MCB 2050 MIDTERM QUESTIONS WITH VERIFIED
ANSWERS
1) What are restriction endonucleases?
2) What do they do (or cut) specificically?
3) What happens to the ends of DNA cut by restriction enzymes? - Answers - 1)
enzymes that cut at specific sequences (restriction sites) within DNA molecules
2) they break phosphodiester bonds that link nucleotides together
3) have single-stranded overhangs; ends are cohesive (complementary, sticky)- they
can reanneal
1) Who discovered restriction enzymes?
2) What do restriction enzymes do for bacteria and how?
3) How is the bacterial genome protected from restriction enzyme degradation? What is
this catalyzed by? - Answers - 1) Smith and Nathans
2) protect genetic material of bacteria from invasion by foreign DNA (viruses) by
restricting their intrusion
3) by methylation of nucleotides within the sequence recognized by the restriction
enzymes; specific methylases after DNA replication
1) How are restriction endonucleases named? Give an example.
2) What do restriction enzymes generally recognize? What do they create as a result?
3) What is a palindrome? Give an example. - Answers - 1) According to the bacterial
strain in which it was originally identified; EcoR1: E.coli strain RY13 restriction enzyme
number 1.
2) palindromic sequences; either staggered (aka cohesive, sticky) or blunt ends
3) a sequence that reads the same in either direction; the DNA is identical but inverted
in a complementary strand
What kind of ends do the following produce:
Sma1
Kpn1
BamH1 - Answers - blunt
3'overhang
5'overhang
How does DNA ligase create a recombinant DNA molecule? - Answers - DNA ligase
joins(ligates) the complementary single stranded ends produced by restriction enzyme
cleavage
What is the generic name of enzymes that recognize palindromic sequences in dsDNA
and cleave the DNA at that location? - Answers - restriction endonucleases
,The kind of ends following restriction endonuclease digestion that results in single
stranded DNA overhangs are called: - Answers - cohesive (or sticky) ends
1) What are cloning vectors used for?
2) Give five elements of cloning vectors
3) What are the three essential components of plasmid vectors? - Answers - 1) isolation
and amplification of DNA sequences
2) -mostly originated from bacteria (some from yeast)
-double-stranded circular DNA
-extra-chromosomal
-replicate independent of cell DNA (plasmids can be multi-copy: ie >1 plasmid/cell)
-small, up to about 10kb (for easy manipulation; maximum insert size usually approx
15kilobase pairs)
3) -an origin of DNA replication (for amplification in bacterial cells)
-a selectable marker: antibiotic resistance gene
-at least one unique RE cleavage site outside of the origin and resistance gene (many
have multiple cloning sites, MCS)
What are the characteristics of the pBluescript cloning vector? - Answers - -bacterial
origin of replication (to allow plasmid replication)
-multiple cloning site (MCS) to allow insertion of foreign DNA
-antibiotic resistance gene for selection of bacteria transformed with the plasmid
-selectable marker (lacZ) to differentiate cells with plasmids containing inserted foreign
DNA from those without (blue/white screening)
-lac promoter to express mRNA (small (3kb) to allow for large insertions)
-all RE sites in the MCS are unique sites in the plasmid
1) What does the bluescript plasmid express?
2) Where is the MCS located?
3) What does insertion of foreign DNA into the MCS do? What is this called? - Answers
- 1) the E.coli lacZ gene (encodes beta-galactosidase)
2) within the plasmid encoded lacZ gene (actually a portion of lacZ)
3) it disrupts the reading frame of the lacZ gene; "insertional inactivation"
1) What is the bluescript blue/white screening strategy called?
2) How is the E.coli lacZ gene involved in this?
3) Describe what happens to a colony containing a plasmid without the insert.
4) Describe what happens to a colony containing a plasmid with the insert. - Answers -
1) insertional inactivation
2) it encodes beta-galactosidase which convert the colourless substrate Xgal into a blue
product
3)- functional lacZ gene
-functional beta-galactosidase enzymes convert X-gal (colourless) to a blue coloured
product
-colonies are blue
4) -non functional lacZ gene
,-functional beta-galactosidase enzyme not made
-colonies are white
1) What are expression plasmids designed for?
