Download Ch.-15-Lecture

Eldra Solomon
Linda Berg
Diana W. Martin
www.cengage.com/biology/solomon
Chapter 15
DNA Technology and Genomics
Albia Dugger • Miami Dade College
Biotechnology
• Studies of DNA sequences reveal the organization of genes
and the relationship between genes and their products
• Recombinant DNA technology allows researchers to splice
together DNA from different organisms in the laboratory
• Molecular modification (genetic engineering) alters an
organism’s DNA to produce new genes with new traits
• Biotechnology includes all commercial or industrial uses of
cells or organisms
15.1 DNA CLONING
LEARNING OBJECTIVES:
• Explain how a typical restriction enzyme cuts DNA molecules
and give examples of the ways in which these enzymes are
used in recombinant DNA technology
• Distinguish among a genomic DNA library, a chromosome
library, and a complementary DNA (cDNA) library; explain why
one would clone the same eukaryotic gene from both a
genomic DNA library and a cDNA library
• Explain how researchers use a DNA probe
• Describe how PCR amplifies DNA in vitro
Recombinant DNA Technology
• Recombinant DNA technology began with genetic studies of
viruses that infect bacteria (bacteriophages)
• Restriction enzymes from bacteria are used to cut DNA
molecules in specific places – a vector molecule transports
the DNA fragment into a cell
• Bacteriophages and plasmids are two examples of vectors
Recombinant DNA Technology (cont.)
• A plasmid with foreign DNA spliced into it (recombinant
plasmid) is introduced into bacteria by transformation
• Once a plasmid enters a cell, it is replicated and distributed to
daughter cells during cell division, producing many identical
copies – the foreign DNA is cloned
Restriction Enzymes
• Restriction enzymes enable scientists to cut DNA from
chromosomes into shorter fragments in a controlled way
• Each restriction enzyme cuts DNA at a specific DNA
sequence (restriction site), such as 5′-AAGCTT-3′
• Many restriction enzymes used for recombinant DNA studies
cut palindromic sequences – the base sequence reads the
same as its complement, in the opposite direction – such as
3′-TTCGAA-5′
Sticky Ends
• Cutting both strands in a staggered fashion produces
fragments with identical, complementary, single-stranded
ends called sticky ends:
5′-A
3′-TTCGA
AGCTT -3′
A-5′
• Sticky ends pair by hydrogen bonding with the
complementary, single-stranded ends of other DNA
molecules that have been cut with the same enzyme
DNA Ligase
• Once the sticky ends of two molecules have been joined, they
are treated with DNA ligase, an enzyme that covalently links
the two DNA fragments to form a stable recombinant DNA
molecule
Cutting DNA with a Restriction Enzyme
Plus HindIII restriction enzyme
Sticky
ends
Fig. 15-1, p. 325
Recombinant DNA
• The DNA to be cloned and plasmid (vector) DNA are cut with
the same restriction enzyme
• The two DNA samples are mixed, and complementary bases
of the sticky ends are bonded
• The result is recombinant DNA
Making Recombinant DNA
DNA of interest from another organism
Plasmid from
a bacterium
1
Clonable DNA fragment
2
Recombinant DNA
3
Fig. 15-2, p. 325
DNA of interest from another organism
Plasmid from
a bacterium
1
Clonable DNA fragment
2
Recombinant DNA
3
Stepped Art
Fig. 15-2, p. 325
Plasmids
• Plasmids used in recombinant DNA technology include
features helpful in isolating and analyzing cloned DNA:
• One or more restriction sites
• Genes that let researchers select cells transformed by
recombinant plasmids
A Plasmid Vector
Aat I
Xba I
Hpa I
E. coli
origin of replication
Cla I
Pvu II
Sal I Bam HI
Sma I
(a) This plasmid vector has many useful features. Researchers constructed it from DNA
fragments they had isolated from plasmids, E. coli genes, and yeast genes. The two origins of
replication, one for E. coli and one for yeast, Saccharomyces cerevisiae , let it replicate
independently in either type of cell. Letters on the outer circle designate sites for restriction
enzymes that cut the plasmid only at that position. Resistance genes for the antibiotics
ampicillin and tetracycline and the yeast URA-3 gene are also shown. The URA-3 gene is
useful when transforming yeast cells lacking an enzyme required for uracil synthesis. Cells
Fig. 15-3a, p. 326
that take up the plasmid grow on a uracil-deficient medium.
Plasmid in Bacterium
Bacterial
chromosome
Bacterium
Plasmid
(b) The relative sizes of a plasmid and
the main DNA of a bacterium.
