Chapter 8 Recombinant DNA Technology - Brazosport College · The Tools of Recombinant DNA Technology •Restriction Enzymes •Bacterial enzymes that cut DNA molecules only at restriction

Chapter 8 Recombinant DNA Technology

MDufilho 10/1/2017 1

The Role of Recombinant DNA Technology in

Biotechnology

• Biotechnology?

• Recombinant deoxyribonucleic acid (DNA)

technology

• Intentionally modifying genomes of organisms for

practical purposes

• Three goals:

• Eliminate undesirable phenotypic traits

• Combine beneficial traits of two or more organisms

• Create organisms that synthesize products humans need

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Bacterial cell

Bacterial

chromosome Plasmid

Isolate plasmid. Gene of interest

DNA containing

gene of interest

Enzymatically cleave

DNA into fragments.

Isolate fragment

with the gene of

interest.

Insert gene into plasmid.

Insert plasmid and gene into

bacterium.

Culture bacteria.

Harvest copies of

gene to insert into

plants or animals.

Harvest proteins

coded by gene.

Eliminate

undesirable

phenotypic

traits.

Create

beneficial

combination

of traits.

Produce vaccines,

antibiotics,

hormones, or

enzymes.

Figure 8.1 Overview of recombinant DNA technology.


The Tools of Recombinant DNA Technology

• Mutagens

• Physical and chemical agents that produce mutations

• Scientists utilize mutagens to:

• create changes in microbes' genomes to change

phenotypes.

• select for and culture cells with beneficial characteristics.

• Mutated genes alone can be isolated



• The Use of Reverse Transcriptase to

Synthesize complementary DNA (cDNA)

• Isolated from retroviruses

• Uses ribonucleic acid (RNA) template to transcribe

molecule of cDNA

• Easier to isolate mitochondrial RNA (mRNA) molecule

for desired protein first

• cDNA generated from mRNA of eukaryotes has introns

removed

• Allows cloning in prokaryotic cells



• Synthetic Nucleic Acids

• Molecules of DNA and RNA produced in cell-free

solutions

• Uses of synthetic nucleic acids:

• Elucidating the genetic code

• Creating genes for specific proteins

• Synthesizing DNA and RNA probes to locate specific

sequences of nucleotides

• Synthesizing antisense nucleic acid molecules

• Synthesizing polymerase chain reaction (PCR) primers



• Restriction Enzymes

• Bacterial enzymes that cut DNA molecules only at

restriction sites

• Restriction site usually sequences palindromes

• Categorized into two groups based on type of cut:

• Cuts with sticky ends

• Cuts with blunt ends


Figure 8.2 Actions of representative restriction enzymes.

Restriction site

(palindrome)

5′ 3′ 5′

5′

5′

5′

3′

3′

3′

3′

5′ 3′ 3′ 5′

5′ 3′ 5′ 3′

Restriction enzyme

Sticky ends Blunt ends

Restriction

enzyme 1

Restriction

enzyme 2

Production of sticky ends

Recombinants using blunt ends

Recombinants using sticky ends

Recombinant DNA molecules

Ligase

Restriction fragments from two different organisms

cut by the same restriction enzyme

Ligase

Production

of blunt ends

Recombinant DNA molecules


Recombinant DNA Technology

Recombinant DNA Technology PLAY


recombinant_dna_subtitle.mp4


• 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

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Antibiotic-

resistance

gene

Restriction

site

mRNA for human

growth hormone (HGH)

Reverse

transcription

cDNA for HGH

Restriction

enzyme

Restriction

enzyme

Plasmid (vector)

Sticky ends

Gene for human

growth hormone

Ligase

Recombinant plasmid

Introduce recombinant

plasmid into bacteria.

Recombinant

plasmid

Inoculate bacteria

on media containing

antibiotic.

Bacteria containing

the plasmid with

HGH gene survive

because they also

have resistance gene.

Bacterial

chromosome

Figure 8.3 An example of the process for producing a recombinant vector.

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• 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

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Genome

Isolate genome

of organism.

Generate fragments using

restriction enzymes.

1 2 3 4 5 6

7 8 9 10 11

Insert each fragment

into a vector.

1 2 3 4 5 6

7 8 9 10 11

Introduce vectors

into cells.

1 2 3

4 5 6

7 8 9

10 11

Culture recombinant cells;

descendants are clones.

1 2 3 4 5 6 7 8 9 10 11

Figure 8.4 Production of a gene library.

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Techniques of Recombinant DNA Technology

• Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)

• Large number of identical molecules of DNA produced

in vitro

• Critical to amplify DNA in variety of situations

• Epidemiologists used PCR to determine that two separate

Ebola outbreaks occurred in Africa in 2014

• Amplified DNA from Bacillus anthracis spores in 2001 to

identify source of spores

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• Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)

• Repetitive process consisting of three steps:

• Denaturation

• Priming

• Extension

• Can be automated using a thermocycler

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• Selecting a Clone of Recombinant Cells

• Must find clone containing DNA of interest

• Probes are used

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• Separating DNA Molecules: Gel

Electrophoresis and the Southern Blot

• Gel electrophoresis:

• Separates molecules based on electrical charge, size,

and shape

• Allows scientists to isolate DNA of interest

• Negatively charged DNA drawn toward positive electrode

• Agarose makes up gel; acts as molecular sieve

• Smaller fragments migrate faster and farther than larger

ones

• Determine size by comparing distance migrated to

standards

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Figure 8.6 Gel electrophoresis.

