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Biotech 2
Bio 2 Lecture 17
| Question | Answer |
|---|---|
| Now that PCR is done, the PCR fragments must be put into a bacterium. How? | Can't be put directly or they'll be degraded by restriction enzymes, so we use restriction enzymes |
| Where are restriction enzyme found? What does it do? | In bacteria. It cuts at a specific DNA sequences called restriction site. Each enzyme recognizes its own specific restriction site. |
| What happens when DNA is cut? | It produces sticky ends that can re-anneal due to H-bonding of staggered ends. |
| What are sticky ends? What is it used in? | Sticky ends are unpaired nucleotides at each end of a strand of DNA. Used in recombinant DNA technology (glue DNA from 2 organisms together) |
| Restriction sites are.. | palindromes (Same sequence when u read it from 5' to 3') (basically looks like the inverse bc they go in dif directions) |
| EcoRI is a... and comes from... | restriction enzymes that comes from E. coli |
| What are the 2 ways a restriction enzyme can cut? | Using EcoRi enzyme (sticky ends) and Sma 1 enzyme (blunt ends) |
| So what do we use? | We use a cloning vector also known as a plasmid |
| What are plasmids? | Plasmids are cicular pieces of DNA that can be naturally occuring |
| What are the key components for a plasmid to be viable? | 1) Origin of replication 2) Multiple cloning sites 3) Selective marker 4) Inducible promoter |
| So what's the general goal of using a plasmid? | We want to insert gene X into the plasmid and then insert the plasmid into the bacterium |
| How is the plasmid put into the host bacteria? | By transformation aka the uptake of DNA from an outside source into a bacteria cell |
| What are the 3 things to do to ensure success of the transformation? | 1) Competent cell 2) Heat shock treatment (Expose bacteria to salt sln and then treat with hot and cold shocks to weaken cell wall) 3) Electroporation (pulse of electricity to open pore of cell membrane) |
| What are selective markers? | Enable certain cells to grow (Ex: ampR gene marker allows bacteria to grow in medium containing ampicilin) |
| What are differential markers? | Distinguishes bacteria (Ex: lac Z makes an enzyme that breaks down X-gal turning the bacterial colony blue) |
| What's the issue with recombinant DNA technique? What's the solution | To allow prokaryotic bacterial cells to express a eukaryotic gene, the eukaryotic gene must be intron-free. So we use reverse transcriptase |
| What is reverse transcriptase? | An enzyme that can catalyze the synthesis of DNA from an RNA template |
| What is cDNA? How? | Complementary DNA, it's the technique used to prepare DNA from RNA. mRNA is isolated from the cytoplasm and cDNA is generated from the mRNA |
| How do we make cDNA? | 1) Isolate eukaryotic mRNA from cytoplasm of cell 2) Expose mRNA to reverse transcriptase 3) Denature the mRNA (leaves ssDNA) 4) Make dsDNA by adding DNA replicatinf molecules using PCR cDNA is the end result and can be carried out in PCR |
| What are DNA libraries? | A collection of DNA fragments from a specific source may be inserted into host cells for storing (one fragment per host) |
| What are the 2 different types of libraries? | 1) Genomic (entire genome inserted into plamids) 2) cDNA library (selected genes) |
| What are the 2 technics that use molecular hybridization? | 1) Southern blot (DNA fragments by gel electrophoresis, ssDNA, radioactive ss-probe) 2) Replica plating (agar plate with bacterial colonies) |
| So give a little review of producing a recombinant DNA | 1) Restriction enzymes 2) Hybridization and ligation 3) Vectors 4) Transformation 5) Selective/Differential markers 6) Cloning to mass produce gene product |
Created by:
Malayka
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