Fig. 15-3b, p. 326
DNA Libraries
• The total DNA in a cell is its genome
• A genomic DNA library is a collection of thousands of DNA
fragments that represent all of the DNA in the genome
• A chromosome library contains all the DNA fragments in
that specific chromosome
• A human genomic DNA library is stored in a collection of
recombinant bacteria, each with a different fragment of DNA
Producing a Genomic DNA Library
or Chromosome Library
Sites of cleavage
Fragment
1
Fragment
2
Fragmen Fragment
t3
4
Human DNA
1
Produce
recombinant
DNA
Gene for
resistance to
antibiotic
2
R
R
R
R
3
Transformation
Bacterium
without plasmid
Bacteria without
plasmid fail to grow.
Bacteria with plasmid live and
multiply to form a colony.
4
Plate with antibioticcontaining medium
Fig. 15-4, p. 327
Locating a Sequence of Interest
• To identify a plasmid containing a sequence of interest, each
plasmid is cloned until there are millions of copies
• A sample of bacterial culture is spread on agar plates so cells
are widely separated – each cell divides many times, forming
a colony of genetically identical clones
• The next task is to determine which colony out of thousands
contains the fragment of interest
DNA Probes
• A segment of DNA that is homologous (identical) to part of the
sequence of interest (DNA probe) can be used to detect the
specific DNA sequence
• The DNA probe is a segment of single-stranded DNA that can
hybridize (attach by base pairing) to complementary base
sequences in target DNA
• DNA that is complementary to that particular probe is
detected
A cDNA Library
• It is possible to clone intact genes and avoid introns by using
DNA copies of mature mRNA to construct complementary
DNA (cDNA)
• Researchers use the enzyme reverse transcriptase to
synthesize single-stranded cDNA, then DNA polymerase to
make the cDNA double-stranded
• A cDNA library is formed using mRNA from a single cell type
as the starting material
Formation of cDNA
1
Exon Intron Exon Intron Exon
DNA in a eukaryotic Transcription
chromosome
Pre-mRNA
RNA processing
(remove introns)
Mature
mRNA
2
mRNA
3
Reverse
transcriptase
Mature
mRNA
cDNA copy
of mRNA
Degraded
RNA
cDNA
4
DNA
polymerase
5
Double-stranded cDNA
Fig. 15-6, p. 329
The Polymerase Chain Reaction
• The polymerase chain reaction (PCR) can be used to
amplify a tiny sample of DNA without cloning in a cell
• PCR uses a heat-resistant DNA polymerase (Taq
polymerase), nucleotides and primers to replicate a DNA
sequence in vitro
• Cycles of denaturing (heating) and replication double the
number of cloned molecules with each cycle
The Polymerase Chain Reaction
Using PCR
• PCR enables researchers to amplify and analyze tiny DNA
samples from a variety of sources, ranging from crime scenes
to archaeological remains
• Example: Investigators have used PCR to analyze
mitochondrial DNA obtained from the bones of Neandertals
KEY CONCEPTS 15.1
• Scientists use DNA technology to produce many copies of
specific genes (gene cloning)
15.2 DNA ANALYSIS
LEARNING OBJECTIVES:
• Distinguish among DNA, RNA, and protein blotting
Gel Electrophoresis
• Gel electrophoresis is used to separate mixtures of certain
macromolecules: proteins, polypeptides, or DNA fragments
• Nucleic acids migrate through the gel toward the positive pole
of the electric field because they are negatively charged due
to their phosphate groups
• DNA fragments are separated by size – small molecules
move farther than large molecules
Gel Electrophoresis
1
–
Standards of
known sizes
placed in well
Direction of
movement
Mixtures
placed in
well
Gel
Buffer
solution
+
Fig. 15-8a, p. 332
2
Anode
Longer molecules
Shorter molecules
Cathode
Fig. 15-8b, p. 332
3
Fig. 15-8c, p. 332
Southern Blot
• DNA separated by gel electrophoresis is denatured and
transferred to a membrane, which picks up DNA like a blotter
picks up ink – this Southern blot is a replica of the gel
• The blot is incubated with a DNA probe, which hybridizes with
any complementary DNA fragments – the probe is detected
by autoradiography or chemical luminescence
Similar Blotting Techniques
• When RNA molecules separated by electrophoresis are
transferred to a membrane and detected using a nucleic acid
probe, the result is called a Northern blot
• When the blot consists of proteins or polypeptides separated
by gel electrophoresis, it is called a Western blot
Southern Blotting Technique
1
2
DNA
5
DNA
fragments
Weight
Absorbent
paper
4
Buffer
solution
Nitrocellulose
filter
Agarose gel
3
Longer DNA
fragments
Gel
Wick
Shorter DNA
fragments
