tenth edition - houston community college · pdf filetenth edition 20 . dna tools and...
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CAMPBELL
BIOLOGY Reece • Urry • Cain • Wasserman • Minorsky • Jackson
© 2014 Pearson Education, Inc.
TENTH EDITION
CAMPBELL
BIOLOGY Reece • Urry • Cain • Wasserman • Minorsky • Jackson
TENTH EDITION
20 DNA Tools and Biotechnology
Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick
The DNA Toolbox • Recently the genome sequences of two extinct
species—Neanderthals and wooly mammoths—have been completed
• Advances in sequencing techniques make genome sequencing increasingly faster and less expensive
Figure 20.1 How can the technique shown in this model speed up DNA sequencing?
Mammoths DNA sequencing
Overview: DNA Terminology • Sequencing of the human genome was completed by
2007. • Sequencing of the genomes of more than 7,000 species
was under way in 2010 • DNA sequencing has depended on advances in
technology, starting with making recombinant DNA • Recombinant DNA: nucleotide sequences from two
different sources, often two species, are combined in vitro into the same DNA molecule
• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes
• Biotechnology: the manipulation of organisms or their genetic components to make useful products
• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes
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DNA sequencing and DNA cloning are valuable tools for genetic engineering
and biological inquiry • The complementarity of the two DNA strands is
the basis for nucleic acid hybridization, the base pairing of one strand of nucleic acid to the complementary sequence on another strand
• Researchers can exploit the principle of complementary base pairing to determine a gene’s complete nucleotide sequence, called DNA sequencing
• The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger
Figure 20.2
(a) Standard sequencing machine
(b) Next-generation sequencing machines
DNA Sequencing identifies the exact nucleotide sequence of a DNA fragment
• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method
• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment
• The DNA sequence can be read from the resulting spectrogram
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Methodology of DNA Sequencing
1. The DNA is denatured to a single strand and is incubated with a Primer, DNA polymerase, and fluorescently labeled dd-nucleotides.
2. Synthesis of a complimentary strand begins at the 3’ end of the primer. The fluorescently labeled ddNTP’s will be added randomly to the 3’ end of the growing strand. This prevents and further elongation of that strand, so each strand has the fluorescent tag of the last ddNTP.
3. The labeled strands are separated by gel electrophoresis using a capillary tube. The shortest strands more the furthest. A fluorescent detector measures the tags.
Figure 20.3 DNA (template strand)
Primer Deoxyribo- nucleotides
Dideoxyribonucleotides (fluorescently tagged)
DNA polymerase
DNA (template strand)
Labeled strands
Direction of movement of strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5′
5′
5′
5′
5′
3′
3′
3′
3′
3′
C T G A C T T C G A C A A
C T G A C T T C G A C A A
T G T T
C T G T T
dATP
G C T G T T
C T G T T
C T G T T
C T G T T
C T G T T
C T G T T
G A C T G A A G C T G T T
A C T G A A G
C T G A A G
T G A A G
C T G T T
G A A G
A A G
A G
ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP
P P P P P P G G
Last nucleotide of longest labeled strand
Last nucleotide of shortest labeled strand
G A C T G A A G C
dd dd
dd dd
dd dd
dd dd
dd
DNA sequencing • “Next-generation sequencing” techniques use
a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
• Thousands or hundreds of thousands of fragments (400–1,000 nucleotides long) are sequenced in parallel
• This is a type of “high-throughput” technology • In “third-generation sequencing,” the
techniques used are even faster and less expensive than the previous
Figure 20.4 Technique
Genomic DNA is fragmented.
Each fragment is isolated with a bead.
Using PCR, 106 copies of each fragment are made, each attached to the bead by 5′ end.
The bead is placed into a well with DNA polymerases and primers.
A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.
If a nucleotide is joined to a growing strand, PPi is released, causing a flash of light that is recorded.
If a nucleotide is not complementary to the next template base, no PPi is released, and no flash of light is recorded.