2) What must they be able to do?
3) What must they contain (3)? - Answers - 1) the expression of proteins in bacteria,
yeast, plant, or animals cells
2) replicate in bacteria in order to amplify the plasmid
3) -a selectable marker to identify bacteria containing the plasmid
-a cloning site located downstream from a promoter appropriate for the host cell to allow
the inserted gene to be expressed
-a selectable marker (antibiotic resistance gene) appropriate for the host cell to allow for
selection of cells that have incorporated the plasmid in their genome
1) Do promoters have directionality?
2) What must eukaryotic expression plasmids contain? - Answers - 1) Yes
2) a promoter upstream of the MCS and a polyA signal downstream of the MCS
1) What does MCS allow for?
2) What do reporter genes code for?
3) What are reporter genes used for?
4) What does luc+=?
5) What is done to the luciferase protein? (2)
6) What is the luciferase protein proportional to?
7) Does the reporter gene have a promoter to control its expression?
8) What happens at the MCS?
9) What is the reporter gene called in this example? - Answers - 1) the insertion of
potential gene regulatory sequences
2) a protein that can be visualized or measured
3) to study gene regulatory sequences after transfection into cells
4) the firefly luciferase gene
5) -it is assayed (converts luciferin to light)
-that light can be measured in a luminometer
6) [light] proportional to [luciferase protein] proportional [luciferase mRNA] which is
proportional to transcriptional activity
7) No
8) insert gene regulatory sequences here
9) firefly luciferase (luc)
Compare the reporter plasmid and expression plasmid (2 for expression, three for
reporter) - Answers - Reporter Plasmid:
-the reporter gene does not have a promoter to control its expression
-MCS: insert regulatory gene sequences here
-the expression level of the reporter gene will tell you the efficiency of the regulatory
sequences that you inserted
Expression Plasmid:
, -promoter to control expression of your gene of interest
-MCS: insert gene of interest (the gene you want to express) here
1) Describe cloning plasmids and give an example.
2) Describe expression plasmids and give an example.
3) Describe reporter plasmids and give an example. - Answers - 1) -for isolation and
amplification of DNA sequences
-contains (i) origin of replication, (ii) selectable marker and (iii) MCS
-example: pBluescript (also has blue/white selection for plasmids without/with inserts)
2) -for the expression of proteins in bacteria, yeast, plant or animal cells
-contains promoter appropriate for target cell located upstream of MCS
-also contains elements found in cloning vector (for cloning, amplification of E.coli)
-example: pCMV, a mammalian cell expression vector
3) -for the measurement of gene regulatory sequences
-contains a reporter gene that when expressed can be quantitatively measured
-MCS is located upstream of the reporter gene
-also contains elements found in cloning vector
-example: pGL3 (contains the luciferase reporter gene)
The name of the part of a cloning vector that has several restriction enzyme sites is
called? - Answers - Multiple Cloning Site (MCS)
In the Bluescript cloning plasmid, the MCS is located within the reading frame of what
gene? - Answers - beta-galactosidase (lacZ)
Bacteria containing the Bluescript plasmid that do not have foreign DNA inserted in the
MCS of the plasmid will be what colour when grown on agar plates containing X-gal? -
Answers - Blue
The kind of plasmid you would use to produce a foreign protein in cells containing the
plasmid is called? - Answers - expression vector
What are the major steps in the construction and screening of DNA libraries and what
happens in each? - Answers - 1) Digestion of vector
-cut vector with restriction enzyme within the multiple cloning site
2) Digestion of genomic DNA
-cut with same restriction enzyme used to cut vector
3) Annealing and ligation
-allow the compatible sticky ends of vector and target DNAs to base pair with each other
-treated annealed DNA with ligase to seal the 'nicks' (broken phosphodiester linkage
between two adjacent nucleotides on the same DNA strand)
4) Transformation
-of E.coli (transfer the DNA into E.coli cells)
5) Selection
-grow the transformed cells on selective media appropriate for the vector (eg selection
media containing ampicillin)
ANSWERS
1) What are restriction endonucleases?
