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Chapter 12
DNA Technology and Genomics
Introduction: DNA and Crime Scene Investigations
A.) 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
GENE CLONING
12.1 Genes can be cloned in recombinant plasmids
A.) 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
B.) 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
§ 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
12.2 Enzymes are used to “cut and paste” DNA
A.) 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
B.) DNA ligase joins DNA fragments together
12.3 Cloned genes can be stored in genomic libraries
A.) A genomic library is a collection of all of the cloned DNA
fragments from a target genome
B.) 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
12.4 Reverse transcriptase can help make genes for cloning
A.) 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
B.) 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
12.5 Nucleic acid probes identify clones carrying specific genes
A.) 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
B.) 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
GENETICALLY MODIFIED ORGANISMS
12.6 Recombinant cells and organisms can
mass-produce gene products
A.) Cells and organisms containing cloned genes are used to
manufacture large quantities of gene products
B.) 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
C.) 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
12.7 CONNECTION: DNA technology has changed the
pharmaceutical industry and medicine
A.) 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
B.) 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
C.) Advantages of recombinant DNA products
– Identity to human protein
– Purity
– Quantity
12.8 CONNECTION: Genetically modified organisms are
transforming agriculture
A.)
Genetically modified (GM) organisms contain one or more
genes introduced by artificial means
B.)
Transgenic organisms contain at least one gene from
another species
C.)
GM plants
D.)
– Resistance to herbicides
– Resistance to pests
– Improved nutritional profile
GM animals
– Improved qualities
– Production of proteins or therapeutics
12.9 Genetically modified organisms raise concerns about
human and environmental health
A.)
Scientists use safety measures to guard against production and
release of new pathogens
B.)
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
C.)
Regulatory agencies address the safe use of biotechnology
12.10 CONNECTION: Gene therapy may someday help treat a
variety of diseases
A.)
Gene therapy aims to treat a disease by supplying a
functional allele
B.)
One possible procedure
C.)
– 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
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
D.)
Challenges
– Safe delivery to the area of the body affected by the disease
– Achieving a long-lasting therapeutic effect
– Addressing ethical questions
DNA PROFILING
12.11 The analysis of genetic markers can produce a DNA
profile
A.)
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
12.12 The PCR method is used to amplify DNA sequences
A.) Polymerase chain reaction (PCR) is a method of amplifying
a specific segment of a DNA molecule
B.)
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
C.)
D.)
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
12.13 Gel electrophoresis sorts DNA molecules by size
A.) 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
12.14 STR analysis is commonly used for DNA profiling
A.) 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
12.15 CONNECTION: DNA profiling has provided evidence in
many forensic investigations
A.)
Forensics
– Evidence to show guilt or innocence
B.)
Establishing family relationships
– Paternity analysis
C.)
Identification of human remains
– After tragedies such as the September 11, 2001, attack on the World Trade Center
D.)
Species identification
– Evidence for sale of products from endangered species
12.16 RFLPs can be used to detect differences in DNA
sequences
A.)
Single nucleotide polymorphism (SNP) is a variation at
one base pair within a coding or noncoding sequence
B.)
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
GENOMICS
12.17 Genomics is the scientific study of whole genomes
A.) 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
B.) 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
12.18 CONNECTION: The Human Genome Project revealed
that most of the human genome does not consist of genes
A.) 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
B.)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
12.19 The whole-genome shotgun method of sequencing a
genome can provide a wealth of data quickly
A.)
B.)
–
–
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
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
12.20 Proteomics is the scientific study of the full set of proteins
encoded by a genome
A.)
Proteomics
– Studies the proteome, the complete set of proteins specified by a
genome
– Investigates protein functions and interactions
B.)
The human proteome may contain 100,000 proteins
12.21 EVOLUTION CONNECTION: Genomes hold clues to
the evolutionary divergence of humans and chimps
A.) 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
You should now be able to
§ 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