin An enzyme that catalyzes thecoagulation of milk used in the production ofcheese


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Researches interested in acquiringavailable sources of specific DNAfragments

Fragments used forDNA study

Looking genomic characteristics

DNA vaccinesLooking at injecting DNA of pathogen toproduce immune response


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Examples of genetically altered plants include Pest resistant plants

Corn, cotton and potatoes

Herbicide resistant plants Soybeans, cotton and corn

Plants with improved nutrient value

Rice

Plants as edible vaccines Bananas and potatoes


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Recombinant DNA Technology and Genetic Engineering