Genetic Engineering

Changing the Living WorldA. Selective Breeding

1. Hybridization

2. Inbreeding

B. Increasing Variation

1. Producing New Kinds of Bacteria

2. Producing New Kinds of Plants

Section 13-1

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Humans use selective breeding to pass desired traits on to the next generation.

While inbreeding assures a set of characteristics it also causes genetic defects as well. (blindness, hip displeasure in dogs and hemophilia in humans)

Wide variation is found in natural populations. Breeders can artificially introduce variation by inducing mutations.

Plants tolerate extra sets of chromosomes. Polyploidy has resulted in new species of plants.

Genetic Engineering

Manipulating DNAA. The Tools of Molecular Biology

1. DNA Extraction

2. Cutting DNA

3. Separating DNA

B. Using the DNA Sequence

1. Reading the Sequence

2. Cutting and Pasting

3. Making Copies

Section 13-2

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13–2

Genetic Engineering

Genetic Engineering

Making changes in the DNA code of a living organism

Restriction Enzyme

An enzyme that divides DNA at a specific sequence of nucleotides.

Gel Electrophoresis

Process to separate out gene fragments in a mixture using an electric current and polarity of the fragments.

Recombinant DNA

Combining DNA from different sources.

Polymerase chain reaction

Process that makes many copies of a particular gene

Genetic Engineering

Genetic Engineering = process in which a gene from the DNA of one organism is removed, and then transferred into the DNA of another organism

Boyle & Cohen (1973) - FIRST people to genetically engineer a new organism: Removed the gene for rRNA from a frog and inserted that gene into the DNA of a bacterium.RESULT: A bacteria that produced large amounts of frog rRNA

Genetic engineering involves the making of a new combination of DNA made from two or more different organisms

Genetic Engineering

IF you can cut out a gene, WHAT do you use to carry that gene into the cell of another organism???

Vector

= agent used to carry the gene of interest into another organism's cell

EXAMPLES of vectors: viruses, yeast or plasmids

PLASMID

= circular DNA molecules in bacteria that

can replicate (copy itself) independently from the single main chromosome in bacteria

Genetic Engineering

Bacterial plasmids are often used to make recombinant DNA.

– plasmids are loops of DNA in bacteria

– restriction enzymes cut plasmid and foreign DNA

– foreign gene inserted into plasmid

Genetic Engineering

Restriction enzymes cut DNA.

• Restriction enzymes act as “molecular scissors.” – come from various types of bacteria– allow scientists to more easily study and manipulate

genes– cut DNA at a specific nucleotide sequence called a

restriction site

Genetic Engineering

• Different restriction enzymes cut DNA in different ways.

– each enzyme has a different restriction site

Genetic Engineering

– some cut straight across and leave “blunt ends”

– some make staggered cuts and leave “sticky ends”

Genetic Engineering

• A restriction map shows the lengths of DNA fragments between restriction sites.

– only indicate size, not DNA sequence

– useful in genetic engineering

– used to study mutations

Genetic Engineering

– Smaller fragments move faster and travel farther than larger fragments.

– Fragments of different sizes appear as bands on the gel.

Gel Electrophoresis

Genetic Engineering

PCR uses polymerases to copy DNA segments.

• PCR makes many copies of a specific DNA sequence in a few hours.

target sequence of DNA

• PCR amplifies DNA samples.• PCR is similar to DNA replication.

Genetic Engineering

PCR is a three-step process.

• PCR uses four materials.– DNA to be copied– DNA polymerase– A, T, C, and G nucleotides– two primers

DNA strands

polymerase

nucleotides

primer 1

primer 2

Genetic Engineering

DNA strands

polymerase

nucleotides

primer 1

primer 2

• The three steps of PCR occur in a cycle.

– heat is used to separate double-stranded DNA molecules

– primers bind to each DNA strand on opposite ends of the segment to be copied

– DNA polymerase binds nucleotides together to form new strands of DNA

Genetic Engineering

Cell TransformationA. Transforming Bacteria

B. Transforming Plant Cells

C. Transforming Animal Cells

Section 13-3

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15–3

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Plasmid

A small circular DNA molecule used in bacteria transformation

Genetic Marker

A gene that identifies the plasmid from the foreign DNA

Genetic Engineering

Human Cell

Gene for human growth hormone

Recombinant DNA

Gene for human growth hormone

Sticky ends

DNA recombination

DNA insertion

Bacterial Cell

Plasmid

Bacterial chromosome

Bacterial cell for containing gene for human growth hormone

Section 13-3Figure 13-9 Making Recombinant DNA

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Genetic Engineering or recombinant DNA

• Connecting or recombining fragments of DNA from different sources– 1. Isolate foreign DNA fragment to be inserted

-Restriction enzymes- cut DNA strands at specific areas

Genetic Engineering

– 2. Attach DNA fragment to a carrierVector- means by which DNA from another species can

be carried into a host cello Biological vectors: viruses, plasmidso Mechanical vectors: micropipette, gene gun

– 3. Transfer into a host organism– Transgenic organism- plants and animals that contain

functional recombinant DNA

Genetic Engineering

Recombinant Examples

• E.Coli bacteria to produce indigo dye-jeans

• Growth hormone- dwarfism• Insulin-diabetes• Hemophilia- sheep milk has

clotting factor• Pest resistant plants

Genetic Engineering

Applications of Genetic EngineeringA. Transgenic Organisms

1. Transgenic Microorganisms

2. Transgenic Animals

3. Transgenic Plants

B. Cloning

Section 13-4

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15–4

Genetic Engineering

Biotechnology is a result of genetic engineering.

