This set of Genetic Engineering Multiple Choice Questions & Answers (MCQs) focuses on “Modifications in PCR – 1”.
1. What are the possibilities which can occur until the temperature has reached for primer annealing?
a) Extension doesn’t starts until the appropriate temperature is reached
b) Extension may start even when the temperature is low
c) At low temperature, there is specific annealing of primer taking place
d) There are more specific products which are generated
Explanation: Until the temperature has reached for primer annealing there are chances of extension by polymerase. It is so because at low temperatures primer can anneal in a non specific manner and thus non specific products are generated.
2. Hot-start PCR is a modification of PCR. Which of the following is not corresponding to it?
a) The basis is that extension is not started until the first cycle reaches its maximum temperature
b) The polymerase is added after the first cycle has reached its maximum temperature or melting temperature
c) It is satisfactory for small number of samples
d) It leads to generation of non specific products
Explanation: Hot-start PCR is a modification which is used in order to overcome the problem of the extension before the maximum temperature of first cycle is reached. Hence, polymerase is added only after the maximum temperature for first cycle is reached. Hence, there are more specific products which are generated because proper annealing of primers has taken place. It is suitable for a small number of samples but not for a large number of samples.
3. An alternative to adding polymerase at later stage is __________
a) Make the polymerase inactive by binding it to an antibody
b) Introduce the polymerase or Magnesium in clay beads
c) Make the polymerase inactive by attaching groups which cause stearic inhindrance
d) Introduction of polymerase or Magnesium in plastic wires
Explanation: An alternative to start the extension at higher temperature is to make the polymerase inactive by binding an antibody to it. The antibody detaches itself at a higher temperature and thus polymerase is activated at higher temperature. Also, the polymerase or Magnesium can be introduced into wax beads and these beads melt at higher temperatures. Magnesium is required for the polymerase to function.
4. The primer annealing temperature is often very low from the maximum temperature. This low temperature leads to some mismatches.
Explanation: The primer annealing temperature is often very low from the maximum temperature. Though the low temperature is for stable binding of the template and the primer but at times it leads to mismatches.
5. Touch-down PCR is another modification. Its characteristics include:
a) Lowering the temperature for primer annealing
b) Primer annealing is done at higher temperatures initially
c) The temperature is abruptly reduced in the second cycle
d) In earlier cycles less stringent conditions are there and in later cycles, more stringent conditions are there
Explanation: It is done in order to overcome the slight mismatch which takes place at lower temperatures. For this, initially the temperature is kept very high and it is reduced in further cycles. As the temperature is reduced, a stage is reduced at which correct primer-template binding is possible but not the incorrect one. In this, in the earlier cycles more stringent conditions are there and in later cycles less stringent conditions are there.
6. If two successive PCR are carried out, it is called as __________
a) Touch-down PCR
b) Hot-start PCR
c) Combined PCR
d) Nested PCR
Explanation: Nested PCR is that in which two PCRs are carried out. In the first PCR, it uses a conventional template and the second PCR is carried out using the product of first PCR as a template.
7. If two successive PCRs are carried out, in which PCR there are chances of having a non-specific product?
a) First PCR
b) Second PCR
c) Both the PCRs
d) It depends on the annealing temperature
Explanation: If two successive PCRs are carried out, there are non-specific products created in the first PCR. But there are least chances that non-specific products also have annealing sites for both the primers in the second PCR. Hence, non specific products are not generated in the second PCR.
8. The process of inserting an amplified PCR product in a vector for cloning is known as __________
a) making library
c) making a hard copy
d) making a PCR based vector
Explanation: The process of inserting an amplified PCR product in a vector for cloning is termed as making a hard copy. It is further maintained by conventional means.
9. How can PCR product be cloned into a vector?
a) It can be done only when PCR products are blunt-ended
b) It can be done only by restriction enzyme digestion
c) Both the methods can be used
d) The blunt-ended approach is favoured
Explanation: PCR product can be cloned into a vector if the DNA molecules are blunt ended or it can also be done by restriction enzyme digestion. In restriction enzyme digestion restriction sites are introduced.
10. Which of the following statement is incorrect regarding the cloning of PCR products?
a) In cloning via restriction enzymes, restriction sites are induced before amplification is carried out
b) The restriction sites are induced in the primers before annealing
c) The intermediate molecules are having restriction sites at both ends
d) The amplified molecules can be cut at both the ends by appropriate enzymes
Explanation: If cloning is done via restriction enzymes, restriction sites are induced before amplification. The restriction sites are induced in the primers before annealing. As the primer binds, the restriction sites are induced at one end of the intermediate molecule. In full length molecules, restriction sites are at both ends. And the amplified molecules can be cut at both the ends by appropriate enzymes.
11. Topoisomerse I is also used for cloning of PCR product at times. Which of the statement holds true for such type of cloning?
a) The restriction site is induced into the vector and the topisomerase enzyme is induced into the PCR primers
b) The topoismerase I is used for cutting both the strands
c) The induction of topoisomerase enzyme is done into the vector in the case it is very small in size
d) The restriction site is induced into the PCR primers and the topoisomerase enzyme is induced into the vector
Explanation: In the case of this type of cloning, the restriction site is induced into the PCR primers and the enzyme is induced into the vector. The enzyme cuts the PCR product at the restriction site and joins it to the vector. Topoisomerase I is responsible for cutting only one strand and Topoisomerase II cuts both the strands.
12. Generally, amplification is carried out between the PCR primers. But if amplification is carried out outside the primers, it is called as __________
a) Inverse PCR
b) Circular PCR
c) Non-conventional PCR
d) In-situ PCR
Explanation: Generally, the amplification is carried out of the region which is flanked by the primers. But in inverse primers amplification is carried out of the region which lies outside the primers.
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