MICR 290 ACTUAL EXAM FULL QUESTIONS
AND CORRECT ANSWERS GRADED A PLUS
●● What are the correct features of a plasmid?
Answer: Circular, double-stranded DNA present in the bacterial
cytoplasm
Many plasmids carry genes that confer antibiotic resistance
●● Why are plasmids useful?
Answer: Plasmids are extremely valuable tools for biotechnology
applications, and recombinant plasmids are used in many different areas
of research. By inserting a sequence of DNA into a plasmid and
transforming a bacterial culture with this recombinant DNA, the culture
can then be used as a "factory" to produce a massive number of copies
of the plasmid. This is one way to produce copies of the DNA sequence
that was inserted into the plasmid (i.e. the insert). The resulting plasmid
can then be purified from the bacterial culture (e.g. using spin columns)
and used in down-stream applications.
●● What are vectors?
Answer: The plasmids used in the lab for cloning purposes are known as
vectors, as they act as "vectors" for the insert that they carry.
,Many of the plasmids used for research purposes are artificial constructs,
combining DNA sequences from several different sources (including
natural bacterial plasmids).
Most empty vectors are relatively small, typically ranging from 2-5 kb
(kilobases) in size; this contrasts natural plasmids, which are often 10-
100 kb. Cloning vectors can be considered to be "stripped down"
versions of natural plasmids, consisting principally of the core
components required for plasmid replication and selection. As long as
these components are not disrupted, it is possible to clone almost any
insert DNA sequence into a plasmid.
●● What are the 3 components found in most cloning vectors?
Answer: 1. Multiple Cloning Site (MCS):
This region of DNA is designed to simplify the process of cloning an
insert into the vector. This region is recognized by several different
restriction enzymes, enzymes which cut DNA. The insert can then be
enzymatically connected to the cut vector.
2. Origin of Replication (Ori):
This sequence of DNA is required for the plasmid to be replicated by
bacteria. This sequence is recognized by the bacterial replication
machinery (i.e. enzymes that replicate DNA), which produce more
copies of the plasmid. The origin of replication plays a role in
determining the copy number of a plasmid, which determines the
number of copies of the plasmid in a cell.
,3. Selectable Marker:
This is a gene that makes it possible to selectively culture the bacteria
that contain the plasmid. Selectable markers tend to be genes that confer
antibiotic resistance. Therefore, in the presence of the antibiotic, bacteria
transformed with the plasmid will survive and bacteria which lack the
plasmid will die.
●● How can we clone insert DNA into a vector?
Answer: A common approach using restriction enzymes and an enzyme
known as DNA ligase.
In this process, the empty vector is cut open using restriction enzymes,
and the insert is connected to the cut vector by DNA ligase.
●● What are restriction enzymes?
Answer: Restriction enzymes, or restriction endonucleases, are enzymes
that cleave phosphodiester linkages, breaking the bond between the
phosphorus atom of the phosphate group and the oxygen of the
deoxyribose sugar. This effectively cuts a strand of DNA into two
smaller strands.
*Very selective: only cleave DNA at these sites
, Different restriction enzymes have different recognition sites (can be
used for cloning purposes)
●● What is the ECOR1 restriction enzyme?
Answer: Recognizes this DNA sequence:
5' - GAATTC - 3'
ECOR1 cutes between G and A: 5' - G - 3' and 5' -AATTC - 3'
●● What does it mean when a recognition site is an inverted repeat
palindrome?
Answer: The sequence is the same on both strands (because DNA is
double stranded).
Example: both strands have the ECOR1 site.
5' - GAATTC - 3'
3' - CTTAAG - 3'
So ECOR1 cleaves between G and A on both the top and bottom strand"
5' - G - 3'
AND CORRECT ANSWERS GRADED A PLUS
●● What are the correct features of a plasmid?
Answer: Circular, double-stranded DNA present in the bacterial
cytoplasm
Many plasmids carry genes that confer antibiotic resistance
●● Why are plasmids useful?
Answer: Plasmids are extremely valuable tools for biotechnology
applications, and recombinant plasmids are used in many different areas
of research. By inserting a sequence of DNA into a plasmid and
transforming a bacterial culture with this recombinant DNA, the culture
can then be used as a "factory" to produce a massive number of copies
of the plasmid. This is one way to produce copies of the DNA sequence
that was inserted into the plasmid (i.e. the insert). The resulting plasmid
can then be purified from the bacterial culture (e.g. using spin columns)
and used in down-stream applications.
●● What are vectors?
Answer: The plasmids used in the lab for cloning purposes are known as
vectors, as they act as "vectors" for the insert that they carry.
,Many of the plasmids used for research purposes are artificial constructs,
combining DNA sequences from several different sources (including
natural bacterial plasmids).
Most empty vectors are relatively small, typically ranging from 2-5 kb
(kilobases) in size; this contrasts natural plasmids, which are often 10-
100 kb. Cloning vectors can be considered to be "stripped down"
versions of natural plasmids, consisting principally of the core
components required for plasmid replication and selection. As long as
these components are not disrupted, it is possible to clone almost any
insert DNA sequence into a plasmid.
●● What are the 3 components found in most cloning vectors?
Answer: 1. Multiple Cloning Site (MCS):
This region of DNA is designed to simplify the process of cloning an
insert into the vector. This region is recognized by several different
restriction enzymes, enzymes which cut DNA. The insert can then be
enzymatically connected to the cut vector.
2. Origin of Replication (Ori):
This sequence of DNA is required for the plasmid to be replicated by
bacteria. This sequence is recognized by the bacterial replication
machinery (i.e. enzymes that replicate DNA), which produce more
copies of the plasmid. The origin of replication plays a role in
determining the copy number of a plasmid, which determines the
number of copies of the plasmid in a cell.
,3. Selectable Marker:
This is a gene that makes it possible to selectively culture the bacteria
that contain the plasmid. Selectable markers tend to be genes that confer
antibiotic resistance. Therefore, in the presence of the antibiotic, bacteria
transformed with the plasmid will survive and bacteria which lack the
plasmid will die.
●● How can we clone insert DNA into a vector?
Answer: A common approach using restriction enzymes and an enzyme
known as DNA ligase.
In this process, the empty vector is cut open using restriction enzymes,
and the insert is connected to the cut vector by DNA ligase.
●● What are restriction enzymes?
Answer: Restriction enzymes, or restriction endonucleases, are enzymes
that cleave phosphodiester linkages, breaking the bond between the
phosphorus atom of the phosphate group and the oxygen of the
deoxyribose sugar. This effectively cuts a strand of DNA into two
smaller strands.
*Very selective: only cleave DNA at these sites
, Different restriction enzymes have different recognition sites (can be
used for cloning purposes)
●● What is the ECOR1 restriction enzyme?
Answer: Recognizes this DNA sequence:
5' - GAATTC - 3'
ECOR1 cutes between G and A: 5' - G - 3' and 5' -AATTC - 3'
●● What does it mean when a recognition site is an inverted repeat
palindrome?
Answer: The sequence is the same on both strands (because DNA is
double stranded).
Example: both strands have the ECOR1 site.
5' - GAATTC - 3'
3' - CTTAAG - 3'
So ECOR1 cleaves between G and A on both the top and bottom strand"
5' - G - 3'