Wells (–)

Electrophoresis

chamber filled with

buffer solution

Agarose gel (+) (50)

(40) (35)

(15) (10)

(5)

A B

C D

E

a

DNA

Movement

of DNA

b

Wire

Lane of DNA

fragments of

known sizes

(kilobase pairs)

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• Separating DNA Molecules: Gel

Electrophoresis and the Southern Blot

• Southern blot

• DNA transferred from gel to nitrocellulose membrane

• Probes used to localize DNA sequence of interest

• Northern blot — similar technique used to detect RNA

• Uses of Southern blot

• Genetic “fingerprinting”

• Diagnosing infectious disease

• Demonstrating the presence of organisms that cannot be

cultured

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• 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

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Figure 8.8a-b Artificial methods of inserting DNA into cells.

Chromosome

Electrical

field applied

Electroporation

Competent cell

Pores in wall and membrane

DNA from

another source

Cell synthesizes

new wall

Recombinant cell

Protoplast fusion

Cell walls

Enzymes remove

cell walls

Protoplasts

Polyethylene

glycol

Cell synthesizes

new wall

Recombinant cell

New cell

Fused protoplasts

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Figure 8.8c-d Artificial methods of inserting DNA into cells.

Gene gun Microinjection

Micropipette

containing DNA

Target cell's

nucleus Target cell

Suction tube

to hold target

cell in place

Plate to stop

nylon projectile

Target cell

Vent Nylon

projectile

Blank .22-

caliber shell

DNA-coated beads

Nylon

projectile

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Applications of Recombinant DNA Technology

• Genetic Mapping

• Locating genes on a nucleic acid molecule

• Until 1970, genes identified by labor-intensive methods

• Simpler and universal methods now available

• Restriction fragmentation

• Provides useful facts concerning metabolism, growth

characteristics, and relatedness to others

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• Microbial Communities 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

• Next-generation sequencing allows for the

determination of all of the members of a microbiome

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• Pharmaceutical and Therapeutic Applications

• Protein synthesis

• Creation of synthetic proteins by bacteria and yeast cells

• Vaccines

• Production of safer vaccines

• Subunit vaccines

• Introduce genes of pathogens into common fruits and

vegetables

• Injecting humans with plasmid-carrying gene from

pathogen

• Humans synthesize pathogen's proteins

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• Genetic screening

• DNA microarrays used to screen individuals for inherited

disease caused by mutations

• Can also identify viral DNA in blood or tissues

• Gene therapy

• Missing or defective genes replaced with normal copies

• Difficult to get a functioning gene into enough cells to

affect the disease

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• 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

• Biomedical Animal Models

• Animals are used in biomedical research to study

diseases and develop new diagnostic and therapeutic

procedures

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• Agricultural Applications

• Production of transgenic organisms

• Recombinant plants and animals altered by addition of

genes from other organisms

• Also called genetically modified organisms (GMOs)

• Herbicide tolerance

• Gene from Agrobacterium tumefaciens conveys

resistance to glyphosate (Roundup)

• Farmers can kill weeds without killing crops

• Salt tolerance

• Scientists have inserted a gene for salt tolerance into

tomato and canola plants

• Transgenic plants survive, produce fruit, and remove salt

from soil

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• Freeze resistance

• Crops sprayed with genetically modified bacteria can

tolerate mild freezes

• Pest resistance

• Bacillus thuringiensis (Bt) toxin

• Naturally occurring toxin only harmful to insects

• Organic farmers use to reduce insect damage to crops

• Gene for Bt toxin inserted into various crop plants

• Genes for Phytophthora resistance inserted into potato

crops

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• Improvements in nutritional value and yield

• Enzyme that breaks down pectin suppressed in some

tomatoes

• Allows tomatoes to ripen on vine and increases shelf

life

• Bovine growth hormone (BGH) allows cattle to gain

weight more rapidly

• Have 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 10/1/2017 MDufilho 30

The Ethics and Safety of Recombinant DNA

Technology

• Long-term effects of transgenic manipulations are

unknown

• Unforeseen problems arise from every new

technology and procedure

• Natural genetic transfer could deliver genes from

transgenic plants and animals into other

organisms

• Transgenic organisms could trigger allergies or

cause harmless organisms to become pathogenic

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Technology

• Studies have not shown any risks to human health

or environment

• Standards imposed on labs involved in

recombinant DNA technology

• Can create biological weapons using same

technology

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Technology

• Ethical issues

• Routine screenings?

• Who should pay?

• Genetic privacy rights?

• Profits from genetically altered organisms?

• Required genetic screening?

• Forced correction of "genetic abnormalities"?

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Chapter 8 Recombinant DNA Technology - Brazosport College · The Tools of Recombinant DNA Technology •Restriction Enzymes •Bacterial enzymes that cut DNA molecules only at restriction

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