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Chapter 12. 0. DNA Technology and Genomics. 0. DNA and Crime Scene Investigations Many violent crimes go unsolved For lack of enough evidence If biological fluids are left at a crime scene DNA can be isolated from them. Investigator at one of the crime scenes (above), Narborough, - PowerPoint PPT Presentation
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Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
PowerPoint Lectures forBiology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon
Lectures by Chris Romero
Chapter 12Chapter 12
DNA Technology and Genomics
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA and Crime Scene Investigations
• Many violent crimes go unsolved
– For lack of enough evidence
• If biological fluids are left at a crime scene
– DNA can be isolated from them
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• DNA fingerprinting is a set of laboratory procedures
– That determines with near certainty whether two samples of DNA are from the same individual
– That has provided a powerful tool for crime scene investigators
Investigator at oneof the crime scenes(above), Narborough,England (left)
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
BACTERIAL PLASMIDS AND GENE CLONING
12.1 Plasmids are used to customize bacteria: An overview
• Gene cloning is one application of DNA technology
– Methods for studying and manipulating genetic material
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Researchers can insert desired genes into plasmids, creating recombinant DNA
– And insert those plasmids into bacteriaBacterium
Bacterialchromosome
Plasmid
1 Plasmidisolated
3 Gene insertedinto plasmid
2 DNAisolated
Cell containing geneof interest
DNAGene ofinterest
Recombinant DNA(plasmid)
4 Plasmid put intobacterial cell
Recombinantbacterium
5 Cell multiplies withgene of interest
Copies of proteinCopies of gene
Clone of cellsGene for pestresistanceinserted intoplants
Gene used to alter bacteriafor cleaning up toxic waste
Protein used to dissolve bloodclots in heart attack therapy
Protein used tomake snow format highertemperature
Figure 12.1
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• If the recombinant bacteria multiply into a clone
– The foreign genes are also copied
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.2 Enzymes are used to “cut and paste” DNA
• The tools used to make recombinant DNA are
– Restriction enzymes, which cut DNA at specific sequences
– DNA ligase, which “pastes” DNA fragments together
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Creating recombinant DNA using restriction enzymes and DNA ligase
Restriction enzymerecognition sequence
G A A T T CC T T A A GDNA1
2
3
4
C T T A AA AT TC
A AT TCG
C T T A AAddition of a DNAfragment fromanother source
Two (or more)fragments sticktogether bybase-pairing
G A AT T CC T TA A G
G A AT T CC T TA A G
5
DNA ligasepastes the strand
Restriction enzymecuts the DNA intofragments
Recombinant DNA molecule
GG
Sticky end
G
Figure 12.2
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.3 Genes can be cloned in recombinant plasmids: A closer look
• Bacteria take the recombinant plasmids from their surroundings
– And reproduce, thereby cloning the plasmids and the genes they carry
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Cloning a gene in a bacterial plasmid 1
Isolate DNAfrom two sources
2Cut both DNAswith the samerestriction enzyme
E.coli
PlasmidDNA
Gene V
Sticky ends
3 Mix the DNAs;they join bybase-pairing
4 Add DNA ligaseto bond the DNA covalently
5 Put plasmid into bacteriumby transformation
Gene VRecombinant DNAplasmid
Recombinant bacterium
6 Clone the bacterium
Bacterial clone carrying manycopies of the human gene
Human cell
Figure 12.3
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.4 Cloned genes can be stored in genomic libraries
• Genomic libraries, sets of DNA fragments containing all of an organism’s genes
– Can be constructed and stored in cloned bacterial plasmids or phages
Recombinantplasmid
Genome cut up withrestriction enzyme
Recombinantphage DNA
or
Bacterialclone
Phageclone
Phage libraryPlasmid libraryFigure 12.4
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.5 Reverse transcriptase helps make genes for cloning
• Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries
– Containing only the genes that are transcribed by a particular type of cell
Cell nucleus
DNA ofeukaryoticgene
Exon Intron Exon Intron Exon
1 Transcription
2 RNA splicing(removes introns)
3 Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand
4 Breakdown of RNA
5 Synthesis of secondDNA strand
RNA transcript
mRNA
Reverse transcriptase
cDNA strand
cDNA of gene(no introns)
Test tube
Figure 12.5
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
CONNECTION
12.6 Recombinant cells and organisms can mass-produce gene products
• Applications of gene cloning include
– The mass production of gene products for medical and other uses
Table 12.6
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Different organisms, including bacteria, yeast, and mammals
– Can be used for this purpose
Figure 12.6
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.7 DNA technology is changing the pharmaceutical industry
• DNA technology
– Is widely used to produce medicines and to diagnose diseases
CONNECTION
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Therapeutic hormones
• In 1982, humulin, human insulin produced by bacteria
– Became the first recombinant drug approved by the Food and Drug Administration
Figure 12.7A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Diagnosis and Treatment of Disease
• DNA technology
– Is being used increasingly in disease diagnosis
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Vaccines
• DNA technology
– Is also helping medical researchers develop vaccines
Figure 12.7B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.8 Nucleic acid probes identify clones carrying specific genes
• DNA technology methods
– Can be used to identify specific pieces of DNA
RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• A nucleic acid probe
– Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA
– Can tag a desired gene in a library
Radioactiveprobe (DNA)
Single-strandedDNA
Mix with single-stranded DNA fromvarious bacterial(or phage) clones
Base pairingindicates thegene of interest
A T C C G A
A T G C G C T T A T C G
A G C
C T
T A
T G
C A
T
A T C C
G A
A G G T A G
G C T A A
Figure 12.8
