Upload
nathan-missler
View
255
Download
3
Embed Size (px)
Citation preview
Copyright © 2009 Pearson Education, Inc.
PowerPoint Lectures forBiology: Concepts & Connections, Sixth EditionCampbell, Reece, Taylor, Simon, and Dickey
Chapter 12Chapter 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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
12.1 Genes can be cloned in recombinant plasmids
Animation: Cloning a Gene
Examples ofgene use
RecombinantDNAplasmid
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Geneof interest
Cell with DNAcontaining geneof interest
Recombinantbacterium
Cloneof cells
Genes may be insertedinto other organisms
Genes or proteinsare isolated from thecloned bacterium
Harvestedproteinsmay be used directly
Examples ofprotein use
Gene of interest
Isolateplasmid
1
IsolateDNA
2
Cut plasmidwith enzyme
3
Cut cell’s DNAwith same enzyme
4
Combine targeted fragmentand plasmid DNA
5
Add DNA ligase,which closesthe circle withcovalent bonds
6
Put plasmidinto bacteriumby transformation
7
Allow bacteriumto reproduce
8
9
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Put plasmidinto bacteriumby transformation
7
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Cloneof cells
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
8
7
RecombinantDNAplasmid
Geneof interest
Recombinantbacterium
Cloneof cells
Genes or proteinsare isolated from thecloned bacterium
Harvestedproteinsmay be used directly
Examples ofprotein use
Put plasmidinto bacteriumby transformation
Allow bacteriumto reproduce
8
7
Genes may be insertedinto other organisms
Examples ofgene 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 togetherCopyright © 2009 Pearson Education, Inc.
Animation: Restriction Enzymes
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 molecule5
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
Copyright © 2009 Pearson Education, Inc.
Recombinantplasmid
Phageclone
Bacterialclone
Phage libraryPlasmid library
or
Recombinantphage DNA
Genome cut up withrestriction enzyme
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine
Advantages of recombinant DNA products
– Identity to human protein
– Purity
– Quantity
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi 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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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)
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
Copyright © 2009 Pearson Education, Inc.
Crime sceneDNA isolated1
Suspect 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 polymeraseCopyright © 2009 Pearson Education, Inc.
Advantages of PCR
– Can amplify DNA from a small sample
– Results are obtained rapidly
– Reaction is highly sensitive, copying only the target sequence
Copyright © 2009 Pearson Education, Inc.
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
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
Copyright © 2009 Pearson Education, Inc.
Video: Biotechnology Lab
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
Copyright © 2009 Pearson Education, Inc.
STR site 1
Crime scene DNA
STR site 2
Suspect’s DNA
Number of short tandemrepeats match
Number of short tandemrepeats do not match
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
Restrictionenzymes added
DNA sample 1 DNA sample 2
Cut
w
z
x
yy
CutCut
w
z
x
yy
Longerfragments
Shorterfragments
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.
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%)
12.20 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
Copyright © 2009 Pearson Education, Inc.
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 vocalizationCopyright © 2009 Pearson Education, Inc.
1.Distinguish between terms in the following groups: restriction enzyme—DNA ligase; GM organism—transgenic organism; genomics—proteomics
2.Define the following terms: cDNA, gel electrophoresis, gene cloning, genomic library, “pharm” animal, plasmid, recombinant DNA, repetitive DNA, vector
3. Describe how genes are cloned
You should now be able to
Copyright © 2009 Pearson Education, Inc.
4.Describe how gene therapy has been attempted and identify challenges to the effectiveness of this treatment approach
5.Distinguish between the use of prokaryotic and eukaryotic cells in producing recombinant DNA products
6.Identify advantages to producing pharmaceuticals with recombinant DNA technology
You should now be able to
Copyright © 2009 Pearson Education, Inc.
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
Copyright © 2009 Pearson Education, Inc.