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DNA Technology and Genomics
Wilson MuseSchoolcraft College
GENE CLONING
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
– Steps in cloning a gene§ Plasmid DNA is isolated
§ DNA containing the gene of interest is isolated
§ Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle
§ DNA with the target gene is treated with the same enzyme and many fragments are produced
§ Plasmid and target DNA are mixed and associate with each other
12.1 Genes can be cloned in recombinant plasmids
§ Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together
§ The recombinant DNA is taken up by a bacterial cell
§ The bacterial cell reproduces to form a clone of cells
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
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Isolateplasmid
IsolateDNA
1
2
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
Combine targeted fragmentand plasmid DNA
5
E. coli bacteriumPlasmid
Bacterialchromosome
Gene of interestDNA
Cell with DNAcontaining geneof interest
Gene of interest
Isolateplasmid
IsolateDNA
Cut plasmidwith enzyme
Cut cell’s DNAwith same enzyme
1
2
3
4
RecombinantDNAplasmid
Geneof interest
Combine targeted fragmentand plasmid DNA
Add DNA ligase,which closesthe circle withcovalent bonds
5
6
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 togetherAnimation: 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
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
Cell nucleus
Isolation of mRNAand addition of reversetranscriptase; synthesisof DNA strand
RNA splicing2
Transcription1
3
Breakdown of RNA4
Synthesis of secondDNA strand
5
mRNA
DNA ofeukaryoticgene
IntronExon
RNAtranscript
Exon Intron Exon
Reverse transcriptase
Test tube
cDNA strandbeing 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
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
RadioactiveDNA probe
Single-strandedDNA
Base pairingindicates thegene of interest
Mix with single-stranded DNA fromgenomic library
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
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
GMsheep
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
– 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
Advantages of recombinant DNA products
Identity to human protein
Purity
Quantity
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
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
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
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 questions
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 similar to DNA Fingerprinting
Can be used to assign paternity or link DNA evidence to a crime scene
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
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
polymerase
– Advantages of PCR– Can amplify DNA from a small sample
– Results are obtained rapidly
– Reaction is highly sensitive, copying only the target sequence
12.12 The PCR method is used to amplify DNA sequences
see animation: http://www.dnalc.org/ddnalc/resources/pcr.html
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
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
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
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
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
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
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
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%)
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
Applied by J. Craig Venter and Celera vs traditional method
Venter’s method
– 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
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. Given alternative splicing and post-translational modifications, the diversity is quite large.
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
How different are we?
Wrap up and Review
• PCR
• Recombinant DNA and Plasmid cloning
• DNA Fingerprinting and Profiling
• DNA sequenceing
Bacterialclone
DNAfragments
Cut Bacterium
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
Bacterialplasmids
(a)
(b)
are copied via
treated with
DNAamplified
via
treated with
DNAsample
(c)
DNAfragments
sorted by size via
Recombinant plasmidsare insertedinto bacteria
Add
(d)
ParticularDNA
sequencehighlighted
(e)
Collectionis called a
(f)
Bacterialplasmids
(a)
(b)
treated with
DNAamplified
via
treated with
DNAsample
are copied via
(c)
DNAfragments
sorted by size via
Recombinant plasmidsare insertedinto bacteria
Add
(d)
ParticularDNA
sequencehighlighted
(e)
Collectionis called a
(f)
(b)
§ Distinguish between terms in the following groups: restriction enzyme—DNA ligase; GM organism—transgenic organism; SNP—RFLP; genomics—proteomics
§ 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
§ Describe how genes are cloned
You should now be able to
§ Describe how a probe is used to identify a gene of interest
§ Describe how gene therapy has been attempted and identify challenges to the effectiveness of this treatment approach
§ Distinguish between the use of prokaryotic and eukaryotic cells in producing recombinant DNA products
§ Identify advantages to producing pharmaceuticals with recombinant DNA technology
You should now be able to
§ Describe the basis for DNA profiling and explain how it is used to provide evidence in forensic investigations
§ Explain how PCR provides copies of a specific DNA sequence
§ Identify ethical concerns related to the use of recombinant DNA technology
§ Describe how comparative information from genome projects has led to a better understanding of human biology
You should now be able to