Term
| List the Steps of in vivo cloning experiment |
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Definition
1. Determine source of target gene NO INTRONS-can use mRNA with reverse transcriptase 2. Select appropriate cloning vector (plasmids only 20kb, bacteriophage or animal virus) 3. Isolate and purify vector and DNA to be cloned 4. Cut Vector and DNA iwth appropriate restriction enzymes. 5. Ligate vector and DNA fragments to be cloned. 6. Transfer vector and DNA fragments to be cloned. Transduction- Virus/bacteriophage deposits DNA Transfection- Free Viral DNA picked up by bacteria or any free DNA picked up by Eukaryotic Cell. Transformation-Free Bacterial DNA picked up by bacteria 7. Check for cloned DNA, potential transcription, or presence of product. |
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Term
| How can you isolate DNA from Eukaryotes WITHOUT Introns? |
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Definition
cDNA (complementary DNA) fragments or DNA produced in vitro by reverse trascriptase of mRNA |
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Term
| What is on the 5' and 3' ends of Eukaryotic DNA? |
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Definition
5'-Methylated guanylyl cap 3'- PolyA Tail |
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Term
| What is the best cloning vector to use for a large piece of DNA? |
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Definition
Use a large virus (i.e. Adeno Virus-30kb), -however could integrate into the wrong chromosome and cause cancer. Plasmids are good for bacteria and yeast but are limited to a miniscule 20kb. |
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Term
| Compare and contrast Adenovirus and Adeno-associated virus. |
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Definition
Adeno Virus is DOUBLE stranded and the associated virus is SINGLE stranded. Both infect dividing and non-dividing cells. |
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Term
| Give an example of a retrovirus and their advantage. |
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Definition
HIV Retroviruses can integrate directly into a host's genome...very stable expression. Disadvantage is that it is small (7.5 kb) and hard to get large titers. |
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Term
What is the function of a restriction enzyme? What are two kinds? |
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Definition
Cut DNA at specific sequences (Generally degrades/cuts up foreign DNA) One that creates Sticky ends and another for Blunt ends |
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Term
| What characteristic determines the velocity at which a DNA fragment moves through an agrose gel? |
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Definition
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Term
| What size of DNA would move fastest. |
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Definition
The smaller, the faster. Larger fragments get caught up in the agrose matrix |
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Term
| What is used to stain a agrose gel? |
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Definition
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Term
Which of the following features have to be present on a plasmid to express the clones? a. Restriction Enzymes b. Stop Codons c. Origin of replication d. Introns e. Promoters |
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Definition
e. Promoters Expression vectors provide a promoter (+/- regulatory elements) to allow for the expression of a cloned gene |
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Term
| (T/F) On agarose gel, the larger DNA fragments run ahead of the smaller fragments because of their larger negative charge. |
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Definition
FALSE Velocity depends on size not charge, and the large DNA fragments go slower b/c they get held up in the agarose matrix. |
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