Genetic Engineering and Recombinant DNA

Genetic Engineering and Recombinant DNA The Origin of Genetic Engineering

Biotechnology - the use of living organisms for practical purposes.

While many believe that biotechnology is a novel concept, it actually began about 10,000 years ago when human populations began selecting and breeding useful plants, animals, fungi, and microorganisms.While the early biotechnology techniques were relatively simple,

modern genetic engineers move genes among all kinds of organisms, including humans, mice, tomatoes, yeasts, and bacteria.

Knowing Biochemical Pathways Helps Molecular Biologists Design Useful Organisms Today, the knowledge of

biochemical pathways in some organisms allows biologists to predict what type of mutation will produce a desired trait.With this in mind, molecular biologists have been With this in mind, molecular biologists have been successful in designing useful organisms by successful in designing useful organisms by inserting or destroying genes that code for inserting or destroying genes that code for proteins involved in specific biochemical proteins involved in specific biochemical pathways.pathways.

What are some examples of this technology?

Knowing Biochemical Pathways Helps Molecular Biologists Design Useful Organisms

For example, Calgene in central California deliberately damaged the gene that controls ethylene production in tomatoes.

Eythlene is responsible for fruit ripening. Since these tomatoes do not

produce ethylene, they will only ripen after the tomato distributor sprays them with ethylene.

This prevents the tomatoes from being picked before they have developed their flavor components.

Such tomatoesSuch tomatoesare termedare termed““Flavor-SaverFlavor-Saver””tomatoes.tomatoes.

How Do Restriction Enzymes Cut Up a Genome?

DNA can be “cut” with special enzymes termed endonucleases.

Endonucleases recognize specific sequences of nucleotides and sever the DNA at these sites.

Endonucleases evolved in bacterial cells as a defense against bacteriophages (bacterial viruses).

When phage DNA enters a bacteria, endonucleases break down the phage DNA (by cutting) in order to restrict viral replication.

Since endonucleases restrict viral replication, they have become known as Restriction Enzymes.

How Do Restriction Enzymes Cut Up a DNA?

Restriction enzymes recognize and “cut” DNA that is foreign to the bacterial cell.

The DNA of the bacterial cell is chemically modified to prevent attack by restriction enzymes.

Restriction Enzymes, therefore, “chop-up” foreign DNA, while leaving the DNA of the bacterial cell unaffected.

How Do Restriction Enzymes Recognize sites for severing?

Since their discovery in 1962, hundreds of restriction enzymes have been identified and isolated from bacterial cells.

These restriction enzymes are extremely specific and work by recognizing short nucleotide sequences in DNA molecules termed RECOGNITION SEQUENCES.

Once these sequences are detected, the restriction enzyme severs the DNA at this point.

EXAMPLE Hae III cuts at the following recognition sequence:

GGCC CCGG

Hae III will cut the DNA every time the above recognition sequence is detected.

The result is a matching set of restriction fragments.

Restriction fragments are pieces of DNA that begin and end with a restriction site.

Hae What?Hae What?

Hae III cuts the DNA each time the recognition sequence repeats itself within a DNA sample.

Mapping A comparison of restriction fragment

sizes allows biologists to construct a restriction map.

Restriction maps demonstrate how the restriction sites are placed within a piece of DNA.

More importantly, biologists can join these fragments into new combinations.

For example, human and mouse fragments can be joined together.

DNA Fingerprinting

Example: Suppose Joe’s DNA has four restriction sites for EcoR1;Example: Suppose Joe’s DNA has four restriction sites for EcoR1;EcoR1 will, therefore, cut Joe’s DNA four timesEcoR1 will, therefore, cut Joe’s DNA four times

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5 fragments result from the action of EcoR1when applied to5 fragments result from the action of EcoR1when applied toJoe’s DNAJoe’s DNA

What is the restriction site for EcoR1?What is the restriction site for EcoR1?

DNA Fingerprinting

Example: Suppose Anisa’s DNA has 3 restriction sites for EcoR1.Example: Suppose Anisa’s DNA has 3 restriction sites for EcoR1.EcoR1 will, therefore, cut Anisa’s DNA three times.EcoR1 will, therefore, cut Anisa’s DNA three times.

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4 DNA segments result4 DNA segments result

EcoR1 cuts Joe’s DNA into 5 fragments and Anisa’s into 4.EcoR1 cuts Joe’s DNA into 5 fragments and Anisa’s into 4.

JoeJoe

AnisaAnisa

Note: In addition to differing in fragment number, the size ofNote: In addition to differing in fragment number, the size ofthe fragments differs as well.the fragments differs as well.

Why is this significant?Why is this significant?

Restriction Enzyme & DNA Fragments

These fragments can now be separated from one another using ELECTROPHORESIS DNA electrophoresis utilizes an

agarose gel and a voltage current to separate the cut DNA fragments from one another.