2) What do they do (or cut) specificically?
3) What happens to the ends of DNA cut by restriction enzymes? - Answers - 1)
enzymes that cut at specific sequences (restriction sites) within DNA molecules
2) they break phosphodiester bonds that link nucleotides together
3) have single-stranded overhangs; ends are cohesive (complementary, sticky)- they
can reanneal
1) Who discovered restriction enzymes?
2) What do restriction enzymes do for bacteria and how?
3) How is the bacterial genome protected from restriction enzyme degradation? What is
this catalyzed by? - Answers - 1) Smith and Nathans
2) protect genetic material of bacteria from invasion by foreign DNA (viruses) by
restricting their intrusion
3) by methylation of nucleotides within the sequence recognized by the restriction
enzymes; specific methylases after DNA replication
1) How are restriction endonucleases named? Give an example.
2) What do restriction enzymes generally recognize? What do they create as a result?
3) What is a palindrome? Give an example. - Answers - 1) According to the bacterial
strain in which it was originally identified; EcoR1: E.coli strain RY13 restriction enzyme
number 1.
2) palindromic sequences; either staggered (aka cohesive, sticky) or blunt ends
3) a sequence that reads the same in either direction; the DNA is identical but inverted
in a complementary strand
What kind of ends do the following produce:
Sma1
Kpn1
BamH1 - Answers - blunt
3'overhang
5'overhang
How does DNA ligase create a recombinant DNA molecule? - Answers - DNA ligase
joins(ligates) the complementary single stranded ends produced by restriction enzyme
cleavage
What is the generic name of enzymes that recognize palindromic sequences in dsDNA
and cleave the DNA at that location? - Answers - restriction endonucleases
,The kind of ends following restriction endonuclease digestion that results in single
stranded DNA overhangs are called: - Answers - cohesive (or sticky) ends
1) What are cloning vectors used for?
2) Give five elements of cloning vectors
3) What are the three essential components of plasmid vectors? - Answers - 1) isolation
and amplification of DNA sequences
2) -mostly originated from bacteria (some from yeast)
-double-stranded circular DNA
-extra-chromosomal
-replicate independent of cell DNA (plasmids can be multi-copy: ie >1 plasmid/cell)
-small, up to about 10kb (for easy manipulation; maximum insert size usually approx
15kilobase pairs)
3) -an origin of DNA replication (for amplification in bacterial cells)
-a selectable marker: antibiotic resistance gene
-at least one unique RE cleavage site outside of the origin and resistance gene (many
have multiple cloning sites, MCS)
What are the characteristics of the pBluescript cloning vector? - Answers - -bacterial
origin of replication (to allow plasmid replication)
-multiple cloning site (MCS) to allow insertion of foreign DNA
-antibiotic resistance gene for selection of bacteria transformed with the plasmid
-selectable marker (lacZ) to differentiate cells with plasmids containing inserted foreign
DNA from those without (blue/white screening)
-lac promoter to express mRNA (small (3kb) to allow for large insertions)
-all RE sites in the MCS are unique sites in the plasmid
1) What does the bluescript plasmid express?
2) Where is the MCS located?
3) What does insertion of foreign DNA into the MCS do? What is this called? - Answers
- 1) the E.coli lacZ gene (encodes beta-galactosidase)
2) within the plasmid encoded lacZ gene (actually a portion of lacZ)
3) it disrupts the reading frame of the lacZ gene; "insertional inactivation"
1) What is the bluescript blue/white screening strategy called?
2) How is the E.coli lacZ gene involved in this?
3) Describe what happens to a colony containing a plasmid without the insert.
4) Describe what happens to a colony containing a plasmid with the insert. - Answers -
1) insertional inactivation
2) it encodes beta-galactosidase which convert the colourless substrate Xgal into a blue
product
3)- functional lacZ gene
-functional beta-galactosidase enzymes convert X-gal (colourless) to a blue coloured
product
-colonies are blue
4) -non functional lacZ gene
,-functional beta-galactosidase enzyme not made
-colonies are white
1) What are expression plasmids designed for?
