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PowerPoint Lectures for
Biology: Concepts & Connections, Sixth EditionCampbell, Reece, Taylor, Simon, and Dickey
Chapter 12 DNA Technology and Genomics
Lecture by Mary C. Colavito
DNA evidence was used to solve a double murder in England
– Showed that two murders could have been committed by the same person
– Showed the innocence of someone who confessed to one of the murders
– Showed the absence of a match in 5,000 men tested when the murderer persuaded another man to donate blood in his name
– Showed a match with the murderer and DNA found with both victims
Introduction: DNA and Crime Scene Investigations
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GENE CLONING
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12.1 Genes can be cloned in recombinant plasmids
Genetic engineering involves manipulating genes for practical purposes
– Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA
– Recombinant DNA is formed by joining DNA sequences from two different sources
– One source contains the gene that will be cloned
– Another source is a gene carrier, called a vector
– Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors
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Steps in cloning a gene
1. Plasmid DNA is isolated
2. DNA containing the gene of interest is isolated
3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle
4. DNA with the target gene is treated with the same enzyme and many fragments are produced
5. Plasmid and target DNA are mixed and associate with each other
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12.1 Genes can be cloned in recombinant plasmids
6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together
7. The recombinant DNA is taken up by a bacterial cell
8. The bacterial cell reproduces to form a clone of cells
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12.1 Genes can be cloned in recombinant plasmids
Animation: Cloning a Gene
12_01CloningAGene_A.html12_01CloningAGene_A.html
Examples of
gene use
Recombinant
DNA
plasmid
E. coli bacteriumPlasmid
Bacterial
chromosome
Gene of interest
DNA
Gene
of interest
Cell with DNA
containing gene
of interest
Recombinant
bacterium
Clone
of cells
Genes may be inserted
into other organisms
Genes or proteins
are isolated from the
cloned bacterium
Harvested
proteins
may be
used directly
Examples of
protein use
Gene
of interest
Isolate
plasmid
1
Isolate
DNA
2
Cut plasmid
with enzyme
3
Cut cell’s DNA
with same enzyme
4
Combine targeted fragment
and plasmid DNA
5
Add DNA ligase,
which closes
the circle with
covalent bonds
6
Put plasmid
into bacterium
by transformation
7
Allow bacterium
to reproduce
8
9
E. coli bacteriumPlasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interestIsolate
plasmidIsolate
DNA
1
2
E. coli bacteriumPlasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmidIsolate
DNA
Cut plasmid
with enzyme
Cut cell’s DNA
with same enzyme
1
2
3
4
E. coli bacteriumPlasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmidIsolate
DNA
Cut plasmid
with enzyme
Cut cell’s DNA
with same enzyme
1
2
3
4
Combine targeted fragment
and plasmid DNA
5
E. coli bacteriumPlasmid
Bacterial
chromosome
Gene of interest
DNA
Cell with DNA
containing gene
of interest
Gene
of interest
Isolate
plasmidIsolate
DNA
Cut plasmid
with enzyme
Cut cell’s DNA
with same enzyme
1
2
3
4
Recombinant
DNA
plasmidGene
of interest
Combine targeted fragment
and plasmid DNA
Add DNA ligase,
which closes
the circle with
covalent bonds
5
6
Recombinant
DNA
plasmidGene
of interest
Recombinant
bacterium
Put plasmid
into bacterium
by transformation
7
Recombinant
DNA
plasmidGene
of interest
Recombinant
bacterium
Clone
of cells
Put plasmid
into bacterium
by transformation
Allow bacterium
to reproduce
8
7
Recombinant
DNA
plasmidGene
of interest
Recombinant
bacterium
Clone
of cells
Genes or proteins
are isolated from the
cloned bacterium
Harvested
proteins
may be
used directly
Examples of
protein use
Put plasmid
into bacterium
by transformation
Allow bacterium
to reproduce
8
7
Genes may be inserted
into other organisms
Examples of
gene use
9
12.2 Enzymes are used to “cut and paste” DNA
Restriction enzymes cut DNA at specific sequences
– Each enzyme binds to DNA at a different restriction site
– Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends”
– Fragments with complementary sticky ends can associate with each other, forming recombinant DNA
DNA ligase joins DNA fragments together
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Animation: Restriction Enzymes
12_02RestrictionEnzymes_A.html12_02RestrictionEnzymes_A.html
Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
4
Two (or more)fragments sticktogether bybase-pairing
Restriction enzymerecognition sequence
1
2
DNA
Restriction enzymecuts the DNA intofragments
Sticky end
3
Addition of a DNAfragment fromanother source
4
Two (or more)fragments sticktogether bybase-pairing
DNA ligasepastes the strands
RecombinantDNA molecule
5
12.3 Cloned genes can be stored in genomic libraries
A genomic library is a collection of all of the cloned DNA fragments from a target genome
Genomic libraries can be constructed with different types of vectors
– Plasmid library: genomic DNA is carried by plasmids
– Phage library: genomic DNA is incorporated into bacteriophage DNA