The DNA samples are placed into the agarose gel (a medium in which the DNA fragments will travel) and the voltage current separates the fragments.

How?

Joe’s DNAJoe’s DNA Anisa’s DNAAnisa’s DNA

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The current is applied and the fragments travel to the + endThe current is applied and the fragments travel to the + enddue to the negatively charged DNA (phosphate).due to the negatively charged DNA (phosphate).

Gel Electrophoresis

Analysis

Joe’s DNAJoe’s DNA Anisa’s DNAAnisa’s DNA

++--

Smaller DNA fragments will travel farther on the gel thanSmaller DNA fragments will travel farther on the gel thanlarger DNA fragments.larger DNA fragments.

Fingerprinting

Since every individual has a unique sequence of bases in their DNA, a unique banding pattern will be generated by electrophoresis for each individual. This is known as a GENETIC FINGERPRINT.

NOTE: Even if two individuals have the same number of restriction sites in their DNA, the size of each fragment will differ and will, therefore, yield a unique banding pattern.

The next slide presents an example:

Forensics

DNA from hairDNA from hairfound on victimfound on victim

DNA from bloodDNA from bloodsample suspect #1sample suspect #1

DNA from bloodDNA from bloodsample suspect #2sample suspect #2

DNA from bloodDNA from bloodsample suspect #3sample suspect #3

All 4 samples areAll 4 samples arecut with the samecut with the samerestriction enzyme.restriction enzyme.

Who did it?Who did it?

Where is the heaviest band?Where is the heaviest band?

Where is the lightest band?Where is the lightest band?

How many restriction sites does the DNA fromHow many restriction sites does the DNA fromsuspect #1 have?suspect #1 have?

How Do Molecular Biologists Use Recombinant DNA? Recombinant DNA - a DNA molecule

consisting of two or more DNA segments that are not found together in nature.

For example, the next slide demonstrates how cells from a tobacco plant are infected with a plasmid carrying a gene for herbicide resistance. The herbicide resistant cells grow into mature plants which produce seeds

containing the resistant gene.

Genetic Engineering and Recombinant DNA

How Do Molecular Biologists Use Recombinant DNA?How Do Molecular Biologists Use Recombinant DNA?

How Do Molecular Biologists Use Recombinant DNA?

Recombinant DNA has provided scientists with:

1) a tool for studying structure, regulation and function of individual genes;

2)a tool for unraveling the molecular bases of molecular diseases;

3)the ability to turn organisms into factories that turn out vast quantities of product (protein or other substance) that these organisms would never make on their own.

How Do Molecular Biologists Join Restriction Fragments Together? Two pieces of DNA from different

sources can be linked together by the enzyme DNA ligase.

DNA ligase is normally used during DNA replication.

DNA ligase is responsible for the linkage of separate pieces of DNA into one continuous strand.

LigaseLigase

This image demonstrateshow ligase can be used to link human and mouse DNA together as well asthe insertion of the human insulin gene into a plasmid causing the bacteria to produce insulin.

How Do Molecular Biologists Express Recombinant DNA in Bacteria and Other Hosts? Molecular biologists face two serious

challenges: 1) To produce large numbers

of particular genes. 2) To induce host cells to

express recombinant genes as usable proteins.

How Can Bacteria Be Induced To Make Great Numbers of Copies of a Gene?

Biologists achieve this goal with the use of plasmids. Plasmids allow bacterial cells to produce large

numbers of copies of a single gene. Using DNA ligase, researchers can link any gene to a plasmid which carries recombinant DNA into cells.

Plasmids are an example of a vector. A vector is anything that spreads genes from one organism to another.

How Can Bacteria Be Induced To Make Eukaryotic Genes?

Eukaryotic DNA contains introns which are base sequences in the pre-mRNA that are not expressed and normally removed by the eukaryotic cell before the mRNA is translated.

Bacterial cells are prokaryotic and, therefore, do not have the required enzymes to recognize and remove the introns.

If they cannot remove introns, they cannot make a mRNA molecule that is translateable and, therefore, cannot directly make eukaryotic genes.

How Can Bacteria Be Induced To Make Eukaryotic Genes?

The solution of intron removal in bacterial cells comes from the action of retroviruses.

Recall that retroviruses contain reverse transcriptase which allows the conversion of RNA to DNA.

Researchers can take mature mRNA (introns have already been removed) and copy it back to DNA with the use of reverse transcriptase.

The resulting DNA is termed complementary DNA (cDNA) and, unlike the genomic DNA, it has no introns.

The image to the left demonstrates how reverse transcriptase is used to copy mature insulin mRNA into DNA.

This DNA can now be joined to a plasmid vector and expressed by a bacterium.

Can Host Cells Be Induced To Express Polypeptides in a Usable Form? Unfortunately, not all eukaryotic

genes can be expressed in bacteria. Such genes code for proteins that must be modified after translation.

