chapter 10 rec dna technology-fall2020

© 2014 Pearson Education, Inc.

CHAPTER 10 Recombinant DNA Technology


The Role of Recombinant DNA Technology in Biotechnology

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


Figure 8.1 Overview of recombinant DNA technology. Bacterial cell

Bacterialchromosome

Plasmid

Isolate plasmid.

DNA containinggene of interest

Gene of interestEnzymatically 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

Createbeneficialcombinationof traits

Produce vaccines,antibiotics,hormones, orenzymes

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3

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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 (Ex. Plasmid curing-

suing acridine-orange mediated mutations)



• The Use of Reverse Transcriptase to Synthesize

cDNA

• Isolated from retroviruses

• Uses RNA template to transcribe molecule of cDNA

• Easier to isolate 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



• Restriction Enzymes

• Bacterial enzymes that cut DNA molecules only at

restriction sites (They are a natural protection against

phages (which attack the bacteria) by slicing their DNA).

• Restriction site sequences usually palindromes

• Categorized into two groups based on type of cut

• Cuts with sticky ends

• Cuts with blunt ends


Figure 8.2 Actions of restriction enzymes.

Restriction site(palindrome)

5¢

Restriction enzyme

Sticky ends

Production of sticky ends

Restrictionenzyme 1

Restrictionenzyme 2

Blunt ends

Productionof blunt ends

Restriction fragments from two different organismscut by the same restriction enzyme

Ligase

Recombinant DNA molecules

Recombinants using blunt ends

Ligase

Recombinants using sticky ends

Recombinant DNA molecules

3¢G A A T T CC T T A A G

5¢ 3¢C C C G G GG G G C C C

5¢ 3¢C C C G G GG G G C C C

5¢ 3¢G T T A A CC A A T T G

5¢ 3¢G T T A A CC A A T T G

5¢ 3¢C C C A A CG G G T T G

5¢ 3¢G T T G G GC A A C C C

5¢ 3¢A A G C T TT T C G A A

5¢ 3¢A A G C T TT T C G A A

AT T C G A

A G C T TA A

T T C G A

GC T T A A

A A T T CG

A G C T TA

+



• 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 An example of the process for producing a recombinant vector.

Antibioticresistancegene

Restrictionsite

mRNA for humangrowth hormone (HGH)

Plasmid (vector)

Restrictionenzyme

Reversetranscription

cDNA for HGH

Restrictionenzyme

Sticky ends

Gene for humangrowth hormone

Ligase

Recombinant plasmid

Introduce recombinantplasmid into bacteria.

Recombinantplasmid

Inoculate bacteriaon media containingantibiotic.

Bacterialchromosome

Bacteria containingthe plasmid withHGH gene survivebecause they alsohave resistance gene.

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2

1

A A GC

TT

T T CGAA

A

AGCT

T

T TCG A

A

A G C T T HGH AHGHA T T C G A

A

AGCTT

T T CGA

A

AG

CT

TAHGHTTCGA

AHGH

AAGCT

T

T T CGA

A

G C T TAHGHTTCGA

AHGHA



• Gene Libraries

• A collection of bacterial or phage clones

• Each clone in library often contains one gene of an

organism's genome

• A genomic library is a collection of the total genomic DNA

from a single organism.


Figure 8.4 Production of a gene library. Genome

Isolate genomeof organism.

Generate fragments usingrestriction enzymes.

Insert each fragmentinto a vector.

Introduce vectorsinto cells.

Culture recombinant cells;descendants are clones.

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2

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4

5

1 2 3 4 5 6 7 8 9 10 11

1 2 3

4 5 6

7 8 9

10 11

1 2 3 4 5 6

7 8 9 10 11

1 2 3 4 5 6

7 8 9 10 11


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 use to amplify genome of unknown

pathogen

• Amplified DNA from Bacillus anthracis spores in 2001 to

identify source of spores (Anthrax infection)



• Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)

• Repetitive process consisting of three steps

• Denaturation

• Priming

• Extension

• Can be automated using a thermocycler


Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA.