– Bacterial artificial chromosome (BAC) library: specialized plasmids can carry large DNA sequences
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Recombinantplasmid
Phageclone
Bacterialclone
Phage libraryPlasmid library
or
Recombinantphage DNA
Genome cut up withrestriction enzyme
12.4 Reverse transcriptase can help make genes for cloning
Complementary DNA (cDNA) is used to clone eukaryotic genes
– mRNA from a specific cell type is the template
– Reverse transcriptase produces a DNA strand from mRNA
– DNA polymerase produces the second DNA strand
Advantages of cloning with cDNA
– Study genes responsible for specialized characteristics of a particular cell type
– Obtain gene sequences without introns
– Smaller size is easier to handle
– Allows expression in bacterial hosts
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Cell nucleus
Isolation of mRNA
and addition of reverse
transcriptase; synthesis
of DNA strand
RNA splicing2
Transcription1
3
Breakdown of RNA4
Synthesis of second
DNA strand
5
mRNA
DNA of
eukaryotic
gene
IntronExon
RNA
transcript
Exon Intron Exon
Reverse transcriptase
Test tube
cDNA strand
being synthesized
cDNA of gene
(no introns)
12.5 Nucleic acid probes identify clones carrying specific genes
Nucleic acid probes bind to cloned DNA
– Probes can be DNA or RNA sequences complementary to a portion of the gene of interest
– A probe binds to a gene of interest by base pairing
– Probes are labeled with a radioactive isotope or fluorescent tag for detection
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12.5 Nucleic acid probes identify clones carrying specific genes
Screening a gene library
– Bacterial clones are transferred to filter paper
– Cells are lysed and DNA is separated into single strands
– A solution containing the probe is added, and binding to the DNA of interest is detected
– The clone carrying the gene of interest is grown for further study
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RadioactiveDNA probe
Single-strandedDNA
Base pairingindicates thegene of interest
Mix with single-stranded DNA fromgenomic library
GENETICALLY MODIFIED ORGANISMS
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12.6 Recombinant cells and organisms can mass-produce gene products
Cells and organisms containing cloned genes are used to manufacture large quantities of gene products
Capabilities of the host cell are matched to the characteristics of the desired product
– Prokaryotic host: E. coli
– Can produce eukaryotic proteins that do not require post-translational modification
– Has many advantages in gene transfer, cell growth, and quantity of protein production
– Can be engineered to secrete proteins
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12.6 Recombinant cells and organisms can mass-produce gene products
Capabilities of the host cell are matched to the characteristics of the desired product
– Eukaryotic hosts
– Yeast: S. cerevisiae
– Can produce and secrete complex eukaryotic proteins
– Mammalian cells in culture
– Can attach sugars to form glycoproteins
– “Pharm” animals
– Will secrete gene product in milk
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12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine
Products of DNA technology
– Therapeutic hormones
– Insulin to treat diabetes
– Human growth hormone to treat dwarfism
– Diagnosis and treatment of disease
– Testing for inherited diseases
– Detecting infectious agents such as HIV
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12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine
Products of DNA technology
– Vaccines
– Stimulate an immune response by injecting
– Protein from the surface of an infectious agent
– A harmless version of the infectious agent
– A harmless version of the smallpox virus containing genes from other infectious agents
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12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine
Advantages of recombinant DNA products
– Identity to human protein
– Purity
– Quantity
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12.8 CONNECTION: Genetically modified organisms are transforming agriculture
Genetically modified (GM) organisms contain one or more genes introduced by artificial means
Transgenic organisms contain at least one gene from another species
GM plants
– Resistance to herbicides
– Resistance to pests
– Improved nutritional profile
GM animals
– Improved qualities
– Production of proteins or therapeutics
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Agrobacterium tumefaciens
DNA containinggene for desired trait
Ti
plasmid Insertion of geneinto plasmid
Recombinant
Ti plasmid
1
Restriction site
Agrobacterium tumefaciens
DNA containinggene for desired trait
Ti
plasmid Insertion of geneinto plasmid
Recombinant
Ti plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Agrobacterium tumefaciens
DNA containinggene for desired trait
Ti
plasmid Insertion of geneinto plasmid
Recombinant
Ti plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Regenerationof plant
3
Plant with new trait
12.9 Genetically modified organisms raise concerns about human and environmental health
Scientists use safety measures to guard against production and release of new pathogens
Concerns related to GM organisms
– Can introduce allergens into the food supply
– FDA requires evidence of safety before approval