2) What must they be able to do?
3) What must they contain (3)? - Answers - 1) the expression of proteins in bacteria,
yeast, plant, or animals cells
2) replicate in bacteria in order to amplify the plasmid
3) -a selectable marker to identify bacteria containing the plasmid
-a cloning site located downstream from a promoter appropriate for the host cell to allow
the inserted gene to be expressed
-a selectable marker (antibiotic resistance gene) appropriate for the host cell to allow for
selection of cells that have incorporated the plasmid in their genome
1) Do promoters have directionality?
2) What must eukaryotic expression plasmids contain? - Answers - 1) Yes
2) a promoter upstream of the MCS and a polyA signal downstream of the MCS
1) What does MCS allow for?
2) What do reporter genes code for?
3) What are reporter genes used for?
4) What does luc+=?
5) What is done to the luciferase protein? (2)
6) What is the luciferase protein proportional to?
7) Does the reporter gene have a promoter to control its expression?
8) What happens at the MCS?
9) What is the reporter gene called in this example? - Answers - 1) the insertion of
potential gene regulatory sequences
2) a protein that can be visualized or measured
3) to study gene regulatory sequences after transfection into cells
4) the firefly luciferase gene
5) -it is assayed (converts luciferin to light)
-that light can be measured in a luminometer
6) [light] proportional to [luciferase protein] proportional [luciferase mRNA] which is
proportional to transcriptional activity
7) No
8) insert gene regulatory sequences here
9) firefly luciferase (luc)
Compare the reporter plasmid and expression plasmid (2 for expression, three for
reporter) - Answers - Reporter Plasmid:
-the reporter gene does not have a promoter to control its expression
-MCS: insert regulatory gene sequences here
-the expression level of the reporter gene will tell you the efficiency of the regulatory
sequences that you inserted
Expression Plasmid:
, -promoter to control expression of your gene of interest
-MCS: insert gene of interest (the gene you want to express) here
1) Describe cloning plasmids and give an example.
2) Describe expression plasmids and give an example.
3) Describe reporter plasmids and give an example. - Answers - 1) -for isolation and
amplification of DNA sequences
-contains (i) origin of replication, (ii) selectable marker and (iii) MCS
-example: pBluescript (also has blue/white selection for plasmids without/with inserts)
2) -for the expression of proteins in bacteria, yeast, plant or animal cells
-contains promoter appropriate for target cell located upstream of MCS
-also contains elements found in cloning vector (for cloning, amplification of E.coli)
-example: pCMV, a mammalian cell expression vector
3) -for the measurement of gene regulatory sequences
-contains a reporter gene that when expressed can be quantitatively measured
-MCS is located upstream of the reporter gene
-also contains elements found in cloning vector
-example: pGL3 (contains the luciferase reporter gene)
The name of the part of a cloning vector that has several restriction enzyme sites is
called? - Answers - Multiple Cloning Site (MCS)
In the Bluescript cloning plasmid, the MCS is located within the reading frame of what
gene? - Answers - beta-galactosidase (lacZ)
Bacteria containing the Bluescript plasmid that do not have foreign DNA inserted in the
MCS of the plasmid will be what colour when grown on agar plates containing X-gal? -
Answers - Blue
The kind of plasmid you would use to produce a foreign protein in cells containing the
plasmid is called? - Answers - expression vector
What are the major steps in the construction and screening of DNA libraries and what
happens in each? - Answers - 1) Digestion of vector
-cut vector with restriction enzyme within the multiple cloning site
2) Digestion of genomic DNA
-cut with same restriction enzyme used to cut vector
3) Annealing and ligation
-allow the compatible sticky ends of vector and target DNAs to base pair with each other
-treated annealed DNA with ligase to seal the 'nicks' (broken phosphodiester linkage
between two adjacent nucleotides on the same DNA strand)
4) Transformation
-of E.coli (transfer the DNA into E.coli cells)
5) Selection
-grow the transformed cells on selective media appropriate for the vector (eg selection
media containing ampicillin)