Denaturation

Priming

Extension

Original DNAmolecule

DNA primerDeoxyribonucleotidetriphosphates

DNA polymerase

Heat to 94°C

Cool to 65°CDNA polymerase

DNA primer

72°C

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3

3¢5¢

3¢

5¢

3¢5¢

3¢5¢

5¢

5¢

Repeat4


Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.


PCR: The Process

PCR: The Process



• 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 (phosphate ion; P3O4-) 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


Electrophoresis chamber filled with buffer solution

Lane of DNA fragments of known sizes (kilobase pairs)

Agarose gel

DNA

Wire

Wells

Movement of DNA

AB

CD

E

a

b

(50)(40)

(35)

(15)(10)(5)

(+)

(–)

Figure 8.6 Gel electrophoresis.



• 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 blots

• Genetic "fingerprSeinting"

• Diagnosis of infectious disease

• Demonstrate presence of organisms that cannot be cultured


Figure 8.7 The Southern blot technique.

Use gel electrophoresis to separate fragments by size; denature DNA into single strands with NaOH.

Incubate with film; radiation exposes film. Develop film.

Absorbent material

Nitrocellulose membrane

GelDNA bands

DNA

DNA molecules

Restriction fragments

Restriction enzymes

The DNA fragments are invisible to the investigators at this stage.

Nitrocellulose membrane with DNA fragments at same locations as in gel (still invisible) is baked to permanently affix DNA.

Side view

Add radioactive probes complementary to DNA nucleotide sequenceof interest.

Absorbent material

Nitrocellulose membrane

Electrophoresis gel

Probes bind to DNA of interest.

Developed film

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• DNA Microarrays

• Consist of molecules of immobilized single-stranded DNA

• Fluorescently labeled DNA washed over array will adhere

only at locations where there are complementary DNA

sequences

• Variety of scientific uses of DNA microarrays

• Monitoring gene expression

• Diagnosis of infection

• Identification of organisms in an environmental sample


Figure 8.8 DNA microarray.



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

• Natural methods

• Transformation

• Transduction

• Conjugation

• Artificial methods

• Electroporation & Heat Shock

• Protoplast fusion

• Injection – gene gun and microinjection


ChromosomePores in wall and membrane

Electrical field applied

ElectroporationCompetent cell

DNA fromanother source

Cell synthesizesnew wall

Recombinant cell

Cell walls

Cellulase Enzymes removecell walls

ProtoplastsProtoplast fusion

Polyethyleneglycol

Fused protoplasts

Recombinant cell

Cell synthesizesnew wall

New wall

Figure 8.9a-b Artificial methods of inserting DNA into cells.

Ex. Hybrid flowers with different colors


Blank .22caliber shell

Nylonprojectile

Vent Plate to stopnylon projectile

DNA-coated beads Target cell

Gene gunNylonprojectile

Micropipette containing DNA

Target cell's nucleus

Target cell

Suction tubeto hold targetcell in place

Microinjection

Figure 8.9c-d Artificial methods of inserting DNA into cells.


Applications of Recombinant DNA Technology

• Genetic Mapping

• Locating genes

• Until 1970, genes identified by labor-intensive methods

• Simpler and universal methods now available

• Restriction fragmentation (RFLP)

• Fluorescent in situ hybridization (FISH)


RFLP


FISH


Figure 8.11 Automated DNA sequencing.



• 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



• 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


Applications of Recombinant DNA Technology• Pharmaceutical and Therapeutic Applications• 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


Applications of Recombinant DNA Technology• Agricultural Applications

• Production of transgenic organisms

• Recombinant plants and animals altered by addition of genes

from other organisms

• Herbicide tolerance (GMO transgenic plants)- Gene from Salmonella

(incorporated in the plant genome) 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


The Ethics and Safety of Recombinant DNA Technology

• Ethical issues

• Routine screenings?

• Who should pay?

• Genetic privacy rights?

• Profits from genetically altered organisms?

• Required genetic screening?

• Forced correction of "genetic abnormalities"?

chapter 10 rec dna technology-fall2020

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