– Exporters must identify GM organisms in food shipments
– May spread genes to closely related organisms
– Hybrids with native plants may be prevented by modifying GM plants
Regulatory agencies address the safe use of biotechnology
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12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases
Gene therapy aims to treat a disease by supplying a functional allele
One possible procedure
– Clone the functional allele and insert it in a retroviral vector
– Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell
– Viral DNA and the functional allele will insert into the patient’s chromosome
– Return the cells to the patient for growth and division
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12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases
SCID (severe combined immune deficiency) was the first disease treated by gene therapy
– First trial in 1990 was inconclusive
– Second trial in 2000 led to the development of leukemia in some patients due to the site of gene insertion
Challenges
– Safe delivery to the area of the body affected by the disease
– Achieving a long-lasting therapeutic effect
– Addressing ethical questionsCopyright © 2009 Pearson Education, Inc.
Insert normal geneinto virus
1
Viral nucleic acid
Retrovirus
Infect bone marrowcell with virus
2
Viral DNA insertsinto chromosome
3
Inject cellsinto patient
4
Bone marrowcell from patient
Bonemarrow
Cloned gene(normal allele)
DNA PROFILING
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12.11 The analysis of genetic markers can produce a DNA profile
DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual
– Compares genetic markers from noncoding regions that show variation between individuals
– Involves amplification (copying) of markers for analysis
– Sizes of amplified fragments are compared
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Crime scene
DNA isolated1Suspect 1 Suspect 2
DNA of selectedmarkers amplified
2
Amplified DNA compared
3
12.12 The PCR method is used to amplify DNA sequences
Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule
Relies upon a pair of primers– Short DNA molecules that bind to sequences at each
end of the sequence to be copied
– Used as a starting point for DNA replication
Repeated cycle of steps for PCR– Sample is heated to separate DNA strands
– Sample is cooled and primer binds to specific target sequence
– Target sequence is copied with heat-stable DNA polymerase
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Advantages of PCR
– Can amplify DNA from a small sample
– Results are obtained rapidly
– Reaction is highly sensitive, copying only the target sequence
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12.12 The PCR method is used to amplify DNA sequences
Cycle 1yields 2 molecules
21 3
GenomicDNA
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
5
Cycle 1yields 2 molecules
GenomicDNA
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
215
3
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
12.13 Gel electrophoresis sorts DNA molecules by size
Gel electrophoresis separates DNA molecules based on size
– DNA sample is placed at one end of a porous gel
– Current is applied and DNA molecules move from the negative electrode toward the positive electrode
– Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel
– DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence
– Each band is a collection of DNA molecules of the same length
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Video: Biotechnology Lab
12_13BiotechLab_SV.mpg12_13BiotechLab_SV.mpg
Mixture of DNAfragments ofdifferent sizes
Completed gel
Longer(slower)molecules
Gel
Powersource
Shorter(faster)molecules
12.14 STR analysis is commonly used for DNA profiling
Short tandem repeats (STRs) are genetic markers used in DNA profiling
– STRs are short DNA sequences that are repeated many times in a row at the same location
– The number of repeating units can differ between individuals
– STR analysis compares the lengths of STR sequences at specific regions of the genome
– Current standard for DNA profiling is to analyze 13 different STR sites
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STR site 1
Crime scene DNA
STR site 2
Suspect’s DNA
Number of short tandemrepeats match
Number of short tandemrepeats do not match
Crime sceneDNA
Suspect’sDNA
12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations
Forensics
– Evidence to show guilt or innocence
Establishing family relationships
– Paternity analysis
Identification of human remains
– After tragedies such as the September 11, 2001, attack on the World Trade Center
Species identification
– Evidence for sale of products from endangered species
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12.16 RFLPs can be used to detect differences in DNA sequences
Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence
Restriction fragment length polymorphism(RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site
– RFLP analysis involves comparison of sizes of restriction fragments by gel electrophoresis
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Restriction
enzymes added
DNA sample 1 DNA sample 2
Cut
w
z
x
yy
CutCut
w
z
x
yy
Longerfragments
Shorterfragments
GENOMICS
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12.17 Genomics is the scientific study of whole genomes
