Genetic Engineering Multiple Questions on “Modifications in Cloning Methods, Linkers, Adaptors and Cassettes”.
1. There are various methods to distinguish whether a colony contains a recombinant or not. One such method is __________
a) blue white screening
b) checking whether replication is taking place or not
c) checking the number of copies
d) looking for the multiple cloning site
Answer: a
Explanation: Blue white screening is a method to know whether a colony is having a recombinant or not. It is also known as alpha complementation method. It is based on the lacZ operon. If there is an insert, the colonies are white in colour and if there is no insert, the colonies are blue in colour.
2. At times it is observed that non-recombinant plasmids are more than that of recombinant plasmids. Choose the correct statement in regard to it.
a) It makes easier to get the recombinant plasmids from the mixture
b) The ratio of insert DNA to vector DNA is reduced to get recombinant plasmids
c) Alkaline phosphatase is used to get recombinant plasmids
d) Alkaline phosphatase method is less reliable in comparison to the ratio method
Answer: c
Explanation: Often the recombination plasmids are less in number as compared to non-recombination plasmids. This makes it difficult to get the recombination plasmids from the mixture. In order to overcome this, the ratio of insert DNA to vector DNA is increased. Another more reliable method is to use the alkaline phosphatase method.
3. Alkaline Phosphatase is used at times and the vector is treated with it. Choose the incorrect statement.
a) It removes 5’ terminal phosphate group from nucleic acids
b) The 5’ phosphate group is required for the ligation to take place
c) Two phosphate bonds should be formed for the complete ligation to take place
d) The ligation between vector and insert won’t take place
Answer: d
Explanation: Alkaline phosphatase is used at times and the vector is treated with it. It is responsible for the removal of phosphatase group from the 5’ end. This group is required for the ligation reaction to take place and at each end two phosphates are required. But as the phosphate groups are removed from the vector, its relegation is not possible now. But as the insert is still having a phosphate group, one strand will form the bond and the ligation reaction will take place.
4. What is the correct time for carrying out the alkaline phosphatase treatment?
a) After the cutting of vector has been done
b) Before the insert DNA and the vector are mixed
c) After the cutting of vector has been done but before the insert DNA and vector are mixed
d) After the mixing of insert DNA and vector
Answer: c
Explanation: The alkaline phosphatase treatment is carried out after the cutting of DNA has been done but before the mixing of insert DNA and vector has been done. There should be no traces of the enzyme left after the insert DNA has been added.
5. ccdB gene is used at times. Choose the correct statement with respect to this gene.
a) It is a control of cell birth gene
b) It is activated in complex vectors to increase the fraction of recombinants
c) If intramolecular ligation takes place this protein is released
d) It is often regarded as control of cell death and birth gene
Answer: b
Explanation: It is an approach to increase the fraction of recombinants. There are some genes which are activated on the self ligation of the molecules and are responsible for the death of host molecules. ccdB is control of cell death gene and its protein is responsible for killing the host molecules when intramolecular ligation takes place.
6. When inserting a DNA fragment, it is possible to have two orientations. If the orientation is controlled, this cloning is referred to as __________
a) forced cloning
b) orientation cloning
c) correct cloning
d) restricted cloning
Answer: a
Explanation: It is possible to insert the DNA fragment into two possible orientations. If somehow it is controlled to insert the fragment in a particular orientation, it is known as forced cloning.
7. What happens if insert DNA is cut with two different restriction enzymes at the ends?
a) It is difficult to insert the fragment
b) The insert can be ligated in any orientation
c) The insert can be ligated in one orientation only
d) The amount of product increases
Answer: c
Explanation: If the DNA is cut with two different enzymes at the ends, it is possible to ligate the fragment in only one orientation. It is so because each end would have a unique sequence to ligate.
8. What is the disadvantage of amplification of using PCR over natural cloning?
a) In PCR, there is no proof reading activity
b) In PCR, small fragments can’t be amplified
c) There is an A incorporated in PCR products, which makes cloning difficult
d) PCR takes more time as compared to natural cloning
Answer: a
Explanation: The biggest disadvantage of using PCR amplification over natural cloning is that there is no proof reading activity in the case of PCR. Thus if an error is induced, it is carried forward and is amplified. Also, if large fragments are to be cloned, natural cloning is preferred over PCR. The incorporated A at ends makes the process of cloning of PCR products easier.
9. TA cloning is a method used for cloning of PCR products. Which of the statement is correct with respect to it?
a) It is based on the fact that a T residue is incorporated at the end of the PCR product
b) ‘A’ residue is incorporated into the end of the vector
c) It gives a low yield
d) It is preferred over blunt end ligation
Answer: d
Explanation: TA cloning is a method used for cloning of PCR products. It is based on the fact that A residue is present at the 3’ end of the PCR product and thus now T residue is incorporated at the end of the vector. It is preferred over blunt end ligation.
10. Choose the incorrect statement in respect to topoisomerase I.
a) It is used to cleave only one DNA strand
b) It leaves at the recognition sequence –C/TCCTT- and is covalently attached to one end of the cleaved molecule
c) The supercoiling of the DNA strand is altered and then it is sealed again by the enzyme
d) It is slower than the normal DNA ligase
Answer: d
Explanation: Topoisomerase I is an enzyme used for cleaving of one DNA strand. It leaves at the recognition sequence –C/TCCTT- and is covalently attached to one end of the cleaved molecule. The supercoiling of the DNA strand is altered and then it sealed by the enzyme again. It is usually faster than DNA ligase. Topoisomerase II is used for cleaving both the strands.
11. Linkers are often used in cloning. Choose the incorrect statement for linkers.
a) These are short chemically synthesized molecules that contain a particular restriction enzyme site within the sequence
b) They are blunt ended molecules
c) They are ligated to staggered ended insert molecules by T4 DNA ligase
d) After treatment with enzyme, both the ends of the linker are staggered
Answer: c
Explanation: Linkers are often used in cloning. These are short chemically synthesized molecules that contain a particular restriction enzyme site within the sequence. They are blunt ended molecules and are ligated to the blunt ended insert molecules by T4 DNA ligase. After treatment with the enzyme, both the ends of the linker are staggered.
12. There is no effect if the insert itself contains the restriction site.
a) True
b) False
Answer: b
Explanation: If the insert itself is having a restriction site, the insert itself is cleaved when the restriction enzyme is cleaved along with the linker. To avoid this, the methylation of the site in the insert is done.
13. Choose the correct statement for adaptors.
a) They are blunt ended at both the ends
b) They are single stranded at both the ends
c) They may be single stranded at one end and other end may be blunt
d) They don’t have extra restriction sites within their sequence
Answer: c
Explanation: Adaptors are molecules which are used for cloning. There is no restriction on the type of end they have. Either of the ends can be blunt or staggered. They may also have extra restriction site within the sequence.
14. If linkers are combined with other features such as a selectable marker, it is called as __________
a) cassette
b) modified linker
c) adaptors
d) induced linker
Answer: a
Explanation: If linkers are combined with the features such as antibiotic resistance, these are called as cassette or cloning cartridges. They may also contain gene expressing signals.