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.9 DNA microarrays test for the expression of many genes at once
• DNA microarray assays
– Can reveal patterns of gene expression in different kinds of cells
CONNECTION
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• DNA microarray
1 mRNAisolated
Reverse transcriptaseand fluorescent DNAnucleotides
2 cDNA madefrom mRNA
4 UnboundcDNA rinsedaway
3 cDNA appliedto wells
DNA microarray
Each well contains DNAfrom a particular gene
Actual size(6,400 genes)
Nonfluorescentspot
Fluorescentspot
cDNA
DNA of anexpressed gene
DNA of anunexpressed gene
Figure 12.9
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.10 Gel electrophoresis sorts DNA molecules by size
+ +
– –
Powersource
Gel
Mixture of DNAmolecules ofdifferent sizes
Longermolecules
Shortermolecules
Completed gel
Figure 12.10
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
How Restriction Fragments Reflect DNA Sequence
• Restriction fragment length polymorphisms (RFLPs)
– Reflect differences in the sequences of DNA samples
Crime scene Suspect
w
x
y y
z
CutCut
Cut
DNA from chromosomes
CCGG
GGCC
ACGG
TGCC
CCGG
GGCC
CCGG
GGCC
Figure 12.11A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• After digestion by restriction enzymes
– The fragments are run through a gel
–
+
Longerfragments
Shorterfragments
x
w
y
z
y
1 2
Figure 12.11B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Using DNA Probes to Detect Harmful Alleles
• Radioactive probes
– Can reveal DNA bands of interest on a gel
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Detecting a harmful allele using restriction fragment analysis 1
2
3
4
5
Restriction fragment preparation
Gel electrophoresis
Blotting
Radioactive probe
Detection of radioactivity(autoradiography)
I II III
I II III
Restriction fragments
Filter paper
Probe
Radioactive, single-stranded DNA (probe)
Film
IIIIII
I
II
IIIFigure 12.11C
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.12 DNA technology is used in courts of law
CONNECTION
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• DNA fingerprinting can help solve crimes
Defendant’sblood
Blood fromdefendant’s clothes Victim’s
blood
Figure 12.12A Figure 12.12B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.13 Gene therapy may someday help treat a variety of diseases
• Gene therapy
– Is the alteration of an afflicted individual’s genes
CONNECTION
Cloned gene(normal allele) 1 Insert normal gene
into virus
2 Infect bone marrowcell with virus
3 Viral DNA insertsinto chromosome
4 Inject cellsinto patient
Bonemarrow
Bone marrowcell from patient
Viral nucleicacid
Retrovirus
Figure 12.13
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Gene therapy
– May one day be used to treat both genetic diseases and nongenetic disorders
• Unfortunately, progress is slow
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.14 The PCR method is used to amplify DNA sequences
• The polymerase chain reaction (PCR)
– Can be used to clone a small sample of DNA quickly, producing enough copies for analysis
1 2 4 8
InitialDNAsegment
Number of DNA moleculesFigure 12.14
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Figure 12.15
GENOMICS CONNECTION
12.15 The Human Genome Project is an ambitious application of DNA technology
• The Human Genome Project, begun in 1990 and now largely completed, involved
– Genetic and physical mapping of chromosomes, followed by DNA sequencing
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• The data are providing insight into
– Development, evolution, and many diseases
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.16 Most of the human genome does not consist of genes
• The haploid human genome contains about 25,000 genes
– And a huge amount of noncoding DNA
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Much of the noncoding DNA consists of repetitive nucleotide sequences
– And transposons that can move about within the genome
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.17 The science of genomics compares whole genomes
• The sequencing of many prokaryotic and eukaryotic genomes
– Has produced data for genomics, the study of whole genomes
CONNECTION
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Besides being interesting themselves
– Nonhuman genomes can be compared with the human genome
Table 12.17
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Proteomics
– Is the study of the full sets of proteins produced by organisms
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.18 Genetically modified organisms are transforming agriculture
GENETICALLY MODIFIED ORGANISMS CONNECTION
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Recombinant DNA technology
– Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid
1
Insertion of geneinto plasmid usingrestriction enzymeand DNA ligase
RecombinantTi plasmid
2
Introductioninto plantcells inculture
3
Regenerationof plant
Plant with new traitT DNA carrying new
gene within plant chromosome
Plant cell
T DNA
Restriction site
Figure 12.18A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Transgenic organisms
– Are those that have had genes from other organisms inserted into their genomes
Figure 12.18B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• A number of important crops and plants
– Are genetically modified
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.19 Could GM organisms harm human health or the environment?
• Development of GM organisms
– Requires significant safety measures
CONNECTION
Figure 12.19A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Genetic engineering involves risks
– Such as ecological damage from GM crops
Figure 12.19B
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.20 Genomics researcher Eric Lander discusses the Human Genome Project
• Genomics pioneer Eric Lander
– Points out that much remains to be learned from the Human Genome Project
CONNECTION
Figure 12.20
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
12.17 The science of genomics compares whole genomes
• The sequencing of many prokaryotic and eukaryotic genomes
– Has produced data for genomics, the study of whole genomes
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
HOMEWORK
Read Cohen’s work for plasmids and answer the questions on the following website
http://www.stanford.edu/class/bio11n/html/week1/papertopic.htm
Read Villa-Komaroff et al. work and answer the questions on the following website
http://www.stanford.edu/class/bio11n/html/week2/papertopic.htm
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
READING
http://www.dnaftb.org/dnaftb/