2015 Pearson Education, Inc. Introduction Hawaiis papaya
industry seemed doomed just a few decades ago. A deadly pathogen
called the papaya ringspot virus (PRV) had spread throughout the
islands. It appeared poised to completely decimate the papaya plant
population. Scientists from the University of Hawaii were able to
rescue the industry by creating new, genetically engineered
PRV-resistant strains of papaya. Today, the papaya industry is once
again vibrant, and the vast majority of Hawaiis papayas are
genetically modified organisms (GMOs).
Slide 2
GMO Genetically Modified Organisms (GMO): When a gene from one
organism is purposely moved to improve or change another organism
in a laboratory, the result is a genetically modified organism
(GMO). It is also sometimes called "transgenic" for transfer of
genes. 84% of corn grown today is genetically modified. Most is
used in processed foods and some are used for animal feed. Some
strains are drought resistant, others are pest resistant, some are
herbicide resistant. 94% of the soy crop in the US is genetically
modified. Very few fresh fruits and vegetables in your local
grocery store are genetically modified. Potatoes are one of the
genetically engineered vegetables available in the United States.
Other genetically modified vegetables that have been approved for
sale in the U.S. are tomatoes, radicchio, zucchini and yellow
squash.
Slide 3
Isnt everything genetically modified????
Slide 4
2015 Pearson Education, Inc. Genes can be cloned in recombinant
plasmids Biotechnology- For thousands of years, humans have used
microbes to make wine and cheese and selectively bred stock, dogs,
and other animals. DNA technology Genetic engineering-
Slide 5
2015 Pearson Education, Inc. Genes can be cloned in recombinant
plasmids Gene cloning- Recombinant DNA One source contains the gene
that will be cloned. Another source is a gene carrier, called a
______. _________ are small, circular DNA molecules that replicate
separately from the much larger bacterial chromosome; they are
often used as vectors.
Slide 6
2015 Pearson Education, Inc. Genes can be cloned in recombinant
plasmids The steps in cloning a gene: 1._____________is isolated.
2.DNA containing the gene of interest is isolated. 3.Plasmid DNA is
treated with a _____________ that cuts in one place, opening the
circle. 4.DNA with the target gene is treated with the same enzyme,
and many fragments are produced. 5.Plasmid and target DNA
__________________.
Slide 7
2015 Pearson Education, Inc. 654321 Plasmid Bacterial
chromosome E. coli bacterium A plasmid is isolated. The plasmid is
cut with an enzyme The cells DNA is isolated. DNA A cell with DNA
containing the gene of interest Gene of interest (gene V) The cells
DNA is cut with the same enzyme. Gene of interest The targeted
fragment and plasmid DNA are combined. DNA ligase is added, which
joins the two DNA molecules. Gene of interest Recombinant DNA
plasmid
Slide 8
2015 Pearson Education, Inc. 7 8 9 Genes may be inserted into
other organisms. Harvested proteins may be used directly. The
recombinant plasmid is taken up by a bacterium through trans-
formation. The bacterium reproduces. Recombinant DNA plasmid
Recombinant bacterium Clone of cells Gene of interest
Slide 9
2015 Pearson Education, Inc. VISUALIZING THE CONCEPT: Enzymes
are used to cut and paste DNA Restriction enzymes recognize a
particular short DNA sequence, called a restriction site, and cut
both strands of the DNA at precise points in the sequence, yielding
pieces of DNA called restriction fragments. Once cut, fragments of
DNA can be pasted together by the enzyme DNA ligase.
Slide 10
2015 Pearson Education, Inc. Animation: Restriction
Enzymes
Slide 11
2015 Pearson Education, Inc. Restriction enzyme Restriction
site DNA Sticky end Gene of interest Sticky end DNA ligase
Recombinant DNA Every restriction enzyme recognizes one specific
nucleotide sequence (its restriction site). A restriction enzyme
always cuts DNA sequences at its restriction site in an identical
manner. A piece of DNA from another source (the gene of interest)
is cut by the same restriction enzyme. The DNA fragments from the
two sources stick together by hydrogen bonding of base pairs. The
enzyme DNA ligase creates new covalent bonds that join the
backbones of the DNA strands. The result is a piece of recombinant
DNA. G AATTC G CTTAA AATTC CTTAA G G G GG G C C C CAATT TTAA GAATTC
CTTAAG
Slide 12
2015 Pearson Education, Inc. Recombinant cells and organisms
can mass-produce gene products Recombinant cells and organisms
constructed by DNA technologies are used to manufacture many useful
products, chiefly proteins. Bacteria are often the best organisms
for manufacturing a protein product because bacteria have plasmids
and phages available for use as gene- cloning vectors, can be grown
rapidly and cheaply, can be engineered to produce large amounts of
a particular protein, and often secrete the proteins directly into
their growth medium.
