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1

Recombinant DNA technology

Involves the use of in vitro molecular techniques to connect different pieces of DNA

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biotechnology

Technologies that involve the use of living organisms, or products from living organisms, as a way to benefit humans

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Transgenic organism

When an organism is given DNA from another organism using such techniques

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gene cloning: two strategies

1. Introducing a gene into a vector, and then propagating the vector in living cells
2. Using the polymerase chain reaction (PCR) to make many copies of a gene in a test tube

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vector

A segment of DNA that can replicate and produce many identical copies of the inserted gene.

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When does vector replication occur?

When it is found within a living host cell

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From what two natural resources were vectors used in gene cloning derived?

1. Some vectors are plasmids
2. Viral vectors

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Plasmid

Small circular pieces of DNA. Derived from R factors and carry antibiotic resistance genes. Contain origin of replication that is recognized by the replication enzymes of the host cell.

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Viral vector

When a chromosomal gene is inserted into a viral genome, the gene will be replicated when the viral DNA is replicated.

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Restriction endonuclease or restriction enzymes

Used to extract a gene from its location within a chromosome, cut DNA

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How do restriction enzymes work?

They bind to a specific base sequence and then cleave the DNA backbone at two defined locations, one in each strand.

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What do restriction enzymes digest DNA into?

Fragments with "sticky ends"

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What are sticky ends?

The ends of these DNA fragments will hydrogen bond to each other because of their complementary sequences. The complementary sequences promote interactions between two different pieces of DNA.

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How is hydrogen bonding between sticky ends only a temporary interaction?

It involves only a few hydrogen bonds between complementary bases.

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How do you set up a permanent connection between two DNA fragments?

The sugar-phosphate backbones within the DNA strands must be covalently linked together by an enzyme known as DNA ligase.

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DNA ligase

Covalently links sugar-phosphate backbones within DNA strands.

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Steps to cloning a gene

1. Chromosomal DNA is isolated and digested with a restriction enzyme
2. A plasmid is cut at one site with the same restriction enzyme
3. The digested chromosomal DNA and plasmid DNA are mixed together and incubated under conditions that will promote the binding of complementary sticky ends.
4. DNA ligase is then added to catalyze the covalent linkage between DNA fragments
5. After libation, DNA is introduced into bacterial cells that have been treated with agents that make them permeable to DNA molecules.

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Hybrid vector

Vector containing a piece of chromosomal DNA

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transformation

When a plasmid vector is used to introduce DNA into bacterial cells that have been treated with agents that make them permeable to DNA molecules

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transfection

When viral vector is introduced into host cell.

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Why are colors of colonies different that contain a recircularized vector versus a hybrid vector?

Blue colonies still have an intact beta-galactosidase gene; white colonies have an inactivated beta-galactosidase gene because of the insertion of a piece of chromosomal DNA

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What two events lead to the amplification of the cloned gene?

1. Bacterial host cell replicates the hybrid vector to produce many copies per cell
2. Bacterial cells divide approximately every thirty minutes, producing a very large population of cells

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Experiment 18A: First Gene-cloning experiment
hypothesis

A piece of DNA carrying a gene can be inserted into a plasmid vector using recombinant DNA techniques. If this recombinant plasmid is introduced into a bacterial host cell, it will be replicated and transmitted to daughter cells, producing many copies of the recombinant plasmid.

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Experiment 18A: First Gene-cloning experiment
Starting materials

Three different strains of E. coli; one strain that did not carry any plasmid and two strains that carried pSC101 or pSC102.

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Experiment 18A: First Gene-cloning experiment
protocol

1. Isolate and purify the two types of plasmid DNA
2. Grow the bacterial cells containing the plasmids
3. Break open the cells.
4. Isolate each type of plasmid DNA by density gradient centrifugation
5. DIgest the plasmid DNAs with EcoRI
6. Mix together the two samples
7. Add DNA ligase
8. Grow E. coli strain that does not carry a plasmid. Treat the cells with CaCl2 to make then permeable to DNA.
9. Add lighted DNA samples to bacterial cells. Most cells do not take up a plasmid but occasionally one will.
10. Plate the cells on growth media containing both tetracycline and kanamycin. Grow overnight to allow growth of visible bacterial colonies.
11. Pick four colonies from the plates. The plasmid found in these colonies is designated in pSC105. Grow the colonies in liquid culture containing radiolabeled deoxyribonucleotides.
12. To isolate the radiolabeled plasmid DNA, break open the cells and subject the DNA to cesium chloride density gradient centrifugation.
13. As a control, subject pSC101 and pSC102 to the same procedure.
14. Digest plasma DNAs with EcoRI, and subject them to gel electrophoresis

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EcoRI

Restriction enzyme that produces sticky ends

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Experiment 18A: First Gene-cloning experiment
Interpreting the data

You had two data sets. One was density from centrifuges fractions. The other was electrophoresis. In the fraction density, we saw that pSC105 had a different density than just the combination of 101 and 102, so it was recombined. Electrophoresis show that pSC105 ended up being pSC101 with just one other fragment from 102 that made the thing resistant to tetracycline and kanamycin. A recombinant molecule was created and put into E coli

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Reverse transcriptase

Enzyme, typically found in viruses, that can use an RNA template to make a copy of DNA

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cDNA

A copy of DNA made from RNA. Made with reverse transcriptase.

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cDNA synthesis: what is a poly-dT primer

Annals to the 3' end of mRNAs