Chapter 16
Recombination DNA and Genetic
Engineering
Cloning DNA
• 1. Cloning is the production of identical copies
• 2. An underground stem sends up new shoots that are
clones
• 3. Members of a bacterial colony on a petri dish are
clones because
• they all came from division of the same cell.
• 4. Human identical twins are clones; the original single
embryo separate to become two individuals.
• 5. Gene cloning is production of many identical
copies of the same gene.
Continue…
• 6. If the inserted gene is replicated and
expressed, we can recover the cloned
gene or protein product.
• 7. Cloned genes have many research
purposes: determining the base sequence
between normal and
• mutated genes, altering the phenotype,
etc.
• 8. Humans can be treated with gene
therapy; alteration of other organisms
forms transgenic organisms.
Clogged Arteries
• Cholesterol does good things for the body,
such as forming membranes and vitamin
D, but it can also combine with lipoproteins
to form atherosclerotic plaques in the walls
of the arteries
– Some persons have genes that cause familial
cholesterolemia
– Gene therapy promises a way to genetically
alter the cells of the liver to keep the levels of
cholesterol in the more normal range
• We can “engineer” genetic changes
through recombinant DNA technology.
– DNA from different species can be cut, spliced
together, and inserted into bacteria, which
then multiply the DNA necessary for protein
production
– Genetic engineering has great promise for
agriculture, medicine, and industry.
Restriction Enzyme
• Bacteria possess restriction enzyme
whose usual function is to cut apart foreign
DNA molecules.
• Each enzyme cut only at sites that
possess a specific base sequence
• The wide variety of restriction enzymes
and their specificity makes it possible to
study the genome of a particular species
Modification Enzymes
• Many times the “sticky ends” that result
from the cut can be used to pair up with
another DNA fragment cut by the same
enzyme.
• DNA fragments produced by restriction
enzymes are treated with DNA ligase to
splice the DNA fragments together to form
a recombinant DNA molecule.
Cloning Vectors for Amplifying DNA
• Plasmids are circular DNA molecules in
bacteria that carry only a few genes and
can replicate independently of the single
“main” chromosomes
• When the plasmids is replicated, any
foreign DNA that might have become
incorporated into it is also replicated
producing a DNA clone.
Continue…
• Modified plasmids that are capable of
accepting, replicating, and delivering DNA
to another host cell are called cloning
vectors
Reverse Transcriptase to Make
cDNA
• Even after a desired gene has been
isolated and amplified, it may not be
translated into functional protein by the
bacteria because introns (noncoding
regions) are still present.
• Researchers minimize this problem by
using cDNA, which is made from “mature
mRNA transcripts.
Continue…
– The cDNA is made from mRNA by reverse
transcriptase
– cDNA can be inserted into a plasmid for
amplification
PCR
• The polymerase chain reaction (PCR) can
be used to make millions of copies of
cDNA
• 1. PCR can create millions of copies of a single gene or a
specific piece of DNA in a test tube.
• 2. PCR is very specific—the targeted DNA sequence can be
less than one part in a million of the total DNA sample;
therefore a
• single gene can be amplified using PCR.
• 3. The polymerase chain reaction (PCR) uses the enzyme
DNA polymerase to carry out multiple replications (a chain
reaction) of
• target DNA.
• 4. PCR automation is possible because heat-resistant DNA
polymerase from Thermus aquaticus, which grows in hot
springs, is an enzyme that withstands the temperature
necessary to separate double stranded DNA.
What are Primers?
• Primers are short nucleotide sequences
that are made in the laboratory.
• They are recognized by DNA polyermases
as the START tags for building
complementary sequences of DNA
dictated by computer programs stored in
the machines.
• DNA FINGERPRINTS ** READ PAGE 257
How is DNA sequenced?
• Current laboratories use automated DNA
sequencing to determine the unknown sequence
of bases in a DNA sample.
• The automated DNA sequencer separates the
sets of fragments by gel electrophoresis.
– The “tag” base at the end of each fragment in the
set is identified by laser beam.
– The computer program in the machine assembles
the information from all the nucleotides in the
sample to reveal the entire DNA sequence
How can you isolate a particular
gene for study?
• Gene Library: a collection of bacteria that
house different cloned DNA fragments
– The library of the entire genome or of cDNA is
free of introns
What are Probes?
• DNA probes, short DNA sequences
assembled from radioactive nucleotides,
can pair with parts of the gene to be
studied
• This nucleic acid hybridization technique
can be used with other procedures to
select cells and their DNA, which may be
of interest to the researcher
Analyzing DNA Segments
• 1. Mitochondria DNA sequences in modern living
populations can decipher the evolutionary history of
human populations.
• 2. DNA fingerprinting is the technique of using
DNA fragment lengths, resulting from restriction
enzyme cleavage and amplified
• by PCR, to identify particular individuals.
• a. DNA is treated with restriction enzymes to cut it into
different sized fragments.
• b. During gel electrophoresis, fragments separate
according to length, resulting in a pattern of bands.
• c. DNA fingerprinting can identify
deceased individuals from skeletal
remains, perpetrators of crimes from blood
or semen
• samples, and genetic makeup of longdead individuals or extinct organisms
• 3. PCR amplification and DNA analysis is
used to:
• a. detect viral infections, genetic disorders,
and
• cancer;
• b. determine the nucleotide sequence of
human
• genes: the Human Genome Project; and
• c. associate samples with DNA of parents
because it is inherited.
