chapter 12 dna technology february 27, 2013. dna technology has led to advances in –creation of...
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Chapter 12DNA Technology
February 27, 2013
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• DNA technology has led to advances in
– creation of genetically modified crops and
– identification and treatment of genetic diseases
– criminology
Biology and Society: DNA, Guilt, and Innocence
© 2013 Pearson Education, Inc.
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RECOMBINANT DNA TECHNOLOGY
• Biotechnology
– is the manipulation of organisms or their components to make useful products and
– has been used for thousands of years to
– make bread using yeast and
– selectively breed livestock for desired traits.
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• DNA technology
– studying and manipulating genetic material,
– modifying specific genes
– moving genes between organisms
RECOMBINANT DNA TECHNOLOGY
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• Recombinant DNA = DNA from 2 sources combined to form a single DNA molecule.
• Genetic engineering = the direct manipulation of genes
• ex. By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities.
RECOMBINANT DNA TECHNOLOGY
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Making Humulin
• In 1982, the world’s first genetically engineered pharmaceutical product was sold.
• Humulin = human insulin
– produced by genetically modified bacteria
– used today by 4 million+ people with
– diabetes.
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Figure 12.3
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• Also makes
– HGH = human growth hormone
– EPO = erythropoietin
– Vaccines
Making Humulin
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Genetically Modified (GM) Foods
• Genetically modified organisms (GMO’s) = organisms that have one or more genes by artificial means.
• Transgenic = organism with a gene from another species.
© 2013 Pearson Education, Inc.
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• In the USA, ~ ½ of the corn crop and ¾ + of the soybean and cotton crops are GM.
Genetically Modified (GM) Foods
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• GM Strawberry have bacterial proteins = natural antifreeze
• GM Potatoes and rice produce harmless proteins; may one day serve as edible vaccines.
Genetically Modified (GM) Foods
© 2013 Pearson Education, Inc.
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• “Golden rice 2”– transgenic rice with genes from daffodils and corn– could help prevent vitamin A deficiency and
resulting blindness.
Genetically Modified (GM) Foods
© 2013 Pearson Education, Inc.
Golden Rice Debate
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“Pharm” Animals
• No transgenic animals are sold as food (yet).
• GM pig has gene for human hemoglobin, which can be used in blood transfusions.
• GM pigs can produce proteins that convert “bad” fatty acids to omega-3 fatty acids.
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How are transgenic organisms created?
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Recombinant DNA Techniques
• Plasmids = workhorses of modern biotechnology
• small, circular DNA molecules
• replicate separately from the larger bacterial chromosome.
• can carry virtually any gene
• act as vectors = move genes from one cell to another
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Figure 12.7
Plasmids
Bacterialchromosome
Remnant ofbacterium
Co
lori
zed
TE
M
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Figure 12.8a
Plasmid
Bacterial cell
Isolate plasmids.
Cut both DNAswith same enzyme.
Isolate DNA.
Geneof
interestOthergenes
DNA fragmentsfrom cell
Mix the DNA fragments and join them together.
DNA
Cell containingthe gene ofinterest
Gene of interest
Recombinant DNA plasmids
1 2
3
4
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Figure 12.8b
5
6
7
Bacteria take up recombinant plasmids.
Recombinant bacteriaBacterial clone
Clone the bacteria.
Find the clone with the gene of interest.
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Figure 12.8c
8
Protein fordissolvingclots
Protein for“stone-washing” jeans
Harvestedproteinsmay beused directly.
The geneand proteinof interestare isolatedfrom thebacteria.
Genes maybe insertedinto otherorganisms.
Some usesof genes
Some usesof proteins
Gene for pestresistance
Genes for cleaning up toxic waste
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Figure 12.8
Plasmid
Bacterial cellIsolate plasmids.1 2
3
4
5
6
7
Cut both DNAswith same enzyme.
Isolate DNA.
Geneof
interestOthergenes
DNA fragmentsfrom cell
DNA
Cell containingthe gene of interest
Mix the DNA fragments and join them together.
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Recombinant bacteriaBacterial clone
Clone the bacteria.
Find the clone with the gene of interest.
A protein is used todissolve blood clotsin heart attacktherapy.
A protein is used to prepare“stone-washed” blue jeans.
Bacteriaproduceproteins,which can be harvestedand used directly.
The geneand proteinof interestare isolatedfrom thebacteria.
Genes maybe insertedinto otherorganisms.
Some usesof genes
Some usesof proteins
A gene for pestresistance isinserted intoplants.
