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| 704440075 | Molecular biology arose from the confluence of which 7 disciplines? | 1. genetics 2. physical chemistry 3. x-ray crystallography 4. biochemistry 5. microbiology 6. bacteriology 7. virology | 0 | |
| 704440076 | When were restriction enzymes discovered? | -in the 1950s | 1 | |
| 704440077 | What is a restriction enzyme? | -an enzyme that cuts DNA at specific recognition nucleotide sequences known as restriction sites | 2 | |
| 704440078 | What is a bacteriophage? | -a virus that infects and replicates within bacteria -kills bacteria in the process | 3 | |
| 704440079 | What two important discoveries occurred at the University of Geneva in 1962? | -bacteria that are resistant to bacteriophages possess an enzyme system that could selectively recognize and destroy foreign phage DNA within the bacterial membrane -resistant bacteria could modify their own chromosomal DNA to prevent self-destruction | 4 | |
| 704440080 | Later, extracts were made from E. coli that were found to efficiently cleave the DNA of bacteriophages. What did these extracts contain? | -non-restriction endonucleases | 5 | |
| 704440081 | What are non-restriction endonucleases? | -enzymes that cut internal regions of DNA of an invading bacteriophage and at the same time protected the DNA of the bacterial host -->these enzymes can both cut the phage DNA and also protect bacterial DNA by modifying it | 6 | |
| 704440082 | Why weren't these initial extracts of any use? | -because the cutting activity of these enzymes was non-specific | 7 | |
| 704440083 | What new type of bacterial endonuclease was discovered at Johns Hopkins in 1970? | -a bacterial endonuclease that could cut at a specific site within the phage DNA, while at the same time protecting the same site on the bacterial host's DNA | 8 | |
| 704440084 | How is the protection of the host's DNA site accomplished? | -through the addition of a methyl group to the site | 9 | |
| 704440085 | From what type of bacterium was this new type of bacterial endonuclease isolated? | -the bacterium haemophilus influenzae | 10 | |
| 704440086 | What was this enzyme called? | -HindII | 11 | |
| 704440087 | How does it cleave DNA? | -predictably -cutting within a specific sequence of base pairs, usually only 4 to 8 base pairs in length | 12 | |
| 704440088 | How can the fragments that result from cutting DNA be separated? | -by size in an electrical field | 13 | |
| 704440089 | How is it then possible to create a "map" of the restriction enzyme sites? | -by finding the size of each fragment | 14 | |
| 704440090 | Is DNA acidic or basic? | -acidic | 15 | |
| 704440091 | Why? | -due to the presence of the phosphate groups | 16 | |
| 704440092 | Can DNA be made to be negatively or positively charged? | -negatively | 17 | |
| 704440093 | How? | -when DNA is surrounded by a slightly basic buffer solution, the hydrogens of the phosphate groups can be removed -this leaves negatively charged phosphate groups | 18 | |
| 704440094 | Why is this important to the process of gel electrophoresis? | -when placed in an electric field, DNA molecules are attracted to the positive pole | 19 | |
| 704440095 | What is the positive pole referred to as? | -the anode | 20 | |
| 704440096 | What is the negative pole referred to as? | -the cathode | 21 | |
| 704440097 | During electrophoresis, how do DNA fragments sort within the gel? | -by size | 22 | |
| 704440098 | Why? | -the longer pieces will encounter more frictional forces than the smaller pieces, meaning that the longer pieces will not be able to travel as far | 23 | |
| 704440099 | Using this information, what can we infer? | -that the distance traveled by each piece is inversely proportional to its molecular weight/number of base pairs in the fragment | 24 | |
| 704440100 | What is electrophoresis gel composed of? | -cross-linked polymer mesh | 25 | |
| 704440101 | How is it made? | -by dissolving agarose (made from seaweed) in water | 26 | |
| 704440102 | How can DNA fragments in different size ranges be separated? | -by adjusting the agarose concentration | 27 | |
| 704440103 | What do low agarose concentrations produce? | -a loose gel that separates large fragments | 28 | |
| 704440104 | What do high agarose concentration s produce? | -a stiff gel that resolves small fragments | 29 | |
| 704440105 | What exact type of gel is used in our experiment? | -a 1% gel | 30 | |
| 704440106 | This gel can effectively separate DNA fragments of what size? | -those between 100 and 5000 nucleotides | 31 | |
| 704440107 | What is a plasmid? | -a small, circular, double-stranded DNA molecule that is found in bacteria -physically separate from chromosomal DNA | 32 | |
| 704440108 | What is the name of the first plasmid to be used routinely in recombinant DNA experiments? This is also the plasmid we will be using in our experiment. | -pBR322 | 33 | |
| 704440109 | What was this plasmid originally derived from? | -E.coli | 34 | |
| 704440110 | It was then artificially constructed to have which two antibiotic resistance genes? | -tet -amp | 35 | |
| 704440111 | What do these confer resistance to? | -tetracycline -ampicillin | 36 | |
| 704440112 | In this experiment, what are we concerned with? | -finding the cutting sites for the three restriction enzymes | 37 | |
| 704440113 | What does each restriction enzyme recognize? | -a different set of base paris on which to act | 38 | |
| 704440114 | What is one reason why restriction enzyme mapping is so important? | -finding restriction enzyme sites is the first step in characterizing a DNA fragment that has been ligated onto a vector/plasmid | 39 | |
| 704440115 | This can then provide information for what? | -DNA sequencing, gene localization, and site-directed mutagenesis | 40 | |
| 782827731 | What did molecular biology borrow from the physical sciences? | -the rigorous use of model systems | 41 | |
| 782827732 | What was the conclusion drawn from the observation of the resistance of some bacteria to invading bacteriophages? | -resistance must be a property of the bacterial cell itself | 42 | |
| 782827733 | What characteristic of DNA does gel electrophoresis take advantage of? | -the fact that, as an organic acid, DNA can be made to be negatively charged | 43 | |
