Term
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Definition
- Small enzymes cleave to foreign DNA after recognizing a specific sequence (which is often palindromic).
- By translating these enzymes in vitro, we have extremely specific DNA-cutting tools for use in the lab.
- "Sticky ends" allow the DNA to rebind after cutting. |
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Term
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Definition
| Copying a biological entity by an artificial method. |
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Term
| Cloning: Plasmid Insertion |
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Definition
1) Plasmid DNA contains a recognition site for a restriction endonuclease.
2) Attach the same recognition site to the gene that will be inserted into the plasmid.
3) A restriction endonuclease makes staggered cuts at each of the recognition sites, creating "sticky ends."
4) Use DNA ligase to catalyze phosphodiester bond, "sealing" the inserted gene.
5) Sticky ends on plasmid and on gene to be inserted bind by complementary base pairing.
6) Insert double-stranded cDNA into a different plasmid.
7) Introduce recombinant plasmids into cells by making the membrane temporarily permeable to large molecules.
8) If the gene included an appropriate promoter, the gene should be expressed in the new cells. |
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Term
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Definition
- Use DNA ligase to catalyze a phosphodiester bond, "sealing" the inserted gene.
- Sticky ends on plasmid and on gene to be inserted bind by complementary base pairing.
- Insert double stranded cDNA into a different plasmid.
- Introduce recombinant plasmids into cells by making the membrane temporarily permeable to large molecules.
- If the gene included an appropriate promoter, the gene should be expressed in the new cells. |
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Term
| Cloning: Using Viral Vectors in Plants |
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Definition
1) Agrobacterium cells contain a Ti (tumor-inducing) plasmid.
2) A section of DNA from the Ti plasmid, called T-DNA, incorporates into the chromosomes of plant cells infected by the bacterium.
3) When transcribed, Ti genes induce the affected cell to begin growing and dividing. The resulting gall encloses an increasing number of Agrobacterium cells. |
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Term
| Cloning: Plant Vector Design |
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Definition
1) Start with normal Ti plasmids.
2) Remove tumor-inducing genes.
3) Add genes for enzymes required for B-carotene synthesis along with promoter that will be activated in endosperm. |
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Term
| Cloning: Taking Advantage of Viral Retrotranscriptase |
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Definition
1) Engineered retrovirus contains recombinant RNA, which has both viral sequences and human sequences.
2) Recombinant genes enter host cell.
3) Viral enzymes make double-stranded DNA version of introduced genes.
4) Recombinant genes are inserted into host chromosome and transcribed. |
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Term
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Definition
1) Stem cells are isolated from the patient's bone marrow and grown in vitro.
2) Recombinant retroviruses carry the normal allele into host cells.
3) Cells that express normal alleles are isolated and implanted into patient. |
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