The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
A T G C
Template strand of DNA
Primer
3′
A 3′ 5′ 5′ T G C
A T G C A T G C A T G C A T G C
Template strand of DNA
Primer
DNA polymerase
dATP dTTP dGTP dCTP
PPi
PPi
C C
C C
A A
A A
T
T
T
G
G G
G
C C
C C
A A
A A
T
T
T
G
G G
G
C C
C C
A A
A A
T
T
T
G
G G
G
C C
C C
A A
A A
T
T
T
G
G G
G
A A A A
C
DNA cloning yields multiple copies of a gene or other DNA segment
• DNA molecules are very long and contain several genes.
• To study a specific gene, scientists make several gene-sized pieces of DNA in identical copies, a process called DNA cloning. – Cloned genes are useful for making several copies of
a particular gene and producing a protein product. • DNA (and gene) cloning utilizes bacteria and their
plasmids. – Plasmids are small circular DNA molecules
that replicate separately from the bacterial chromosome
Gene cloning involves using bacteria to make multiple copies of a gene.
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Gene cloning involves using bacteria to make multiple copies of a gene
• DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell
• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA
• This results in the production of multiple copies of a single gene
• A plasmid used to clone a foreign gene is called a cloning vector
• Bacterial plasmids are widely used as cloning vectors because they are readily obtained, easily manipulated, easily introduced into bacterial cells, and once in the bacteria they multiply rapidly
• Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes
Figure 20.5
Using Restriction Enzymes to Make Recombinant DNA
• $ ---Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites. eg. EcoRI, BamHI, HindIII
• A restriction enzyme usually makes many cuts in DNA, yielding restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other DNA fragments.
• Most of restriction enzymes are bacterial origin.
• DNA ligase is an enzyme that seals the bonds between restriction fragments
Sticky ends Blunt ends
EcoRI PstI SmaI
EcoRI= Escherichia coli PstI= Providencia stuartii SmaI= Serratia mercescens
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Figure 20.6 Bacterial plasmid
Restriction site
DNA
Restriction enzyme cuts the sugar-phosphate backbones at each arrow.
1
Base pairing of sticky ends produces various combinations.
2
DNA ligase seals the strands.
3
Sticky end
Fragment from different DNA molecule cut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
5′
5′
5′ 5′
5′ 5′
5′
5′
3′
3′
3′
3′ 3′
3′
3′
3′
3′
3′ 3′
3′
3′
3′
3′
3′
5′ 5′ 5′
5′ 5′ 5′
5′
5′
G A A T T C G A A T T C
G A A T T C G A A T T C
Recombinant plasmid
GAATTC CTTAAG
Gel Electrophoresis and Southern Blotting
• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
• $ --- This technique uses a gel to separate nucleic acids or proteins by size
• A current is applied that causes charged molecules to move through the gel
• Molecules are sorted into “bands” by their size • Smaller DNA (or proteins) migrate through the gel much easier (and
faster) than larger DNA/protein. • To check the recombinant plasmid, researchers might cut the products
again using the same restriction enzyme • To separate and visualize the fragments produced, gel
electrophoresis would be carried out • This technique uses a gel made of a polymer to separate a mixture of
nucleic acids or proteins based on size, charge, or other physical properties © 2014 Pearson Education, Inc.
Figure 20.7
Anode Cathode
Wells
Gel
Mixture of DNA mol- ecules of different sizes
(a) Negatively charged DNA molecules move toward the positive electrode.
Power source
(b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel.
Restriction fragments (size standards)
Methods for Analysis of DNA – Negative charge of molecule causes DNA to move
toward positive pole – Rate of movement is dependent on size of fragment –
larger fragments move more slowly
• Southern blotting combines gel electrophoresis separation of DNA fragments with nucleic acid hybridization
• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the separated DNA fragments after they have been transferred to a paper-like membrane i.e. DNA immobilized on a “blot” of gel.
Southern Blotting
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DNA + restriction enzyme
3 2 1
4
TECHNIQUE
I Normal β-globin allele
II Sickle-cell allele
III Heterozygote
Restriction fragments Nitrocellulose
membrane (blot)
Heavy weight
Gel
Sponge
Alkaline solution Paper
towels
II I III
II I III II I III
Preparation of restriction fragments
Gel electrophoresis DNA transfer (blotting)
Radioactively labeled probe for β-globin gene
Nitrocellulose blot
Probe base-pairs with fragments
Fragment from sickle-cell β-globin allele
Fragment from normal β- globin allele
Film over blot
Hybridization with labeled probe Probe detection 5
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
• The polymerase chain reaction (PCR) can produce millions of copies of a specific target segment of DNA
• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
• The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase obtained from hot-spring bacteria.