Using the Genetic Strips
• Microorganisms can produce useful
substances such as human insulin and
blood-clotting factors.
• Genetically engineered bacteria can clean
up messes such as oil spills
• Gene may help use devise counterattacks
against rapidly mutating pathogens
Transgenic Bacteria
• 1. Bacteria are grown in large vats called bioreactors.
• a. Foreign genes are inserted and the product is
harvested. (insulin, hepatitis B vaccine, t-PA, and
human growth hormone)
• 2. Transgenic bacteria have been produced to protect
and improve the health of plants (frost resistance,
pesticides)
• 3. Transgenic bacteria can degrade substances (oileating bacteria, bio-filters)
• 4. Transgenic bacteria can produce chemical
products. (phenylalanine for aspartame sweetener)
Designer Plants (Transgenic
Plants)
• Regenerating plants from cultured cells
– Botanist are searching the world for seeds from
the wild ancestors of potatoes, corn, etc.
• They are worry is that there is too little
diversity in the few strains now used for food
crops
– Many plant species can be regenerated from
cultured cells.
– Useful mutations, such as resistance to a
toxin, can be identified
– Foreign genes now give cotton, corn, and
potato strains ability to produce an insect
toxin
– Plants are being engineered to produce
human proteins including hormones, clotting
factors, and antibodies in their seeds;
antibodies made by corn deliver radioisotopes
to tumor cells and a soybean engineered
antibody can treat genital herpes.
How are Genes Transferred Into
Plants?
• An early experiment showed that a
plasmid from a bacterium that normally
causes tumors in plants could be modified
by replacing the tumor gene with the
desirable genes
Gene Transfer in Animals
• Supermice and Biotech Barnyards
– In 1982, the rat gene for somatotropin
production was introduced into mouse eggs;
the mice which subsequently expressed the
rat gene grew larger than their littermates
– Farm animals may be used to produce TPA
for diminishing the severity of heart attacks or
CFTR is in the diminishing of cystic fibrous
• 1. Animal use requires methods to insert genes
into eggs of animals.
• a. It is possible to micro-inject foreign genes into
eggs by hand.
• b. Vortex mixing places eggs in an agitator with
DNA and silicon-carbide needles that make tiny
holes which the DNA can enter.
• c. Using this technique, many types of animal
eggs have been injected with bovine growth
hormone (bGH) to produce larger fishes, cows,
pigs, rabbits, and sheep.
Gene Pharming
Gene pharming is the use of transgenic
farm animals to produce pharmaceuticals;
obtainable from the milk of females.
• a. Genes for therapeutic proteins are
inserted into animal’s DNA; animal’s milk
produces proteins.
• b. Drugs obtained through gene pharming
are planned for the treatment of cystic
fibrosis, cancer,blood diseases, and other
disorders.
Cloning Transgenic Animals
• 1. For many years, it was believed that adult
vertebrate animals could not be cloned; the
cloning of Dolly in 1997 demonstrated this
can be cone.
• 2. Cloning of an adult vertebrate would
require that all genes of an adult cell be
turned on again.
• 3. Cloning of mammals involves injecting a
2n nucleus adult cell into an enucleated
egg.
• 4. The cloned eggs begin development in
vitro and are then returned to host mothers
until the clones are born.
• 5. Somatic Cell Nuclear Transfer –
cloning from adult cells (Dolly)
Animal Organs as
Biotechnology Products
• 1. It may be possible to use genetically
engineered pigs to serve as a source of
organs for human transplant.
• 2. Scientists are learning how to stimulate
human cells to construct organs in the
laboratory.
Mapping the Human Genome
• Human Genome Initiative is dependent on
technology
• The information gained will give insights
into genetic disorders and utimately
provide gene therapy
• New field of genomics will be concerned
with mapping and sequencing the
genomes as well understanding the
evolutionary relationships
Safety Issues
• Genetically Engineered bacteria have “fail
safe” genes included in the DNA which are
supposed to be lethal if the bacteria
escapes into a non-lab environment
• General public is concerned about
organisms being released that are not
“natural” and may endanger human lives
Biotechnology in a Brave New
World
• Microarray or gene chips can reveal a
stunning amount of information about an
individual DNA
Gene Therapy
• Has been successful in a trail against
SCID-X1
• Eugenic engineering is idea of being able
to select desirable humans traits
Send in the Clones
• Xenotransplantation is the transferring of
an organ from one species to another
• Pigs can be engineered to lack certain
genes that would cause rejection problems
when their organs are transplanted to
humans
Weighing the Benefits and Risks
• Some say we should never alter the DNA
of any organisms; others say we already
have
• What would be problems with genetic
engineering over the long haul?
• Scenario 1: Melissa is a happy 5 year old
who is loved by her family. She becomes
ill and is diagnosed with childhood
leukemia. A desperate search ensues to
find a bone marrow donor whose type
matches Melissa. After a year of
searching, Melissa’s outlook is grim. Her
family decides to clone Melissa so that her
clone could be the bone marrow donor.
Do you think this is a god idea? Why or
why not.
• Scenario 2: A well-loved horse named
Barbero breaks his leg in a race. Many
people were praying for his well being and
thousands of dollars were spent trying to
get him to recover. Mail and flowers
poured into the animal hospital and stable
where Barbero lived. Also, after a year of
poor recovery, the decision was made to
euthanize Barbero. The owners save
sample of his DNA so that Barbero can be
cloned. Do you think they should clone
him? Why or why not.