MICR 290 EXAM QUESTIONS WITH 100%
CORRECT ANSWERS!!
What is cloning?
Cloning refers to the process of inserting a sequence of DNA (also called an insert) into a
plasmid. The resulting plasmid is a form of recombinant DNA, meaning that it is an artificial (i.e.
laboratory-produced) construct combining DNA sequences from two or more different sources.
It refers to the process of inserting a sequence of DNA into a vector. The insert may come from
various sources (e.g. from DNA purified from bacteria), but is most commonly produced by
polymerase chain reaction.
Plasmids used for cloning are known as cloning vectors, and are designed to simplify the process
of transferring an insert into the empty vector. If the goal is to produce a protein encoded by the
insert DNA (i.e. the insert is a gene), a specific kind of cloning vector known as an expression
vector must be used.
What are the correct features of a plasmid?
Circular, double-stranded DNA present in the bacterial cytoplasm
Many plasmids carry genes that confer antibiotic resistance
Why are plasmids useful?
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?
,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?
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?
,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?
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?
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?
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'
, 3' - CTTAA -5'
and
5' - AATTC - 3'
3' - G - 5'
*THIS CREATES A 4 BASE-PAIR OVERHAND = 'Sticky Ends'*
What are sticky ends?
They are overhangs after being cleaves by the restriction enzymes.
The sticky end on one strand is complementary to the sticky end on the other strand.
Even though the phosphodiester bond was broken by restriction enzymes, the sticky ends can
interact through base-pair interactions (so they will be held in close proximity).
Do all restriction enzymes generate sticky ends?
NO.
For example, the enzyme Smal recognizes 5' - CCCGGG - 3'
Which creates the inverted repeat palindrome
3' - GGGCCC - 5'
Smal will cleave between the CCC and GGG
BUT there is no overhang which creates BLUNT ENDS (no base pair interactions)
What is ligation?
CORRECT ANSWERS!!
What is cloning?
Cloning refers to the process of inserting a sequence of DNA (also called an insert) into a
plasmid. The resulting plasmid is a form of recombinant DNA, meaning that it is an artificial (i.e.
laboratory-produced) construct combining DNA sequences from two or more different sources.
It refers to the process of inserting a sequence of DNA into a vector. The insert may come from
various sources (e.g. from DNA purified from bacteria), but is most commonly produced by
polymerase chain reaction.
Plasmids used for cloning are known as cloning vectors, and are designed to simplify the process
of transferring an insert into the empty vector. If the goal is to produce a protein encoded by the
insert DNA (i.e. the insert is a gene), a specific kind of cloning vector known as an expression
vector must be used.
What are the correct features of a plasmid?
Circular, double-stranded DNA present in the bacterial cytoplasm
Many plasmids carry genes that confer antibiotic resistance
Why are plasmids useful?
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?
,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?
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?
,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?
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?
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?
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'
, 3' - CTTAA -5'
and
5' - AATTC - 3'
3' - G - 5'
*THIS CREATES A 4 BASE-PAIR OVERHAND = 'Sticky Ends'*
What are sticky ends?
They are overhangs after being cleaves by the restriction enzymes.
The sticky end on one strand is complementary to the sticky end on the other strand.
Even though the phosphodiester bond was broken by restriction enzymes, the sticky ends can
interact through base-pair interactions (so they will be held in close proximity).
Do all restriction enzymes generate sticky ends?
NO.
For example, the enzyme Smal recognizes 5' - CCCGGG - 3'
Which creates the inverted repeat palindrome
3' - GGGCCC - 5'
Smal will cleave between the CCC and GGG
BUT there is no overhang which creates BLUNT ENDS (no base pair interactions)
What is ligation?