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Figure 20.8
Target sequence
Genomic DNA
Technique
Denaturation
5′
5′
5′
5′
3′
3′
3′
3′ Annealing
Extension
Cycle 1 yields
2 molecules
Primers
New nucleotides
1
2
3
Cycle 2 yields
4 molecules
Cycle 3 yields 8 molecules; 2 molecules
(in white boxes) match target
sequence
PCR
• PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
• The resulting clones are sequenced and error-free inserts selected
Figure 20.9 DNA fragments obtained by PCR with restriction sites matching those in the cloning vector
Cut with same restriction enzyme used on cloning vector A gene that makes bacterial
cells resistant to an antibiotic is present on the plasmid.
Cloning vector (bacterial plasmid)
Only cells that take up a plasmid will survive
Mix and ligate
Recombinant DNA plasmid
Bacterial Expression Systems • Several technical difficulties hinder expression of cloned
eukaryotic genes in bacterial host cells • To overcome differences in promoters and other DNA
control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter
• Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
• Researchers can avoid this problem by using cDNA, complementary to the mRNA, which contains only exons
Eukaryotic DNA Cloning and Expression Systems
• Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes
• Even yeasts may not possess the proteins required to modify expressed mammalian proteins properly.
• The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems – YACs behave normally in mitosis and can carry more
DNA than a plasmid – Eukaryotic hosts can provide the post-translational
modifications that many proteins require • In such cases, cultured mammalian or insect cells may
be used to express and study proteins
• One method of introducing recombinant DNA into eukaryotic cells is electroporation-applying a brief electrical pulse to create temporary holes in plasma membranes
• Alternatively, scientists can inject DNA into cells using microscopically thin needles
• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination
• Eukaryotic expression is a good way to test the function of a specific protein
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Storing Cloned Genes in DNA Libraries Genomic library is made by cloning DNA fragments from an
entire genome into four different kinds of vector which depend on size of clone.
1. A genomic library is made by cloning DNA fragments from an entire genome into plasmids then growing the plasmids in bacteria. Each plasmid-containing bacterial cell carries copies of a particular segment of the genome.
2. A genomic library can also be made using bacteriophages ( Phage).
3. A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert used to make genomic libraries. BACs are another type of vector used in DNA library construction.
4. A yeast artificial chromosome (YAC) is a large plasmid used to make genomic libraries.
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Bacterial clones
Recombinant plasmids
Recombinant phage DNA
or
Foreign genome cut up with restriction enzyme
(a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones
Phage clones
Large plasmid Large insert with many genes
BAC clone
Cross-Species Gene Expression and Evolutionary Ancestry
• The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
• For example, Pax-6 is a gene that directs formation of a vertebrate eye; the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
• The Pax-6 genes in flies and vertebrates can substitute for each other
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Biologists use DNA technology to study gene expression and function
• Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
• Analyzing Gene Expression- The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made Labeled nucleic acid probes that hybridize with mRNAs can provide information about the time or place in the organism at which a gene is transcribed.
Studying the Expression of Single Genes • Changes in the expression of a gene during embryonic
development can be tested using the following method to compare mRNA from different developmental stages. 1. Northern blotting-Scientists carry out gel
electrophoresis on samples of mRNA from different stages of development, or following treatment using drugs or growth factors, etc. Then the separated mRNA is transferred to a nitrocellulose membrane, and then allow the mRNAs on the membrane to hybridize with a labeled probe that recognizes a specific mRNA.