Slide 13
2015 Pearson Education, Inc. Recombinant cells and organisms
can mass-produce gene products Yeast cells are eukaryotes, are easy
to grow, have long been used to make bread and beer, can take up
foreign DNA and integrate it into their genomes, and are often
better than bacteria at synthesizing and secreting eukaryotic
proteins.
Slide 14
2015 Pearson Education, Inc. Recombinant cells and organisms
can mass-produce gene products Mammalian cells must be used to
produce glycoproteins, proteins with chains of sugars attached.
Examples include human erythropoietin (EPO), which stimulates the
production of red blood cells, factor VIII to treat hemophilia, and
tissue plasminogen activator (TPA), used to treat heart attacks and
strokes.
Slide 15
2015 Pearson Education, Inc.
Slide 16
Recombinant cells and organisms can mass-produce gene products
Pharmaceutical researchers are currently exploring the mass
production of gene products by whole animals or plants. Recombinant
animals are difficult and costly to produce and may be cloned to
produce more animals with the same traits.
Slide 17
2015 Pearson Education, Inc. CONNECTION: DNA technology has
changed the pharmaceutical industry and medicine DNA technology,
including gene cloning, is widely used to produce medicines and to
diagnose diseases. Therapeutic hormones produced by DNA technology
include insulin to treat diabetes, human growth hormone to treat
dwarfism, and tissue plasminogen activator (TPA), a protein that
helps dissolve blood clots and reduces the risk of subsequent heart
attacks.
Slide 18
2015 Pearson Education, Inc. CONNECTION: DNA technology has
changed the pharmaceutical industry and medicine DNA technology is
used to test for inherited diseases, detect infectious agents such
as HIV, and produce vaccines, harmless variants (mutants) or
derivatives of a pathogen that stimulate the immune system to mount
a lasting defense against that pathogen, thereby preventing
disease.
Slide 19
2015 Pearson Education, Inc. CONNECTION: Genetically modified
organisms are transforming agriculture Since ancient times, people
have selectively bred agricultural crops to make them more useful.
DNA technology is quickly replacing traditional breeding programs
to improve the productivity of agriculturally important plants and
animals. Genetically modified organisms (GMOs) contain one or more
genes introduced by artificial means. Transgenic organisms contain
at least one gene from another species.
Slide 20
2015 Pearson Education, Inc. CONNECTION: Genetically modified
organisms are transforming agriculture The most common vector used
to introduce new genes into plant cells is a plasmid from the soil
bacterium Agrobacterium tumefaciens called the Ti plasmid.
Slide 21
2015 Pearson Education, Inc. Plant cell 213 Agrobacterium
tumefaciens DNA containing the gene for a desired trait Ti plasmid
Restriction site The gene is inserted into the plasmid. Recombinant
Ti plasmid The recombinant plasmid is introduced into a plant cell.
DNA carrying the new gene The plant cell grows into a plant. A
plant with the new trait
Slide 22
2015 Pearson Education, Inc. CONNECTION: Genetically modified
organisms are transforming agriculture GMO crops may be able to
help a great many hungry people by improving food production, shelf
life, pest resistance, and the nutritional value of crops. Golden
Rice, a transgenic variety created in 2000 with a few daffodil
genes, produces yellow grains containing beta-carotene, which our
body uses to make vitamin A.
Slide 23
2015 Pearson Education, Inc. CONNECTION: Genetically modified
organisms are transforming agriculture Genetic engineers are now
creating plants that make human proteins for medical use.
Pharmaceutical trials currently under way involve using modified
rice to treat infant diarrhea, corn to treat cystic fibrosis,
safflower to treat diabetes, and duckweed to treat hepatitis.
Although promising, no plant-made drugs intended for use by humans
have been approved or sold.
