BIOLOGY 283 Chapter Notes - Chapter 14.2-6: Reverse Transcription Polymerase Chain Reaction, Reverse Transcriptase, Cloning
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Why does a template-dependent DNA polymerase require a primer to initiate DNA synthesis?
DNA polymerase require 5' phosphate to add a new nucleotide | |
DNA polymerase require 3' hydroxyl group to add a new nucleotide |
DNA polymerase require energy by hydrolizing the primers |
Which of the following enzymes is template independent?
Reverse transcriptase | |
Klenow fragment |
DNA polymerase | |
Terminal deoxynucleotidyl transferase |
RNA polymerase |
The natural function of a DNA ligase enzyme is:
Joining together DNA sequences with compatible cohesive ends from two different organisms to yield a recombinant DNA molecule | |
Sealing single stranded nicks in double stranded DNA molecules |
Joining together two blunt ended fragments of DNA within a cell | |
Joining together two single stranded DNA molecules |
The natural function of 3' -> 5' exonuclease activity of a DNA polymerase is to:
Remove the 5' end of the DNA strand that is being copied | |
Remove damaged nucleotide from the template strand |
Remove nucleotides from the end of DNA to generate blunt ends | |
Remove incorrect nucleotides from the newly synthesized DNA |
Which technique is used to resolve the different sizes of DNA fragments?
Gene cloning | |
PCR |
DNA sequencing | |
Gel electrophoresis |
Which of the following vectors does not contain bacterial origin of replication (oriC)?
plasmid | |
cosmid |
bacterial artificial chromosome | |
lambda vector |
Which process of bacteriophage is not used for gene cloning?
Concatemer formation | |
Lytic cycle |
Lysogenic cycle | |
viral packaging |
What vector would be best suited for creating a contig of bovine (cattle) chromosome 10?
lambda vector | |
Cosmid vector |
P1 vector | |
Plasmid |
E. coli takes up plasmid DNA by which of the following methods?
Transduction | |
Transformation |
Conjugation | |
Transfection |
The following times and temperatures are an example of the steps for PCR. You can use the Figure to help you answer the following questions.
Why is the first step is carried out at 94°C?
To degrade the template | |
To denature the template |
To activate the polymerase | |
To facilitate the primer binding |
What happens in the reaction when the temperature shifts to 55°C during cycling?
the DNA polymerase will carry out DNA synthesis by extending the annealed primers | |
the DNA polymerase will finish DNA synthesis |
the primers anneal to the single-stranded regions of the DNA | |
the primers anneal to the double-stranded regions of the DNA |
During cycling, what occurs when the temperature is at 72°C?
the primers anneal to the double-stranded regions of the DNA | |
the DNA polymerase will carry out DNA synthesis by extending the annealed primers |
the primers anneal to the single-stranded regions of the DNA | |
the DNA polymerase will finish DNA synthesis |
You designed a set of primers to amplify 1 kb DNA with polymerase chain reaction. By which cycle of polymerase chain reaction, we would expect to see the first double stranded DNA with expected size?
The end of cycle 1 | |
The end of cycle 2 |
The end of cycle3 | |
The end of cycle 4 |
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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