For example, most membrane proteins require modifications that can only be made in eukaryotic hosts.

How Do Researchers Make Multiple Copies of Recombinant DNA? Researchers need enormous

quantities of a gene in order to sequence it, detect mutations or study how proteins interact with the gene to influence gene expression.

Cloning and PCR (Polymerase Chain Reaction) allow researchers to make millions of copies of a particular gene.

How Do Researchers Make How Do Researchers Make Multiple Copies of Recombinant Multiple Copies of Recombinant

DNA?DNA?CloningCloning simply involves the introduction of a single recombinantDNA (gene and plasmid) molecule into a bacterial host cell.

The plasmid can induce the host cell to make many copies of the gene it carries and, in addition, researchers can induce the bacterial cell to divide rapidly.

As the bacteria divide, the recombinant DNA multiplies.

PCR allows researchers to produce multiple numbers of individual DNA sequences in a very short period of time.

In PCR:1) The selected DNA segment is heated causing the two

strands to separate.2) The DNA is cooled and two short nucleotide sequences

termed primers bind to the complementary DNA strands.3) DNA polymerase then copies each strand until the

researcher stops the reaction by again raising the temperature.

4) Increasing the temperature repeats the process.

How Do Biologists Find the Right DNA Sequence in a Recombinant DNA Library?

A gene library is a collection of restriction fragments from a single genome.

Such a library is only useful to researchers if they can find the gene they are interested in.

Two tools are used to find specific genes:

1) hybridization probes - short segments of single stranded DNA that binds to and detects the gene in question.

2) antibodies - detect and bind with specific proteins in colonies of bacteria containing recombinant DNA.

The following slides demonstrates the use of each technique.

Genetic Engineering and Recombinant DNA

Note that the hybridizationprobe locates specific DNA sequences while antibodieslocate the protein productof the same sequence.

Figure 13-4Figure 13-4

Genetically Engineered Bacteria and Eukaryotic Cells Can Make Useful Proteins

Genetic reprogramming using recombinant DNA technology allows the production of an extraordinary number of products.

For example: insulin growth hormone ingredients for processed foods enzymes used to produce valuable molecules or destroy

pollutants enzymes in laundry soap Vaccines New proteins researchers are currently developing new

antibodies that can interfere with disease processes

Gene Therapy: Products of Recombinant DNA Can Be Released

Directly into the Body from Engineered Somatic Cells Gene Therapy - The insertion of therapeutic

genes into an individual so that their products act to modulate a particular phenotype.

One strategy associated with gene therapy involves the removal of cells from the body, engineering them to produce the desired effect, and then implanting them back into the body of the individual.

For example, researchers are now experimenting with the insertion of genes for clotting factor into cells that are then implanted into individuals suffering from hemophilia.

This allows the body to produce clotting factor and alleviate symptoms associated with hemophilia.

RECOMBINANT DNA CAN GENETICALLY ALTER ANIMALS AND PLANTS Organisms that carry

recombinant DNA are termed transgenic organisms and the added DNA is termed a transgene.

How Do Researchers Produce a Transgenic Mammal?

For a gene to be expressed, researchers must put the transgene into the zygote before the beginning of embryonic development.

If this is performed successfully, all of the cells of the organism will contain the desired DNA.

To date, researchers have been successful in producing transgenic mice, pigs, goats, and sheep.

How Do Researchers Produce a Transgenic Mammal? The engineering of transgenic animals

faces serious obstacles: 1) they must be made one at a time; 2) in knockouts (animals in which a

particular gene has been inactivated), recombinant genes are inserted at random and may not function as researchers hope.

In spite of these obstacles, such animals can provide clues about how previously mysterious proteins function in the body.

The Genetic Engineering of Plants Is Easier Than That of Animals

Plant advantages: 1) they are easier to clone than animal cells; 2) they can be grown in vast fields which allows massive

production of desired products; 3) they have the potential to be extremely lucrative.

ex: If the Flavor-Saver tomato becomes popular, the inventors will gain a virtual monopoly in the tomato market.

Molecular biologists can genetically engineer plants that can: synthesize animal or plant proteins; resist herbicides; resist infection by plant viruses.

What Are the Environmental Risks of Recombinant DNA?

The long-term consequences are unknown. Some argue that severe ecological effects will

result. For example, genetically

engineered plants may eventually transfer their engineered genes into other plants.

Will pesticide resistant genes inserted into a crop plant be transferred to unrelated pest plants creating herbicide resistant weeds?

The Application of Recombinant DNA Technology Poses Moral Questions for Society Diagnosis of genetic disease is far in advance

of treatment. Under such a situation, people may know that

they have a genetic disease, but will not be able to do anything about it.

Will biologists try to modify genes that affect characteristics other than those responsible for disease?

Will future societies try to produce more intelligent citizens?

Will future societies try to produce fewer aggressive people?