2. In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
3. Reverse transcriptase-polymerase chain reaction (RTPCR)
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Figure 20.10
Head
50 µm
Thorax Abdomen
Segment boundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5′ 3′
3′ 3′
3′
5′ 5′
5′ TAACGGTTCCAGC ATTGCCAAGGTCG
CTCAAGTTGCTCT GAGTTCAACGAGA
Cells expressing the wg gene
Cells expressing the en gene
wg mRNA en mRNA
• Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive than Northern blotting and is useful for comparing amounts of specific mRNAs in several samples at the same time
• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest
• The products are run on a gel and the mRNA of interest identified
RT-PCR
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Figure 20.11
DNA in nucleus
mRNAs in cytoplasm
Reverse transcriptase Poly-A tail mRNA
DNA strand
Primer (poly-dT)
5′
5′
5′
5′
3′
3′ 3′
3′ A A A A A A
A A A A A A
T T T T T
T T T T T
5′ 3′ 5′
3′
DNA polymerase
1. mRNA is isolated and reverse transcriptase is added to the tube. 2. RT makes the first DNA strand. Then the mRNA is degraded. 3. DNA polymerase syn- thesizes the second DNA strand using an added primer. 4. The resulting cDNA carries the complete coding sequence of the gene.
A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell
A cDNA library
represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells
cDNA
Figure 20.12
cDNA synthesis mRNAs
cDNAs
PCR amplification Primers
Specific gene
Gel electrophoresis
Results Embryonic stages 1 2 3 4 5 6
Technique 1
2
3
Studying the Expression of Interacting Groups of Genes
• Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
Genome-wide expression studies are made possible by the use of DNA microarray assays
• A DNA microarray consists of tiny amounts of a large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid, also called a DNA chip.
• Ideally, these fragments represent all the genes of an organism.
• The DNA fragments on a microarray are tested for hybridization with cDNA molecules that have been prepared from the mRNAs in particular cells of interest and labeled with fluorescent dyes.
• In addition to uncovering gene interactions and providing clues to gene function, DNA microarray assays may contribute to a better understanding of certain diseases and suggest new diagnostic techniques or therapies. – Comparing patterns of gene expression in cancer tumors and
noncancerous tissue has resulted in more effective treatment protocols.
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Figure 20.13
Genes expressed in first tissue.
Genes expressed in second tissue.
Genes expressed in both tissues.
Genes expressed in neither tissue.
DNA microarray (actual size)
Each dot is a well containing identical copies of DNA fragments that carry a specific gene.
►
• With rapid and inexpensive sequencing methods available, researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
• By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
Determining Gene Function • One way to determine function is to disable the
gene and observe the consequences • Using in vitro mutagenesis, mutations are
introduced into a cloned gene, altering or destroying its function
• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype
• Gene expression can also be silenced using RNA interference (RNAi)
• Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA
• In humans, researchers analyze the genomes of many
people with a certain genetic condition to try to find nucleotide changes specific to the condition
• These genome-wide association studies test for genetic markers, sequences that vary among individuals
• SNPs (single nucleotide polymorphisms), single nucleotide variants, are among the most useful genetic markers
• SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
• SNPs are rarely directly involved in the disease; they are most often in noncoding regions of the genome
Figure 20.14
DNA
SNP Normal allele
Disease-causing allele
A
T
C
G
Cloned organisms and stem cells are useful for basic research and
other applications • Organismal cloning produces one or more
organisms genetically identical to the “parent” that donated the single cell
• A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely, or under certain conditions can differentiate into one or more types of specialized cells
Cloning Plants: Single-Cell Cultures
• In plants, cells can dedifferentiate and then give rise to all the specialized cell types of the organism
• A totipotent cell, such as this, is one that can generate a complete new organism
• Plant cloning is used extensively in agriculture
Cloning a Eukaryotic Gene in a Bacterial Plasmid
• In gene cloning, a plasmid is used as a cloning vector
• A cloning vector is a circular DNA molecule that can carry foreign DNA into a host cell and replicate there
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Figure 20.15
Cross section of carrot root
Small fragments
Fragments were cultured in nutrient medium; stirring caused single cells to shear off into the liquid.
Single cells free in suspension began to divide.
Embryonic plant developed from a cultured single cell.
Plantlet was cultured on agar medium. Later it was planted in soil.