Buffer
6
7
Radioactive
probe solution
Fig. 15-9, p. 333
Restriction Fragment Length Polymorphisms
(RFLPs)
• Random DNA mutations and recombination result in
individuals with different lengths of fragments produced by a
given restriction enzyme
• These restriction fragment length polymorphisms
(RFLPs) can be used to determine how closely related
different members of a population are
• A genetic polymorphism exists if individuals of two or more
discrete types, or “morphs,” are found in a population
RFLP Analysis
• RFLP analysis has been used for paternity testing and
analyzing evidence found at crime scenes
• RFLP analysis helped map the exact location of gene
mutations, such as the mutation that causes cystic fibrosis
• Today, RFLP analysis is rapidly being replaced by newer
methods, such as automated DNA sequencing
Rapid DNA Sequencing
• Automated DNA-sequencing machines connected to
powerful computers let scientists sequence huge amounts of
DNA quickly and reliably
Automated DNA-Sequencing Results
Fig. 15-11, p. 335
Sequencing Entire Genomes
• Advances in sequencing technology have made it possible for
researchers to study the nucleotide sequences of entire
genomes in a wide variety of organisms
• The Human Genome Project, which sequenced the 3 billion
base pairs of the human genome, was completed in 2001
• DNA sequence information is stored in large computer
databases, many of which are accessed through the Internet
KEY CONCEPTS 15.2
• Biologists study DNA using gel electrophoresis, DNA blotting,
automated sequencing, and other methods
15.3 GENOMICS
LEARNING OBJECTIVES:
• Describe three important areas of research in genomics
• Explain what a DNA microarray does
• Define pharmacogenetics and proteomics
Genomics
• Genomics is the study of the entire DNA sequence of an
organism’s genome to identify all the genes, determine their
RNA or protein products, and how the genes are regulated
• Structural genomics: mapping and sequencing
• Functional genomics: functions of genes and nongene
sequences
• Comparative genomics: comparing genomes of different
species (evolution)
• Metagenomics: analyzing communities of microorganisms
instead of individual organisms
RNA Interference (RNAi)
• RNA interference (RNAi) can be used to quickly determine
the function of a specific gene by inactivating the gene
• A short stretch of RNA complementary to part of the DNA
sequence being examined is put into cells to silence the gene
• Biologists observe any changes in phenotype to help
determine the function of the missing protein
Gene Targeting
• In gene targeting, the researcher chooses and “knocks out”
(inactivates) a single gene in an organism
• Knockout mice are used to study the roles of proteins in
numerous diseases
• A knockout gene is cloned and introduced into mouse
embryonic stem cells (EScells), which are injected into
early mouse embryos
DNA Microarrays
• DNA microarrays provide a way to study patterns of gene
expression in a variety of organisms
• Each spot in a DNA microarray contains copies of a singlestranded cDNA molecule, placed on a glass slide or chip
• cDNA molecules from two cell populations are tagged with
different-colored fluorescent dyes and added to the array
• The array fluoresces at spots where hybridization occurs
DNA Microarray Analysis
1
Treated cell
Untreated
(control)
cell
2
Mature mRNA
Mature mRNA
Reverse transcriptase
Reverse transcriptase
cDNA copy of mRNA
cDNA copy of mRNA
3
cDNA
mRNA
(discard)
cDNA
mRNA
(discard)
4
Laser 1
Laser 2
5
Emissions
6
Fig. 15-12, p. 337
Genome Sequencing
for Other Species
• Comparison of the DNA sequences and chromosome
organization of related genes from different species helps
identify elements essential for their functions
• If a human gene has an unknown function
• researchers can often deduce its role by studying the
equivalent gene in another species, such as a mouse or
rat
New Fields of Science
• bioinformatics (biological computing)
• Storage, retrieval, and comparison of nucleotide or amino
acid sequences within and among species
• pharmacogenetics
• Gene-based medicine; studies how genetic variation
among patients affects the action of drugs in individuals
• proteomics
• Study of all proteins expressed by a cell at a given time
KEY CONCEPTS 15.3
• Genomics is an emerging field that comprises the structure,
function, and evolution of genomes
15.4 APPLICATIONS OF
DNA TECHNOLOGIES
LEARNING OBJECTIVE:
• Describe at least one important application of
recombinant DNA technology in each of the following
