BIOMG 3300 - Unit 9 Study Questions
Solved 100% Correct
Describe the five general procedures in DNA cloning
a. Obtaining the DNA segment to be cloned
b. Selecting a small molecule of DNA capable of autonomous
replication c. Joining two DNA fragments
d. Moving recombinant DNA from the test tube to the host organism e.
Selecting or identifying host cells that contain recombinant DNA
Thousands of restriction endonucleases have been discovered. Are
restriction enzymes made by eukaryotes or prokaryotes? What is their function in vivo?
Restriction enzymes are made by prokaryotes, their function in vivo is to cleave DNA
molecules at specific base sequences,
Name a restriction endonuclease that
generates sticky ends and one that generates blunt ends.
Sticky End Endonuclease: EcoRI, Blunt End Endonuclease: PvuII
When two DNA fragments generated by the same restriction endonuclease are mixed
together, the complementary ends pair and form hydrogen bonds (a process called
annealing), DNA ligase then forms phosphodiester bonds that covalently join the fragments,
and the restriction endonuclease cleavage site is restored.
DNA ligase seals the nicks that are present once the two pieces of DNA recombine
Define plasmid
a circular DNA molecule that replicates separately from the host chromosome
Define transformation
the introduction of small plasmids into bacterial cells
Why do plasmids always contain the selectable marker gene?
, They contain an antibiotic resistance in order to make it easier to have a population of only
bacteria that have taken up the plasmid. This is done by putting in this antibiotic resistance
gene and then having plating the bacteria on the antibiotic so only cells with the plasmid
survive
Why do plasmids always contain the origin of replication?
The origin of replication is the sequence where replication of the plasmid is initiated by
cellular enzymes. It is required for the plasmid to be able to be replicated.
Why do plasmids always contain unique recognition sequences for restriction enzymes?
The unique recognition sequences are required in order to incorporate the DNA of interest
into the plasmid in order to perform transformation.
Illustrate how plasmids are used to clone
DNA.
- Plasmids are first cleaved by a restriction endonuclease
- Then the foreign DNA cleaved by the same endonuclease will combine with the Plasmid
- DNA ligase seals them up
- The cells of interest are transformed then grown on agar plates with compounds that help
the selectivity of the bacteria
Discuss the importance of each region of the
plasmid (polylinker sequence, P site, O site, ori).
Polylinker sequence is the place with a bunch of restriction enzyme sites where our gene will
be inserted, the P site is where RNA polymerase binds, the O site is where the repressor binds,
the ori is the origin of replication
Discuss the restriction-modification system
In site directed mutagenesis, restriction endonucleases are used in order to splice the plasmid
and insert the new piece of DNA in, whereas in oligonucleotide-directed mutagenesis, the
plasmid is denatured and the plasmid is annealed with the mutation, after this the DNA
polymerase replicates the rest of the plasmid and the parental strands are cleaved so the newly
formed strands combine to one another
Why is it necessary to have a promoter in the expression vector when using cDNA inserts?
Discuss why is it desirable to have a regulatable promoter in an expression vector.
If the promoter is not regulated, it can kill the cell by making a ton of it. This is bad.
Distinguish oligonucleotide directed mutagenesis (b) from the synthetic insert method (a).
Describe how DNA primers and probes of a specific sequence are synthesized.
Solved 100% Correct
Describe the five general procedures in DNA cloning
a. Obtaining the DNA segment to be cloned
b. Selecting a small molecule of DNA capable of autonomous
replication c. Joining two DNA fragments
d. Moving recombinant DNA from the test tube to the host organism e.
Selecting or identifying host cells that contain recombinant DNA
Thousands of restriction endonucleases have been discovered. Are
restriction enzymes made by eukaryotes or prokaryotes? What is their function in vivo?
Restriction enzymes are made by prokaryotes, their function in vivo is to cleave DNA
molecules at specific base sequences,
Name a restriction endonuclease that
generates sticky ends and one that generates blunt ends.
Sticky End Endonuclease: EcoRI, Blunt End Endonuclease: PvuII
When two DNA fragments generated by the same restriction endonuclease are mixed
together, the complementary ends pair and form hydrogen bonds (a process called
annealing), DNA ligase then forms phosphodiester bonds that covalently join the fragments,
and the restriction endonuclease cleavage site is restored.
DNA ligase seals the nicks that are present once the two pieces of DNA recombine
Define plasmid
a circular DNA molecule that replicates separately from the host chromosome
Define transformation
the introduction of small plasmids into bacterial cells
Why do plasmids always contain the selectable marker gene?
, They contain an antibiotic resistance in order to make it easier to have a population of only
bacteria that have taken up the plasmid. This is done by putting in this antibiotic resistance
gene and then having plating the bacteria on the antibiotic so only cells with the plasmid
survive
Why do plasmids always contain the origin of replication?
The origin of replication is the sequence where replication of the plasmid is initiated by
cellular enzymes. It is required for the plasmid to be able to be replicated.
Why do plasmids always contain unique recognition sequences for restriction enzymes?
The unique recognition sequences are required in order to incorporate the DNA of interest
into the plasmid in order to perform transformation.
Illustrate how plasmids are used to clone
DNA.
- Plasmids are first cleaved by a restriction endonuclease
- Then the foreign DNA cleaved by the same endonuclease will combine with the Plasmid
- DNA ligase seals them up
- The cells of interest are transformed then grown on agar plates with compounds that help
the selectivity of the bacteria
Discuss the importance of each region of the
plasmid (polylinker sequence, P site, O site, ori).
Polylinker sequence is the place with a bunch of restriction enzyme sites where our gene will
be inserted, the P site is where RNA polymerase binds, the O site is where the repressor binds,
the ori is the origin of replication
Discuss the restriction-modification system
In site directed mutagenesis, restriction endonucleases are used in order to splice the plasmid
and insert the new piece of DNA in, whereas in oligonucleotide-directed mutagenesis, the
plasmid is denatured and the plasmid is annealed with the mutation, after this the DNA
polymerase replicates the rest of the plasmid and the parental strands are cleaved so the newly
formed strands combine to one another
Why is it necessary to have a promoter in the expression vector when using cDNA inserts?
Discuss why is it desirable to have a regulatable promoter in an expression vector.
If the promoter is not regulated, it can kill the cell by making a ton of it. This is bad.
Distinguish oligonucleotide directed mutagenesis (b) from the synthetic insert method (a).
Describe how DNA primers and probes of a specific sequence are synthesized.