Adult plant
Cloning Animals: Nuclear Transplantation
• In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
• Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg
• However, the older the donor nucleus, the lower the percentage of normally developing tadpoles
Figure 20.16 Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ- entiated cell
Fully differ- entiated (intestinal) cell
Donor nucleus trans- planted
Enucleated egg cell
Donor nucleus trans- planted Egg with donor nucleus
activated to begin development Results
Most develop into tadpoles.
Most stop developing before tadpole stage.
DNA technology applications for transgenic animals
• DNA technology permits the genetic engineering of transgenic animals, which speeds up the selective breeding process.
• The goals of creating a transgenic animal are often the same as the goals of traditional breeding—for instance, to make a sheep with better quality wool, a pig with leaner meat, or a cow that will mature in a shorter time.
• Scientists might, for example, identify and clone a gene that leads to the development of larger muscles (muscles make up most of the meat we eat) in one variety of cattle and transfer it to other cattle or even to sheep.
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Cloning of Mammals using Nuclear Transplantation- Reproductive Cloning of
Mammals • In 1997, Scottish researchers announced the birth of Dolly,
a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
• These researchers cultured donor mammary cells in a nutrient-poor medium and then fused these cells with enucleated sheep eggs.
• The resulting diploid cells divided to form early embryos, which were implanted into surrogate mothers.
• Out of several hundred implanted embryos, one successfully completed normal development, and Dolly was born.
• Later analyses showed that Dolly’s chromosomal DNA was identical to that of the nucleus donor.
• Dolly’s mitochondrial DNA came from the egg donor. • Dolly’s premature death in 2003, as well as her arthritis,
led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 20.17 Technique
1 2
3
4
5
6
Mammary cell donor
Results
Egg cell donor
Egg cell from ovary
Cultured mammary cells
Nucleus removed
Cells fused
Nucleus from mammary cell
Grown in culture
Early embryo
Implanted in uterus of a third sheep
Surrogate mother
Embryonic development Lamb (“Dolly”)
genetically identical to mammary cell donor
• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs
• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”
• Cloned animals do not always look or behave exactly the same
Figure 20.18 CC, the first cloned cat (right), and her single parent
Faulty Gene Regulation in Cloned Animals
• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects
• Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
Figure 20.19
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bone cells
or White blood cells
Stem Cells of Animals Stem cells are relatively unspecialized cells that can both reproduce indefinitely and, under certain conditions, differentiate into one or more specialized cell types
Embryonic and Adult Stem Cells • Many early embryos contain stem cells capable
of giving rise to differentiated embryonic cells of any type
• In culture, these embryonic stem cells reproduce indefinitely
• Depending on culture conditions, they can be made to differentiate into a variety of specialized cells
• Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace non reproducing cells as needed
Stem Cells of Animals • A stem cell is a relatively unspecialized cell that can
reproduce itself indefinitely and differentiate into specialized cells of one or more types
• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types (pluripotent)
• The adult body also has stem cells, which replace non reproducing specialized cells
• With the addition of specific growth factors, cultured stem cells from adult animals have been made to differentiate into multiple types of specialized cells.
• This technology may provide cells for the repair of damaged or diseased organs, such as insulin-producing pancreatic cells for people with diabetes or certain kinds of brain cells for people with Parkinson’s disease or Huntington’s disease.
Figure 20.20
Cells that can generate all embryonic cell types
Cultured stem cells
Cells that generate a limited number of cell types
Different culture conditions
Liver cells Nerve cells Blood cells
Different types of differentiated cells
Embryonic stem cells
Adult stem cells
• Embryonic stem (ES) cells are
pluripotent, capable of differentiating into many different cell types
• The ultimate aim of research with stem cells is to supply cells for the repair of damaged or diseased organs
• ES cells present ethical and political issues
Induced Pluripotent Stem (iPS) Cells • Researchers can treat differentiated cells, and
reprogram them to act like ES cells • Researchers used retroviruses to induce extra
copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
• iPS cells can perform most of the functions of ES cells
• iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
Figure 20.21 Stem cell Precursor cell Experiment
Skin fibroblast cell
Four “stem cell” master regulator genes were introduced, using the retroviral cloning vector.