Genomics is the study of an organism’s complete set of genes and their interactions
– Initial studies focused on prokaryotic genomes
– Many eukaryotic genomes have since been investigated
Evolutionary relationships can be elucidated
– Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans
– Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast
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12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes
Goals of the Human Genome Project (HGP)
– To determine the nucleotide sequence all DNA in the human genome
– To identify the location and sequence of every human gene
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12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes
Results of the Human Genome Project– Humans have 21,000 genes in 3.2 billion nucleotide
pairs
– Only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs
– The remaining 88.5% of the DNA contains– Control regions such as promoters and enhancers
– Unique noncoding DNA
– Repetitive DNA
– Found in centromeres and telomeres
– Found dispersed throughout the genome, related to transposable elements that can move or be copied from one location to another
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RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)
RepetitiveDNA unrelated totransposableelements(15%)
UniquenoncodingDNA (15%)
Introns andregulatorysequences(24%)
Exons (regions of genes coding for proteinor giving rise to rRNA or tRNA) (1.5%)
12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly
Three stages of the Human Genome Project
– A low-resolution linkage map was developed using RFLP analysis of 5,000 genetic markers
– A physical map was constructed from nucleotide distances between the linkage-map markers
– DNA sequences for the mapped fragments were determined
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12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly
Whole-genome shotgun method
– Restriction enzymes were used to produce fragments that were cloned and sequenced
– Computer analysis assembled the sequence by aligning overlapping regions
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Chop up withrestriction enzyme
Chromosome
DNA fragments
Sequencefragments
Alignfragments
Reassemblefull sequence
12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome
Proteomics
– Studies the proteome, the complete set of proteins specified by a genome
– Investigates protein functions and interactions
The human proteome may contain 100,000 proteins
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12.21 EVOLUTION CONNECTION: Genomes hold clues to the evolutionary divergence of humans and chimps
Comparisons of human and chimp genomes
– Differ by 1.2% in single-base substitutions
– Differ by 2.7% in insertions and deletions of larger DNA sequences
– Human genome shows greater incidence of duplications
– Genes showing rapid evolution in humans
– Genes for defense against malaria and tuberculosis
– Gene regulating brain size
– FOXP2 gene involved with speech and vocalization
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Bacterialclone
DNAfragments
CutBacterium
Recombinantbacteria
Genomic library
RecombinantDNA
plasmids
Cut
Plasmids
Longer fragmentsmove slower
Powersource
Shorter fragmentsmove faster
Mixture of DNA fragments
A “band” is acollection of DNAfragments of oneparticular length
DNA attracted to +pole due to PO4
– groups
Bacterial
plasmids
(a)
(b)
are copied via
treated with
DNA
amplified
via
treated with
DNA
sample
(c)
DNA
fragments
sorted by size via
Recombinant plasmids
are inserted
into bacteria
Add
(d)
Particular
DNA
sequence
highlighted
(e)
Collection
is called a(f)
Bacterial
plasmids
(a)
(b)
treated with
DNAamplified
via
treated with
DNA
sample
are copied via
(c)
DNA
fragments
sorted by size via
Recombinant plasmids
are inserted
into bacteria
Add
(d)
Particular
DNA
sequence
highlighted
(e)
Collection
is called a(f)
(b)
1.Distinguish between terms in the following groups: restriction enzyme—DNA ligase; GM organism—transgenic organism; SNP—RFLP; genomics—proteomics
2.Define the following terms: cDNA, gel electrophoresis, gene cloning, genomic library, “pharm” animal, plasmid, probe, recombinant DNA, repetitive DNA, reverse transcriptase, STR, Taq polymerase, vector, whole-genome shotgun method
3.Describe how genes are cloned
You should now be able to
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4.Describe how a probe is used to identify a gene of interest
5.Describe how gene therapy has been attempted and identify challenges to the effectiveness of this treatment approach
6.Distinguish between the use of prokaryotic and eukaryotic cells in producing recombinant DNA products
7.Identify advantages to producing pharmaceuticals with recombinant DNA technology
You should now be able to
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8. Describe the basis for DNA profiling and explain how it is used to provide evidence in forensic investigations
9. Explain how PCR provides copies of a specific DNA sequence
10. Identify ethical concerns related to the use of recombinant DNA technology
11. Describe how comparative information from genome projects has led to a better understanding of human biology
You should now be able to
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