| 782827734 | What is the distance traveled by a DNA fragment in agarose gel inversely proportional to? | -molecular weight/the number of base pairs in each fragment -->smaller restriction fragments will migrate relatively far from the origin compared with the larger fragments | 44 | |
| 782827735 | What four restriction enzymes were used in our experiment? | -EcoR1 -HincII -PvuII -Hinc III | 45 | |
| 782827736 | What was Hinc III used for? | -to make the ladder | 46 | |
| 782937728 | What type of DNA was used to make the ladder in this experiment? | -lambda DNA | 47 | |
| 782937729 | What are the three basic components of the loading dye in the experiment? | -blue pigment -violet pigment -glycerol | 48 | |
| 782937730 | What is the role of glycerol in the experiment? | -the glycerol is heavier than the running buffer solution -as a result, it helps to weigh down the restriction digests within the wells | 49 | |
| 782937731 | What is the function of the pigment in the experiment? | -the pigments tell us where our restriction digests are within the agarose gel so that we can turn off the electric current before our fragments run off the end of the plate | 50 | |
| 782937732 | How many different buffers did we use in this experiment? | -three | 51 | |
| 782937733 | Why did we add buffer to every restriction digest tube? | -the addition of buffer helps to limit changes to the pH of the solution -enzymes function at a very specific pH -thus, the addition of buffer helps to ensure that our enzymes remain functional | 52 | |
| 782937734 | Why did we add buffer when we cast our electrophoresis gel? Why not just use water? | -the buffer has a pH of 8, and is therefore able to remove hydrogen atoms from the phosphate groups within DNA -this leaves the DNA negatively charged and capable of moving toward the anode within the electric field -we cannot use pure water because it has a pH of 7 -therefore, it is unable to remove hydrogen atoms from the phosphate groups of DNA | 53 | |
| 782937735 | Why is ethidium bromide (EtBr) added to the agarose gel? | -to stain DNA | 54 | |
| 782937736 | Why is EtBr considered to be a hazardous material? | -ethidium cation unwinds DNA, creating openings where it can insert itself into the strand -these structural changes can result in functional modifications, including the inhibition of transcription, replication, and repair processes -as a result, EtBr is a suspected carcinogen, mutagen, and teratogen | 55 | |
| 782937737 | What is a teratogen? | -an agent that causes the production of physical defects in the developing embryo. | 56 | |
| 782937738 | How does EtBr stain DNA? | -EtBr is a planar molecule -as a result, it is capable of unwinding DNA and creating openings where it can insert itself into the strand -this process is known as intercalation -after intercalation occurs, exposure to UV light causes the DNA to fluoresce with an orange color, thus allowing it to be viewed within an agarose gel | 57 | |
| 782937739 | Why did we use a running buffer in the electrophoresis chamber? Why didn't we just use water? | -buffer provides ions that are capable of carrying a current -buffer offers a path of least resistance -->water is not capable of generating a current, water offers a path of greater resistance | 58 | |
| 782937740 | In order to run our electrophoresis chambers, why did we cover the gels with just a little buffer; why not use quite a lot? | -if we used a large amount of buffer, the current would pass through the buffer itself--not the agarose gel -a thin layer helps to ensure that the current runs through the agarose gel, which results in separation of the DNA fragments | 59 | |
| 782937741 | How do the pigments in the loading dye tell you when to stop the current? | -one of the pigments travels at the same rate as the smallest fragment, and one travels at the same rate as the largest fragment | 60 | |
| 782937742 | How long did we electrophorese the digests for? | -until the leading pigment ran down the gel to between the numbers four and five | 61 | |
| 782937743 | What would happen if we ran the gel with the electrodes reversed? | -if the electrodes were reversed, then the electric field would be reversed as well -the DNA would no longer migrate to the bottom of the gel; instead, it would run out of the top of the gel into the buffer solution -the sample would most likely be lost | 62 | |
| 782937744 | What causes smearing within the gels? | -the production of very small fragments that are similar in length -because of their similarity in length, they appear very close together within a gel, and may be indistinguishable | 63 | |
| 782937745 | What causes smearing? | -contamination -incorrect buffer concentration -too much running buffer -DNA run too fast or too slow -gel not the right thickness | 64 | |
| 782937746 | What are two reasons that extra bands may appear in some of the lanes? | 1. small amounts of DNA might not have been completely digested by the restriction enzymes; as a result, these bands may be longer or shorter than the brightest bands within the lane 2. a sample may have spilled or leaked over from one well into a neighbor well | 65 | |
| 782937747 | What organism does "EcoR1" come from? | -E. coli | 66 | |
| 782937748 | What organism does HincII come from? | -Haemophilus influenzae | 67 | |
| 782937749 | What organism does PvuII come from? | -Proteus vulgaris | 68 | |
| 782937750 | Do enzymes work best at a high or low heat? | -high heat | 69 | |
| 782937751 | What is agarose made from? | -seaweed | 70 | |
| 782937752 | What equation describes the relationship between the distance traveled by a piece of DNA and the number of base pairs? | distance traveled ∝ 1/log of base pairs | 71 | |
| 782956115 | When performing digest problems, what does "bright" indicate? | -the presence of two pieces of DNA of the same length | 72 | |
| 782956116 | What does "very bright" indicate? | -the presence of three pieces of DNA of the same length | 73 | |
| 782956117 | What does "very very bright" indicate? | -the presence of four pieces of DNA of the same length | 74 | |
| 783066081 | When drawing a restriction enzyme map, what should always be included at the center of the plasmid? | -the plasmid name and size (in base pairs) | 75 |