Induced pluripotent stem (iPS) cell
Oct3/4 Sox2
c-Myc Klf4
The practical applications of DNA-based biotechnology affect our lives
in many ways • Many fields benefit from DNA technology
and genetic engineering. • Medical Applications:- • One benefit of DNA technology is identification of human
genes in which mutation plays a role in genetic diseases • Researchers use microarray assays or other tools to
identify genes turned on or off in particular diseases • The genes and their products are then potential targets for
prevention or therapy
Diagnosis and Treatment of Diseases
• Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation
• SNPs may be associated with a disease-causing mutation
• SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
• Cloned disease genes include those for sickle-cell disease, hemophilia, cystic fibrosis, Huntington’s disease, and Duchenne muscular dystrophy (DMD).
Human gene therapy • Gene therapy—introducing genes into an afflicted
individual for therapeutic purposes—holds great potential for treating genetic disorders caused by a single defective gene.
• In theory, a normal allele of the defective gene could be inserted into dividing somatic cells of the tissue affected by the disorder, such as bone marrow cells.
• Bone marrow cells, which include the stem cells that give rise to all the cells of the blood and immune system, are prime candidates for gene therapy.
• 10 young patients with severe combined immunodeficiency (SCID), which is caused by a single defective gene, have been treated in gene therapy trials. – After two years, nine of these patients showed
significant improvement. However, three of the patients subsequently developed leukemia and one died.
• Gene therapy provokes both technical and ethical questions.
Figure 20.22 Cloned gene 1
2
3
4
Insert RNA version of normal allele into retrovirus or other viral vector.
Viral RNA
Viral capsid
Let virus infect bone marrow cells that have been removed from the patient and cultured.
Viral DNA carrying the normal allele inserts into chromosome.
Bone marrow cell from patient
Inject engineered cells into patient.
Bone marrow
• Transgenic animals are made by introducing genes from one species into the genome of another animal
• Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use
• “Pharm” plants are also being developed to make human proteins for medical use
Protein Production by “Pharm” Animals and Plants
Figure 20.23 Goat milk can produce silk to make bullet proof vest.
Pharmaceutical Products • Advances in DNA technology and genetic
research are important to the development of new drugs to treat diseases
Synthesis of Small Molecules for Use as Drugs • The drug imatinib is a small molecule that
inhibits overexpression of a specific leukemia-causing receptor
• This approach is feasible for treatment of cancers in which the molecular basis is well-understood
Protein Production in Cell Cultures • Host cells in culture can be engineered to
secrete a protein as it is made, simplifying the task of purifying it.
• Cells in culture can also be used to produce vaccines, which stimulate the immune system to defend against specific pathogens.
• This is useful for the production of insulin, human growth hormones, and vaccines
Genetic profiles may be important forensic evidence
• Body fluids or small pieces of tissue may be left at the scene of a violent crime or on the clothes of the victim or assailant.
• DNA testing, in contrast, can identify the guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins).
• Genetic markers that vary in the population can be analyzed for a given person to determine that individual’s unique set of genetic markers, or genetic profile. Genetic profiles can be analyzed using RFLP(Restriction Fragment Length Polymorphism)analysis or STR (short tandem repeats).
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STR’s • STRs (short tandem repeats) are variations in the
lengths number of repeats of specific DNA sequences in specific regions of the genome.
• PCR is used to amplify particular STRs using sets of primers that are labeled with different colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis.
• Forensic scientists test only a few selected portions of the DNA—about 13 STR markers.
• The probability that two people (who are not identical twins) would have exactly the same set of STR markers is somewhere between one chance in 10 billion and one in several trillion.
• As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
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Figure 20.24 (a) Earl Washington just before his release in 2001, after 17 years in prison.
(b) These and other STR data (not shown) exonerated Washington and led Tinsley to plead guilty to the murder.
Source of sample
STR marker 1
STR marker 2
STR marker 3
Semen on victim 17,19 13,16 12,12
Earl Washington 16,18 14,15 11,12
Kenneth Tinsley 17,19 13,16 12,12
Microorganisms can be used for environmental cleanup
• Many bacteria can extract heavy metals, such as copper, lead, and nickel, from their environments and incorporate the metals into compounds such as copper sulfate or lead sulfate, which are readily recoverable.
