chapter 08: recombinant dna technology

© 2014 Pearson Education, Inc.

CHAPTER 08: RECOMBINANT DNA TECHNOLOGY


The Role of Recombinant DNA Technology in Biotechnology

•  Biotechnology – the use of microorganisms to

make practical products

•  Recombinant 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


Bacterial cell

Bacterial chromosome

Plasmid

Isolate plasmid.

DNA containing gene of interest

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

1

2

3

4

5

6

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 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

•  Restriction site sequences usually palindromes

•  Categorized into two groups based on type of cut

•  Cuts with sticky ends

•  Cuts with blunt ends


Restriction site (palindrome)

5ʹ

Restriction enzyme

Sticky ends

Production of sticky ends

Restriction enzyme 1

Restriction enzyme 2

Blunt ends

Production of blunt ends

Restriction fragments from two different organisms cut by the same restriction enzyme

Ligase

Recombinant DNA molecules

Recombinants using blunt ends

Ligase

Recombinants using sticky ends

Recombinant DNA molecules

3ʹ G A A T T C C T T A A G

5ʹ 3ʹ C C C G G G G G G C C C

5ʹ 3ʹ C C C G G G G G G C C C

5ʹ 3ʹ G T T A A C C A A T T G

5ʹ 3ʹ G T T A A C C A A T T G

5ʹ 3ʹ C C C A A C G G G T T G

5ʹ 3ʹ G T T G G G C A A C C C

5ʹ 3ʹ A A G C T T T T C G A A

5ʹ 3ʹ A A G C T T T T C G A A

A T T C G A

A T T C G A

G C T T A A

+

Figure 8.2 Actions of restriction enzymes.


Recombinant DNA Technology

Recombinant DNA Technology

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•  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


Antibiotic resistance gene

Restriction site

mRNA for human growth hormone (HGH)

Plasmid (vector)

Restriction enzyme

Reverse transcription

cDNA for HGH

Restriction enzyme

Sticky ends

Gene for human growth hormone

Ligase

Recombinant plasmid

Introduce recombinant plasmid into bacteria.

Recombinant plasmid

Inoculate bacteria on media containing antibiotic.

Bacterial chromosome

Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.

4

3

2

1

A T

A

A

T T

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

A

A G

T T C

G H

T T

A

G H

A

A G

T T C

G H

T T

A

H G

H

A

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



•  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


Genome

Isolate genome of organism.

Generate fragments using restriction enzymes.

Insert each fragment into a vector.

Introduce vectors into cells.

Culture recombinant cells; descendants are clones.

1

2

3

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

Figure 8.4 Production of a gene library.


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



•  Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)

•  Repetitive process consisting of three steps

•  Denaturation

•  Priming

•  Extension

•  Can be automated using a thermocycler


Polymerase Chain Reaction (PCR): Overview

Polymerase Chain Reaction (PCR): Overview

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PCR: Components


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

Denaturation

Priming

Extension

Original DNA molecule

DNA primer Deoxyribonucleotide triphosphates

DNA polymerase

Heat to 94°C

Cool to 65°C

DNA polymerase

DNA primer

72°C

1

2

3

3ʹ 5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ 5ʹ

5ʹ

5ʹ

Repeat 4


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


PCR: The Process

PCR: The Process

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•  Selecting a Clone of Recombinant Cells

•  Must find clone containing DNA of interest

•  Probes are used



•  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


Electrophoresis chamber filled with buffer solution

Lane of DNA fragments of known sizes (kilobase pairs)

Agarose gel

DNA

Wire

Wells

Movement of DNA

A B

C D

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


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

Gel DNA 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 sequence of interest.

Absorbent material

Nitrocellulose membrane

Electrophoresis gel

Probes bind to DNA of interest.

Developed film

1

2

3

4

5

Figure 8.7 The Southern blot technique.



•  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

•  Protoplast fusion

•  Injection – gene gun and microinjection


Chromosome Pores in wall and membrane

Electrical field applied

Electroporation

Competent cell DNA from another source

Cell synthesizes new wall

Recombinant cell

Cell walls

Enzymes remove cell walls

Protoplasts

Protoplast fusion

Polyethylene glycol

Fused protoplasts

Recombinant cell

Cell synthesizes new wall

New wall

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


Blank .22 caliber shell

Nylon projectile

Vent Plate to stop nylon projectile

DNA-coated beads Target cell

Gene gun Nylon projectile

Micropipette containing DNA

Target cell's nucleus

Target cell

Suction tube to hold target cell in place

Microinjection

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


Applications of Recombinant DNA Technology

•  Genetic Mapping

•  Locating genes on a nucleic acid molecule

•  Provides useful facts concerning metabolism, growth

characteristics, and relatedness to others



•  Genetic Mapping

•  Locating genes

•  Until 1970, genes identified by labor-intensive methods

•  Simpler and universal methods now available

•  Restriction fragmentation

•  Fluorescent in situ hybridization (FISH)


Figure 8.10 Fluorescent in situ hybridization (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



•  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


Figure 8.12 DNA fingerprinting.



•  Pharmaceutical and Therapeutic Applications •  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



•  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 a 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 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


Figure 8.13 Genetically modified papaya plants.



•  Agricultural Applications •  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

•  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


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


The Ethics and Safety of Recombinant DNA 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


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 08: recombinant dna technology

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