Slide 24
2015 Pearson Education, Inc. CONNECTION: Gene therapy may
someday help treat a variety of diseases Gene therapy is the
alteration of a diseased individuals genes for therapeutic
purposes. One possible procedure is the following: 1.A gene from a
healthy person is cloned, converted to an RNA version, and then
inserted into the RNA genome of a harmless virus. 2.Bone marrow
cells are taken from the patient and infected with the recombinant
virus. 3.The virus inserts a DNA version of its genome, including
the normal human gene, into the cells DNA. 4.The engineered cells
are then injected back into the patient.
Slide 25
2015 Pearson Education, Inc. Healthy person 1234 Cloned gene
(normal allele) An RNA version of a healthy human gene is inserted
into a retrovirus. RNA genome of virus Retrovirus Bone marrow cells
are infected with the virus. Viral DNA carrying the human gene
inserts into the cells chromosome. Bone marrow cell from the
patient Bone marrow The engineered cells are injected into the
patient.
Slide 26
2015 Pearson Education, Inc. The analysis of genetic markers
can produce a DNA profile DNA profiling is the analysis of DNA
samples to determine whether they came from the same individual. In
a typical investigation involving a DNA profile: 1.DNA samples are
isolated from the crime scene, suspects, victims, or other
evidence, 2.selected markers from each DNA sample are amplified
(copied many times), producing a large sample of DNA fragments, and
3.the amplified DNA markers are compared, providing data about
which samples are from the same individual.
Slide 27
2015 Pearson Education, Inc. Crime scene 231 Suspect 1 Suspect
2 DNA is isolated. The DNA of selected markers is amplified. The
amplified DNA is compared.
Slide 28
2015 Pearson Education, Inc. The PCR method is used to amplify
DNA sequences Polymerase chain reaction (PCR) is a technique by
which a specific segment of a DNA molecule can be targeted and
quickly amplified in the laboratory.
Slide 29
2015 Pearson Education, Inc. The PCR method is used to amplify
DNA sequences PCR relies upon a pair of short primers, which are
chemically synthesized, single-stranded DNA molecules with
sequences that are complementary to sequences at each end of the
target sequence. One primer is complementary to one strand at one
end of the target sequence. The second primer is complementary to
the other strand at the other end of the sequence. The primers thus
bind to sequences that flank the target sequence, marking the start
and end points for the segment of DNA being amplified.
Slide 30
2015 Pearson Education, Inc. The PCR method is used to amplify
DNA sequences The basic steps of PCR are as follows: 1.The reaction
mixture is heated to separate the strands of the DNA double
helices. 2.The strands are cooled. As they cool, primer molecules
hydrogen-bond to their target sequences on the DNA. 3.A heat-stable
DNA polymerase builds new DNA strands by extending the primers in
the 53 direction. These three steps are repeated over and over,
doubling the amount of DNA after each three-step cycle.
Slide 31
2015 Pearson Education, Inc. 3355 3355 3355 33 55 55 3355 3355
55 3355 3355 3355 3355 Cycle 1 yields two molecules Cycle 2 yields
four molecules Cycle 3 yields eight molecules Additional Cycles
Sample DNA Target sequence PrimerNew DNA Heat separates DNA
strands. Primers bond with ends of target sequences. DNA polymerase
adds nucleotides. 12 3
Slide 32
2015 Pearson Education, Inc. The PCR method is used to amplify
DNA sequences Devised in 1985, PCR has had a major impact on
biological research and biotechnology. PCR has been used to amplify
DNA from fragments of ancient DNA from a mummified human, a
40,000-year-old frozen woolly mammoth, a 30-million-year-old plant
fossil, and DNA from fingerprints or from tiny amounts of blood,
tissue, or semen found at crime scenes.
Slide 33
2015 Pearson Education, Inc. Gel electrophoresis sorts DNA
molecules by size Many DNA technology applications rely on gel
electrophoresis, a method that separates macromolecules, usually
proteins or nucleic acids, on the basis of size, electrical charge,
or other physical properties.
Slide 34
2015 Pearson Education, Inc. Gel electrophoresis sorts DNA
molecules by size Gel electrophoresis can be used to separate DNA
molecules based on size as follows: 1.A DNA sample is placed at one
end of a porous gel. 2.Current is applied, and DNA molecules move
from the negative electrode toward the positive electrode.