• Genetically engineered microbes may become important in both mining minerals (especially as ore reserves are depleted) and cleaning up highly toxic mining wastes.
• Biotechnologists are also trying to engineer microbes that can be used in wastewater treatment plants to degrade chlorinated hydrocarbon.
• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
•
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Biofuels can supplement or replace fossil fuels
• Biofuels from crops such as corn, soybeans, and cassava have been proposed as a supplement or even replacement for fossil fuels.
• To produce “bioethanol,” starch is converted to sugar and then fermented by microorganisms; a similar process can produce “biodiesel” from plant oils. Either product can be mixed with gasoline or used alone to power vehicles.
• Critics charge that the environmental effects of growing these crops makes their total impact greater than that of fossil fuels.
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Agricultural Applications • DNA technology is being used to improve agricultural
productivity and food quality • Genetic engineering of transgenic animals speeds up the
selective breeding process • Beneficial genes can be transferred between varieties or
species • Agricultural scientists have endowed a number of crop
plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for
introducing new genes into plant cells • Genetic engineering in plants has been used to transfer
many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops
Figure 20-25
Site where restriction enzyme cuts
T DNA
Plant with new trait
Ti plasmid
Agrobacterium tumefaciens
DNA with the gene of interest
Recombinant Ti plasmid
TECHNIQUE
RESULTS
Safety and Ethical Questions Raised by DNA Technology
• Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
• As biotechnology continues to change, so does its use in agriculture, industry, and medicine
• National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
Ethical Questions Raised by DNA Technology
• The increasing speed and decreasing cost with which genome sequences of individual people can be determined are raising significant ethical questions.
• Who should have the right to examine someone else’s genetic information?
• How should a person’s genetic information be used?
• Should a person’s genome be a factor in suitability for a job or eligibility for insurance?
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Safety and Ethical Questions Raised by DNA Technology
• Most public concern about possible hazards
centers on genetically modified (GM) organisms used as food like GM Corn, Golden Rice.
• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives.
• Other worries include the possibility that transgenic protein products might cause allergic reactions.
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Producing Clones of Cells Carrying Recombinant Plasmids
• Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:
The hummingbird genomic DNA and a bacterial plasmid are isolated – Both are digested with the same restriction enzyme – The fragments are mixed, and DNA ligase is added
to bond the fragment sticky ends – Some recombinant plasmids now contain
hummingbird DNA – The DNA mixture is added to bacteria that have
been genetically engineered to accept it – The bacteria are plated on a type of agar that selects
for the bacteria with recombinant plasmids – This results in the cloning of many hummingbird
DNA fragments, including the β-globin gene
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restriction site
Sticky ends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carrying plasmids
RESULTS
Colony carrying non- recombinant plasmid with intact lacZ gene
One of many bacterial clones
Colony carrying recombinant plasmid with disrupted lacZ gene
Screening a Library for Clones Carrying a Gene of Interest
• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene
• This process is called nucleic acid hybridization • The DNA probe can be used to screen a large number of clones
simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be
cultured
Gene of interest Nucleic acid probe
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Fig. 20.7
Probe DNA
Radioactively labeled probe
molecules
Film
Nylon membrane
Multiwell plates holding library clones
Location of DNA with the complementary sequence
Gene of interest
Single-stranded DNA from cell
Nylon membrane
TECHNIQUE
•
Screening a Library for Clones
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DNA technology allows us to study the
sequence, expression, and function of a gene
• DNA cloning allows researchers to – Compare genes and alleles between
individuals – Locate gene expression in a body – Determine the role of a gene in an organism
• Several techniques are used to analyze the DNA of genes
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• Restriction fragment analysis- DNA fragments produced by restriction enzyme digestion of a DNA molecule are separated/ or sorted by gel electrophoresis.
• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene if the nucleotide difference alters a restriction site.
• Variations in DNA sequence are called polymorphisms
• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)
Restriction Fragment Analysis
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Figure 20.10
Normal β-globin allele
Sickle-cell mutant β-globin allele
Large fragment
Normal allele
Sickle-cell allele
201 bp 175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of the β-globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp 175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
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