The restriction enzyme EcoR1 makes staggered cuts at 6 nucleotide palindromic sequences while the enzyme HaeIII makes blunt end cuts in middle of 4 nucleotide sequences. If a given DNA sample is digested by these two enzymes in separate reactions which one would generate more fragments? Explain your reasoning.
The restriction enzyme EcoR1 makes staggered cuts at 6 nucleotide palindromic sequences while the enzyme HaeIII makes blunt end cuts in middle of 4 nucleotide sequences. If a given DNA sample is digested by these two enzymes in separate reactions which one would generate more fragments? Explain your reasoning.
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We have four DNA sequences that we need to digest (cut up), and have 6 restriction enzymes in stock in our lab. We want to find out which restriction enzyme we can use on each sample.
For each sequence, please indicate which restriction enzyme(s) would cut within each sequence. Each sequence may be cut by one, more than one, or no restriction enzymes.
Following the sequences are two questions. Please choose from the same list of enzymes to indicate the correct answer to these questions.
| Restriction enzyme | Digestion pattern |
| EcoRI | 5' G|AATTC 3' 3' CTTAA|G 5' |
| HindIII | 5' A|AGCTT 3' 3' TTCGA|A 5' |
| BamHI | 5' G|GATCC 3' 3' CCTAG|G 5 |
| AluI | 5' AG|CT 3' 3' TC|GA 5' |
| SmaI | 5' CCC|GGG 3' 3' GGG|CCC 5' |
| HbaI | 5' GCG|C 3' 3' C|GCG 5' |
(The | symbol indicates the specific cut sites.)
Each answer may be used once, more than once, or not at all.
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View/perform/read ALL THREE of the following prior to answeringthe questions.
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)
http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)
http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)
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From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA
| IV, V, I, II, III |
| III, II, IV, V, I |
| III, IV, V, I, II |
| II, III, V, IV, I |
| I, II, IV, III, V |
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Plasmids (or vectors) are important in biotechnology becausethey are
| a vehicle for the insertion ofrecombinant DNA into bacteria. |
| surfaces for respiratoryprocesses in bacteria. |
| recognition sites onrecombinant DNA strands. |
| surfaces for protein synthesisin eukaryotic recombinants. |
| proviruses incorporated intothe host DNA |
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Plasmids are put into bacterial cells by
| restriction enzymes |
| DNA ligase |
| binding of cohesive stickyends |
| transformation |
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Restriction enzymes usually
| cut donor DNA evenly so smoothedges result |
| cut donor DNA but do notaffect plasmids |
| make staggered cuts atspecific sequences in DNA in both donor DNA and plasmid |
| are used in ligating plasmidsinto bacterial host cells |
| more than one of theabove |
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After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.
| Restriction enzyme |
| DNA Ligase |
| Reverse transcriptase |
| DNA polymerase |
| Helicase |
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It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?
| All organisms haveribosomes. |
| All organisms have the samegenetic code. |
| All organisms are made up ofcells. |
| All organisms have similarnuclei. |
| All organisms have transferRNA. |
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Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to
| cut the plasmid withrestriction enzyme X and insert the fragments cut with Y into theplasmid. |
| cut the plasmid with enzyme Xand then insert the gene into the plasmid. |
| cut the DNA again withrestriction enzyme Y and insert these fragments into the plasmidcut with the same enzyme. |
| cut the plasmid twice withrestriction enzyme Y and ligate the two fragments onto the ends ofthe human DNA fragments cut with restriction enzyme X. |
| insert the fragments cut withX directly into the plasmid without cutting the plasmid. |
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Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?
| They are used to make proteinsusing a cloned gene. |
| They contain a promotor. |
| They are the first plasmidtype used to clone a gene. |
| They contain aterminator. |
| More than one of the above isfalse. |
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What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?
| If the eukaryotic genecontains introns the bacteria will not remove them and theresulting amino acid sequence will be different that that made by aeukaryote. |
| The bacteria may not fold theprotein correctly. |
| The bacteria may degrade theprotein. |
| All of the above are potentialproblems. |
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Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.
| True |
| False |
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Question 111 pts
Gene cloning is used to do all of the following except
| Make insulin |
| Making genetically identicalanimals (e.g. Dolly the sheep) |
| Make vaccines |
| Perform Gene Therapy |
| Making genetically engineeredplants |
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