Term
| What are the three steps in making recombinant insulin? |
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Definition
1. Isolate the gene in the human genome
2. Put the gene into E. coli
3. Have E. coli produce large amounts of insulin |
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Term
| What role do restriction enzymes play in genetic engineering? How do cells prevent restriction enzymes? |
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Definition
Restriction enzymes slice DNA at cleavage sites. It recognizes these sites by looking for paliondromes between the two strands. It does not, however, cut straight down across the two strands; sticky ends are produced.
Restriction enzymes are often used to destroy foreign DNA, cells protect their own genome by methylating palindrome sites, preventing the enzyme from cutting. |
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Term
| How is human DNA inserted into plasmids for bacteria uptake during transformation? What is a recombinant plasmid? |
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Definition
Human DNA and plasmids are both cut by the same restriction enzymes. They are then mixed and complimentary sticky ends attach with the help of DNA ligase. This creates a recombinant plasmid.
These plasmids are then introduced to bacteria cultures for uptake. |
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Term
| How are colonies containing the insulin gene separated from other colonies who also uptook plasmids through nucleic acid hybridization? |
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Definition
Only some cells took up recombinant plasmids. Nucleic acid hybridization begins by transfering colonies to multiwell plates, then nylon membranes.
The bacteria is treated and its DNA denatured, the DNA strands separate. These ssDNA stick to the nylon. Then, a nucleic acid probe (a synthetic, radioactive ssDNA complementary to the insulin gene) is added to the nylon membrane.
After washing off the excess probe and subjecting the nylon to X-ray film, you can see where the probes bound to DNA. The wells of nylon that contain probes mean that the corresponding well of the multiwell plate contains bacteria with the insulin gene. Grow these bacteria and express the gene.
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Term
| A plasmid from ___bacterium, called the ___ plasmid, contains ___ DNA, which is introduced to a gene that prevents Roundup from killing crops. This plasmid is then introduced to crops. |
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Definition
| A plasmid from agrobacterium, called the Ti plasmid, contains T DNA, which is introduced to a gene that prevents Roundup from killing crops. This plasmid is then introduced to crops. |
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Term
| What is the benefit of using Polymerase Chain Reaction (PCR) to replicate DNA? |
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Definition
| It requires a small amount of DNA. |
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Term
| What are the three steps of PCR? |
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Definition
1. Denaturation - Separate DNA strands
2. Annealing - Synthetic primers attach to the target promoters
3. Extension - complimentary DNA strands are created using heat-allowing DNA polymerase
Over time more and more of the desired fragment will be replicated compared to the entire DNA strand. |
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Term
| How can you separate the desired DNA fragment from the entire DNA strand after duplication in PCR? |
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Definition
Put the DNA in an agarose gel and submit it to a cathode (negative charge). The DNA will migrate to the anode, with the smaller fragments making it farther because they are less hindered.
You can see where the DNA ends up by soaking the gell in flourescent dye that binds the DNA. |
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Term
| What are STRs? How are they used to identity someone in forensics? |
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Definition
STR stands for short tandem repeats. These are a type of simple sequence reptitive DNA located across different loci on chromosomes. The sequences surrounding STRs are ussually the same for everyone. PCR is used to make a large amount of DNA found at a crime scene.
You can then run the fragments per gel to find out the number of repeat units for each STR at each locus. The chances of two people having the same profile is less than 1/1B. |
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Term
| How does DNA sequencing work? |
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Definition
A synthetic primer and a DNA polymerase are mixed with an unkown DNA single strand along with dNTPs and ddNTPs (dideoxy- (minus 2 oxygens)). ddNTPs lack the hydroxyl group at the 3' carbon, so they end the chain. They are also flourescently tagged different colors.
The unkown sample is submitted to PCR and what ends up being made are complimentary strands of the unkown DNA with lengths differing by 1 base. Since they are tagged, you can tell what the original strand of DNA was. |
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