A gene is used to alterbacteria for cleaningup toxic waste.
8
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A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
• Recombinant DNA is produced by combining two ingredients:
1. a bacterial plasmid and
2. the gene of interest.
• To combine these ingredients, a piece of DNA must be spliced into a plasmid.
© 2013 Pearson Education, Inc.
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• DNA gets spliced by
– using restriction enzymes, which cut DNA at specific restriction sites
– Producing restriction fragments with “sticky ends”
– DNA ligase connects the DNA fragments into one piece by forming new bonds
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
© 2013 Pearson Education, Inc.
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Figure 12.9-1
Recognition site (recognition sequence)for a restriction enzyme
Restrictionenzyme
Sticky end
Sticky end
DNA
A restriction enzyme cuts the DNA into fragments.
1
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Figure 12.9-2
Recognition site (recognition sequence)for a restriction enzyme
Restrictionenzyme
Sticky end
Sticky end
DNA
A DNA fragment is added from another source.
A restriction enzyme cuts the DNA into fragments.
1
2
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Figure 12.9-3
Recognition site (recognition sequence)for a restriction enzyme
Restrictionenzyme
Sticky end
Sticky end
DNA
A DNA fragment is added from another source.
A restriction enzyme cuts the DNA into fragments.
Fragments stick together bybase pairing.
1
2
3
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Figure 12.9-4
Recognition site (recognition sequence)for a restriction enzyme
Restrictionenzyme
Sticky end
Sticky end
DNA
DNAligase
Recombinant DNA molecule
A DNA fragment is added from another source.
A restriction enzyme cuts the DNA into fragments.
Fragments stick together bybase pairing.
DNA ligase joins the fragments into strands.
1
2
3
4
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How is DNA used in forensics?
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Biology and Society: DNA, Guilt, and Innocence
• DNA profiling = analysis of DNA samples to determine whether the samples come from the same individual.
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Figure 12.13-1
DNA isolated Crime scene Suspect 1 Suspect 21
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Figure 12.13-2
DNA isolated
DNA amplified
Crime scene Suspect 1 Suspect 21
2
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Figure 12.13-3
DNA isolated
DNA amplified
DNA compared
Crime scene Suspect 1 Suspect 21
2
3
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Investigating Murder, Paternity, and Ancient DNA
• DNA profiling can be used to
– Test guilt of suspected criminals,
– identify tissue samples of victims
– resolve paternity cases
– identify contraband animal products
– trace the evolutionary history of organisms
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NAU’s Innocence Project
Arizona Innocence Project
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Figure 12.14
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DNA Profiling Techniques The Polymerase Chain Reaction (PCR)
• The polymerase chain reaction (PCR)
– quickly copies a specific segment of DNA
– generates enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling.
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Figure 12.15
InitialDNAsegment
Number of DNA molecules1 2 4 8
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• Short tandem repeats (STRs) are
– short sequences of DNA
– repeated many times together in the genome
• STR analysis
– method of DNA profiling
– Compares lengths of STR sequences
Short Tandem Repeat (STR) Analysis
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Figure 12.16
Crime scene DNA
Suspect’s DNA
Same number ofshort tandem repeats
Different numbers ofshort tandem repeats
STR site 1 STR site 2
AGAT
AGAT GATA
GATA
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Figure 12.17-1
Mixture of DNAfragments ofdifferent sizes
Powersource
Gel electrophoresis = method for sorting DNA fragments by size
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Figure 12.17-2
Mixture of DNAfragments ofdifferent sizes
Powersource
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Figure 12.17-3
Band of longest(slowest) fragments
Band of shortest(fastest) fragments
Mixture of DNAfragments ofdifferent sizes
Powersource
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Figure 12.18
Amplifiedcrime sceneDNA
Amplifiedsuspect’sDNA
Longerfragments
Shorterfragments
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Figure 12.UN02
Crime scene Suspect 1 Suspect 2
DNA
Polymerase chainreaction (PCR)amplifies STRsites
LongerDNAfragments
ShorterDNAfragments
DNA fragments compared by gel electrophoresis
Gel
(Bands of shorter fragments move faster toward the positive pole.)
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Gene Therapy = giving “good” gene to patientthat lacks it.
History of Gene Therapy
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Figure 12.UN03
RNA versionof a normalhuman gene
Virus withRNA genome
Bonemarrow
A normal human gene is transcribedand translated in a patient, potentiallycuring the genetic disease permanently