chapter 10 rec dna technology-fall2020
TRANSCRIPT
© 2014 Pearson Education, Inc.
CHAPTER 10 Recombinant DNA Technology
© 2014 Pearson Education, Inc.
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
© 2014 Pearson Education, Inc.
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
12
3
4
5
6
© 2014 Pearson Education, Inc.
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)
© 2014 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
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
+
© 2014 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
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.
4
3
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
© 2014 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• 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.
© 2014 Pearson Education, Inc.
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.
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
© 2014 Pearson Education, Inc.
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)
© 2014 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• Multiplying DNA in vitro: The Polymerase
Chain Reaction (PCR)
• Repetitive process consisting of three steps
• Denaturation
• Priming
• Extension
• Can be automated using a thermocycler
© 2014 Pearson Education, Inc.
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
1
2
3
3¢5¢
3¢
5¢
3¢5¢
3¢5¢
5¢
5¢
Repeat4
© 2014 Pearson Education, Inc.
Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.
© 2014 Pearson Education, Inc.
PCR: The Process
PCR: The Process
© 2014 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
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.
© 2014 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
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
1
2
3
4
5
© 2014 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
Figure 8.8 DNA microarray.
© 2014 Pearson Education, Inc.
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 & Heat Shock
• Protoplast fusion
• Injection – gene gun and microinjection
© 2014 Pearson Education, Inc.
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
© 2014 Pearson Education, Inc.
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.
© 2014 Pearson Education, Inc.
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)
© 2014 Pearson Education, Inc.
RFLP
© 2014 Pearson Education, Inc.
FISH
© 2014 Pearson Education, Inc.
Figure 8.11 Automated DNA sequencing.
© 2014 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• 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
© 2014 Pearson Education, Inc.
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
© 2014 Pearson Education, Inc.
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
© 2014 Pearson Education, Inc.
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"?