3.Shorter DNA fragments move through the gel matrix more quickly
and travel farther through the gel. 4.DNA fragments appear as
bands, visualized through staining or detecting radioactivity or
fluorescence. 5.Each band is a collection of DNA molecules of the
same length.
Slide 35
2015 Pearson Education, Inc. Gel Power source A mixture of DNA
fragments of different sizes Completed gel Longer (slower)
molecules Shorter (faster) molecules
Slide 36
2015 Pearson Education, Inc. Short tandem repeat analysis is
commonly used for DNA profiling Repetitive DNA consists of
nucleotide sequences that are present in multiple copies in the
genome. Short tandem repeats (STRs) are short nucleotide sequences
that are repeated in tandem, composed of different numbers of
repeating units in individuals, that are used in DNA profiling. STR
analysis compares the lengths of STR sequences at specific sites in
the genome and typically analyzes 13 sites scattered in the
genome.
Slide 37
2015 Pearson Education, Inc. STR site 1 STR site 2 Crime scene
DNA AGATGATA The number of short tandem repeats match The number of
short tandem repeats do not match GATA AGAT Suspects DNA
Slide 38
2015 Pearson Education, Inc. Amplified crime scene DNA
Amplified suspects DNA Longer STR fragments Shorter STR
fragments
Slide 39
2015 Pearson Education, Inc. CONNECTION: DNA profiling has
provided evidence in many forensic investigations DNA profiling is
used to determine guilt or innocence in a crime, settle questions
of paternity, and probe the origin of nonhuman materials.
Slide 40
2015 Pearson Education, Inc. RFLPs can be used to detect
differences in DNA sequences Geneticists have cataloged many
single-base-pair variations in the genome. Such a variation found
in at least 1% of the population is called a single nucleotide
polymorphism (SNP, pronounced snip). SNPs occur on average about
once in 100 to 300 base pairs in the human genome, in the coding
sequences of genes and in noncoding sequences between genes.
Slide 41
2015 Pearson Education, Inc. RFLPs can be used to detect
differences in DNA sequences SNPs may alter a restriction sitethe
sequence recognized by a restriction enzyme. Such alterations
change the lengths of the restriction fragments formed by that
enzyme when it cuts the DNA. A sequence variation of this type is
called a restriction fragment length polymorphism (RFLP, pronounced
rif-lip). Thus, RFLPs can serve as genetic markers for particular
loci in the genome.
Slide 42
2015 Pearson Education, Inc. Cut Restriction enzymes added
Sample 1 DNA sample 1 Longer fragments Shorter fragments w Sample 2
DNA sample 2 x y y z yy z x w CCGGCCGG CCGGCCGG CCGGCCGG GC GC GC
TGCCTGCC ACGGACGG
Slide 43
2015 Pearson Education, Inc. You should now be able to
1.Explain how plasmids are used in gene cloning. 2.Explain how
restriction enzymes are used to cut and paste DNA into plasmids.
3.Explain how DNA technology has helped to produce insulin, growth
hormone, and vaccines. 4.Explain how genetically modified organisms
(GMOs) are transforming agriculture. 5.Describe the benefits and
risks of gene therapy in humans.
Slide 44
2015 Pearson Education, Inc. You should now be able to
6.Describe the benefits and risks of gene therapy in humans.
7.Describe the basic steps of DNA profiling. 8.Explain how PCR is
used to amplify DNA sequences. 9.Explain how gel electrophoresis is
used to sort DNA and proteins. 10.Explain how short tandem repeats
are used in DNA profiling. 11.Explain how restriction fragment
analysis is used to detect differences in DNA sequences.
Slide 45
2015 Pearson Education, Inc. Figure 12.UN02 A mixture of DNA
fragments A band is a collection of DNA fragments of one particular
length Longer fragments move slower Shorter fragments move faster
Power source DNA is attracted to + pole due to PO 4 groups
Slide 46
2015 Pearson Education, Inc. Figure 12.UN03 DNA amplified via
Bacterial plasmids DNA sample treated with DNA fragments sorted by
size via Recombinant plasmids are inserted into bacteria Add
Particular DNA sequence highlighted are copied via treated with (a)
(b) (c) (d) (e)