Transgenic Microorganisms

Used in disease research. They are easy to grow and reproduce rapidly.

Transgenic Animals

Used to improve the food supply by making animals stronger

Transgenic Plants

Genetically modified plants increase the food supply, and additionally are able to produce a naturally occurring herbicide.

Genetic Engineering

Cloning occurs in nature.

– bacteria (binary fission)– some plants (from roots)– some simple animals (budding, regeneration)

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Mammals can be cloned through a process called nuclear transfer.

– nucleus is removed from an egg cell– nucleus of a cell from the animal to be cloned is

implanted in the egg

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Cloning has potential benefits.

– organs for transplant into humans– save endangered species

Cloning raises concerns.– low success rate– clones “imperfect” and less healthy than original animal– decreased biodiversity

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A donor cell is taken from a sheep’s udder. Donor

Nucleus

These two cells are fused using an electric shock.

Fused Cell

The fused cell begins dividing normally.

EmbryoThe embryo is placed in the uterus of a foster mother.Foster

Mother

The embryo develops normally into a lamb—Dolly

Cloned Lamb

Egg Cell

An egg cell is taken from an adult female sheep.

The nucleus of the egg cell is removed.

Section 13-4Figure 13-13 Cloning of the First Mammal

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Genetic engineering produces organisms with new traits.

• A transgenic organism has one or more genes from another organism inserted into its genome.

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• Transgenic bacteria can be used to produce human proteins.– gene inserted into plasmid – plasmid inserted into bacteria– bacteria express the gene

• Transgenic plants are common in agriculture.– transgenic bacteria

infect a plant– plant expresses

foreign gene– many crops are now

genetically modified(GM)

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• Transgenic animals are used to study diseases and gene functions.

– transgenic mice used to study development and disease

– gene knockout mice used to study gene function

Genetic Engineering

• Human Molecular GeneticsA. Human DNA Analysis

1. Testing for Alleles

2. DNA Fingerprinting

B. The Human Genome Project1. Rapid Sequencing

2. Searching for Genes

3. A Breakthrough for Everyone

C. Gene Therapy

D. Ethical Issues in Human Genetics

Section 14-3

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14-3

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Testing for Alleles:

Alleles responsible for genetic disorders have a different DNA sequences than the normal counterpart.

Genetic testing: Genetic tests have been developed to spot the defective sequence. Other tests detect changes in restriction enzyme cutting sites or differences in lengths of normal and abnormal alleles.

Prenatal Testing

Amniocentesis = sample of fluid

surrounding the fetus is taken with a long, thin needle

Cells from the fluid can be grown in culture and

karyotype prepared

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Human Genome Project:

Started in 1990.

Objective: To sequence all human DNA.

Finished in June 2000.

First markers were found that allowed the DNA to be broken down to smaller segments.

Second: Private companies used a shot gun approach to sequence random segments.

Third: computers were used to find overlapping regions between the fragments to place them in order.

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• Gene sequencing is determining the order of DNA nucleotides in genes or in genomes.

• The genomes of several different organisms have been sequenced.

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Technology allows the study and comparison of both genes and proteins.

• Bioinformatics is the use of computer databases to organize and analyze biological data.

• DNA microarrays are used to study the expression of many genes at once.

• Proteomics is the study and comparison of proteins.

Genetic Engineering

DNA FingerprintsRestriction Fragment Length Polymorphism (RFLP) - used to

identify base sequences in our DNA

Restriction enzymes are used to cut DNA into fragments that have SPECIFIC lengths.

DNA of different people contain different nucleotides sequences – restriction enzymes cut the DNA from different people into pieces of DIFFERENT lengths.

Separate the pieces by gel electrophoresis - DNA hasnegative charge :smaller pieces move faster than larger pieces

This produces a series of bands = DNA fingerprint!! May use blood, hair, semen, bone - any cell with DNA.

Genetic Engineering

A DNA fingerprint is a type of restriction map.

• DNA fingerprints are based on parts of an individual’s DNA that can by used for identification.– based on noncoding regions of DNA– noncoding regions have repeating DNA sequences– number of repeats differs between people– banding pattern on a gel is a DNA fingerprint

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– Individual probabilities are multiplied to find the overall probability of two DNA fingerprints randomly matching.

– Several regions of DNA are used to make DNA fingerprints.

1 1 1 1500 90 120 5,400,000 1 chance in 5.4 million peoplex x = =

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• DNA fingerprinting is used in several ways.

– evidence in criminal cases

– paternity tests– immigration requests– studying biodiversity

– tracking genetically modified crops

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• Gene Therapy:• An absent or faulty gene is replaced by a normal

working gene.• Vector: Benign virus is modified with the working

gene then introduced into the body to start replicating the gene.

• Mixed results to date, it is still a high risk, experimental procedure.

• Ethical Issues:• Disease cure is generally accepted.• Bioengineering to produce “ideal” human is not and

becomes a societal discussion and decision question.

Genetic Engineering

Genetic screening can detect genetic disorders.

• Genetic screening involves the testing of DNA.– determines risk of having

or passing on a geneticdisorder

– used to detect specificgenes or proteins

– can detect some genesrelated to an increasedrisk of cancer

– can detect some genesknown to cause geneticdisorders

DMD N

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• Several experimental techniques are used for gene therapy.

– genetically engineered viruses used to “infect” a patient’s cells

– insert gene to stimulate immune system to attack cancer cells

– insert “suicide” genes into cancer cells that activate a drug

Genetic Engineering

• Scientists have concerns about some uses of genetic engineering.– possible long-term health effects of eating GM foods– possible effects of GM plants on ecosystems and

biodiversity