fields: medicine, DNA fingerprinting, and transgenic
organisms
DNA Technology in Medicine
• Genetic tests determine whether an individual has a
particular genetic mutation associated with disorders such as
Huntington’s disease, hemophilia, cystic fibrosis, Tay-Sachs
disease, breast cancer, and sickle cell anemia
• Gene therapy uses specific DNA to treat a genetic disease
by correcting the genetic problem
Genetically Modified Proteins
• Recombinant DNA techniques are used to produce medical
proteins in genetically altered bacteria or other organisms
• Human insulin
• Human growth hormone (GH)
• Tissue-engineered skin grafts, cartilage, and other tissues
• Human clotting factor VIII
• Recombinant vaccines
• influenza A, hepatitis B, polio, HPV
DNA Fingerprinting
• The analysis of DNA fragments unique to an individual is
known as DNA fingerprinting
• Today, DNA fingerprinting relies on PCR amplification,
restriction enzyme digestion, and Southern blot hybridization
to detect molecular markers
• The most useful markers are short tandem repeats (STRs) –
short sequences of repetitive DNA, up to 200 nucleotide
bases, with a simple pattern such as GTGTGTGTGT
DNA Fingerprinting
• DNA fingerprints from
a crime scene
(middle), along with
DNA profiles of seven
suspects
• Which DNA profile
matches blood from
the crime scene?
1
2
3 From blood 4
at crime
scene
5
6
7
Fig. 15-13, p. 340
Applications of DNA Fingerprinting
1. Analyzing evidence found at crime scenes (forensic
analysis)
2. Identifying mass disaster victims
3. Proving parentage in dogs for pedigree registration
purposes
4. Identifying human cancer cell lines
5. Studying endangered species in conservation biology
6. Tracking tainted foods
7. Studying the genetic ancestry of human populations
8. Clarifying disputed parentage
9. Exonerating prisoners wrongfully convicted of a crime
Transgenic Organisms
• Plants and animals in which foreign genes have been
incorporated are called transgenic organisms
• Transgenic animals are usually produced by injecting DNA of
a particular gene into the nucleus of a fertilized egg or ES cell
Transgenic Organisms in Research
• Transgenic animals are
used in research areas
such as regulation of
gene expression
• A mouse with two
copies of the GH gene,
grew to more than
double normal size
Fig. 15-14, p. 341
Transgenic Animals and
Genetically Modified Proteins
• “Pharming”
• Certain transgenic
animals produce milk
containing foreign
proteins of therapeutic
or commercial
importance – such as
human lactoferrin
Fig. 15-15, p. 341
Genetically Modified (GM) Crops
• The United States is the world’s top producer of transgenic or
genetically modified (GM) crops
• Globally, 51% of the soybean crop, 31% of corn, 13% of
cotton, and 5% of canola are GM crops
• GM plants are resistant to insect pests, viral and fungal
diseases, heat, cold, herbicides, salty or acidic soil, or drought
• GM plants may also be engineered to increase nutrition, or to
produce medically important proteins
Transgenic Corn: Resistant to Drought
(a) Note the poor yield in genetically unmodified
corn plants used as a control.
Fig. 15-16a, p. 342
(b) Genetically modified corn plants withstood
drought better than the unmodified corn.
Fig. 15-16b, p. 342
Concerns About Health Effects
• Some people are concerned about the health effects of
consuming foods derived from GM crops
• There is also ongoing controversy as to whether GM foods
should be labeled
• In 1996, the U.S. Court of Appeals upheld the FDA position
that labeling should not be required
KEY CONCEPTS 15.4
• DNA technology and genomics have wide applications, from
medical to forensic to agricultural
15.5 DNA TECHNOLOGY HAS
RAISED SAFETY CONCERNS
LEARNING OBJECTIVE:
• Describe safety issues associated with recombinant DNA
technology and explain how these issues are being
addressed
SAFETY CONCERNS
• When recombinant DNA technology was introduced,
scientists considered potential concerns:
• An organism with undesirable environmental effects might
be accidentally produced
• New strains of bacteria or other organisms might be
difficult to control
• So far, no evidence suggests that researchers have
accidentally cloned hazardous genes
Risk Assessment
• DNA technology in agriculture offers many potential benefits
• However, molecular modification poses some risks, such as
the risk that genetically modified plants and animals could
pass their foreign genes to wild relatives
KEY CONCEPTS 15.5
• Scientists must assess the risks of each new recombinant
organism