Biochemistry 2280A Lecture Notes - Chain Termination, Restriction Map, Ampicillin
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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 of recombinant DNA intobacteria. |
surfaces for respiratory processes in bacteria. |
recognition sites on recombinant DNA strands. |
surfaces for protein synthesis in eukaryotic recombinants. |
proviruses incorporated into the host DNA |
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Plasmids are put into bacterial cells by
restriction enzymes |
DNA ligase |
binding of cohesive sticky ends |
transformation |
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Restriction enzymes usually
cut donor DNA evenly so smooth edges result |
cut donor DNA but do not affect plasmids |
make staggered cuts at specific sequences in DNA in both donorDNA and plasmid |
are used in ligating plasmids into bacterial host cells |
more than one of the above |
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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 have ribosomes. |
All organisms have the same genetic code. |
All organisms are made up of cells. |
All organisms have similar nuclei. |
All organisms have transfer RNA. |
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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 with restriction enzyme X and insert thefragments cut with Y into the plasmid. |
cut the plasmid with enzyme X and then insert the gene into theplasmid. |
cut the DNA again with restriction enzyme Y and insert thesefragments into the plasmid cut with the same enzyme. |
cut the plasmid twice with restriction enzyme Y and ligate thetwo fragments onto the ends of the human DNA fragments cut withrestriction enzyme X. |
insert the fragments cut with X directly into the plasmidwithout 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 proteins using a cloned gene. |
They contain a promotor. |
They are the first plasmid type used to clone a gene. |
They contain a terminator. |
More than one of the above is false. |
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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 gene contains introns the bacteria will notremove them and the resulting amino acid sequence will be differentthat that made by a eukaryote. |
The bacteria may not fold the protein correctly. |
The bacteria may degrade the protein. |
All of the above are potential problems. |
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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 identical animals (e.g. Dolly thesheep) |
Make vaccines |
Perform Gene Therapy |
Making genetically engineered plants |
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