This set of Protein Engineering Multiple Choice Questions & Answers (MCQs) focuses on “New Protein Molecule Design – Error Prone PCR”.
1. Which of the following technique is used to alter the function of an enzyme without the need for exhaustive structural and functional information?
a) Slow evolution
b) Indirect evolution
c) Rational protein design
d) Directed evolution
View Answer
Explanation: Directed evolution is used to alter the function of an enzyme without the need for exhaustive structural and functional information. It can be used to change substrate specificity, either changing the enzyme to recognize a different substrate or by making subtle changes in cases in which the substrate is slightly different.
2. Directed evolution screens for new enzyme activities by constructing a library of different enzymes derived from the same original protein.
a) False
b) True
View Answer
Explanation: The above statement is true. Directed evolution screens for new enzyme activities by constructing a library of different enzymes derived from the same original protein. Random mutagenesis during amplification of the gene creates a library of slightly different genes and, when expressed, slightly different proteins.
3. Who discovered PCR?
a) Charles Chamberland
b) Linus Pauling
c) Robert brown
d) Kary Mullis
View Answer
Explanation: Kary Mullis discovered PCR in 1983. It is an in-vitro method of enzymatic synthesis of specific DNA fragments. Charles Chamberland invented autoclave in 1884. Robert Brown discovered the nucleus.
4. Which of the following technique is used to amplify a precise fragment of DNA from a complex mixture of starting material called template DNA?
a) Northern Blotting
b) Southern Blotting
c) Western Blotting
d) PCR
View Answer
Explanation: PCR is a technique that is used to amplify a precise fragment of DNA from a complex mixture of starting material called as template DNA. It is a very simple technique for characterizing, analyzing and synthesizing DNA from virtually any living organism (plant, animal, virus, or bacteria).
5. Which of the following component is not required in a basic PCR?
a) DNA template
b) Two primers
c) Thermostable DNA polymerase
d) Initiation and Elongation Factors
View Answer
Explanation: A basic PCR requires a DNA template that contains the gene to be amplified, two primers, and a thermostable DNA polymerase like Taq, Vent, Pfu, etc. It does not require Initiation and Elongation Factors.
6. Which of the following is not supposed to be present in the PCR reaction mixture solution?
a) Deoxynucleotide triphosphates (dNTPs)
b) Magnesium ions
c) Buffer
d) Calcium ions
View Answer
Explanation: Calcium ions are not supposed to be present in the PCR reaction mixture solution. Deoxynucleotide triphosphates (dNTPs), magnesium ions, and buffers are supposed to be present in the PCR reaction mixture solution. dNTPs are the building blocks from which the DNA polymerase synthesizes a new DNA strand. Buffers are required for optimal activity and stability of the DNA polymerase enzyme. Magnesium ions are necessary for maximum Taq polymerase activity.
7. PCR machine is also known as thermo-cycler.
a) False
b) True
View Answer
Explanation: The above statement is true. PCR machine is also known as thermo-cycler. It maintains constant reaction temperature throughout the cycles. The purpose of a PCR is to amplify a specific DNA or RNA fragment.
8. What is the temperature of the reaction mixture in the initiation step of PCR?
a) 70-72°C
b) 100-110°C
c) 68-70°C
d) 94-96°C
View Answer
Explanation: The temperature of the reaction mixture in the initiation step of PCR is kept at 94-96°C for a few minutes. The function of this step is to initiate the breaking of the hydrogen bonds in DNA strands. This step is also called a denaturation step.
9. Which of the following technique allows, the initiation of DNA amplification, starting with tiny amounts of the parent molecule, and produces considerable amounts of mutated genes?
a) Southern Blotting
b) Western Blotting
c) PCR
d) Error-prone PCR
View Answer
Explanation: Error-prone PCR technique allows, the initiation of DNA amplification, starting with tiny amounts of the parent molecule, and produces considerable amounts of mutated genes. Error-prone PCR is a variant of PCR. It is a technique used to generate randomized genomic libraries.
10. In error-prone PCR the Taq polymerase anneals incompatible base-pairs to each other during amplification.
a) False
b) True
View Answer
Explanation: The above statement is true. In error-prone PCR the Taq polymerase anneals incompatible base-pairs to each other during amplification. The working principle of this technique is based on the ability of Taq polymerase to anneal incompatible base-pairs to each other during amplification, under imperfect PCR conditions.
11. What is the temperature of the reaction mixture in the second (annealing) step of PCR?
a) 100-110°C
b) 90-94°C
c) 70-72°C
d) 50-65°C
View Answer
Explanation: The temperature of the reaction mixture in the second (annealing) step of PCR is 50-65°C. The reaction mixture is kept at this temperature for 20-40 seconds. This allows annealing of the primers to the single-stranded DNA template. Typically, the annealing temperature is 3-5°C below the melting temperature (Tm) of the primers used.
12. Which of the following cannot produce errors in error-prone PCR?
a) High concentration of dATPs in the reaction mixture
b) High concentration of Mg+2
c) High concentration of dTTPs in the reaction mixture
d) Taq polymerase with proof-reading ability
View Answer
Explanation: Taq polymerase with proof-reading ability does not produce errors in error-prone PCR. High concentration of dATPs in the reaction mixture, high concentration of Mg+2, and high concentration of dTTPs in the reaction mixture can produce errors in error-prone PCR.
13. If we start with a single double-stranded DNA molecule in PCR amplification, then how many molecules of dsDNA would be obtained after 23 cycles of PCR amplification?
a) 23,258,369
b) 5,456,987
c) 7,256,458
d) 8,388,608
View Answer
Explanation: The number of dsDNAmolecules that would be obtained after 23 cycles is 8,388,608.
Formula: A=2n x N°
Where n=number of cycles of amplification,
N°=Initial number of DNA molecules ,
A=number of DNA molecules after n cycles.
A=223 x 1
A=8,388,608.
14. What is the temperature of the reaction mixture in the extension/elongation step of PCR?
a) 90-94°C
b) 50-65°C
c) 100-110°C
d) 68-72°C
View Answer
Explanation: The temperature of the reaction mixture in the extension/elongation step of PCR is 68-72°C. Taq polymerase has its optimum activity at 75-80°C. The temperature of this step depends on the DNA polymerase used.
15. If we start with 10 double-stranded DNA molecules in PCR amplification, then how many molecules of dsDNA would be obtained after 10 cycles of PCR amplification?
a) 10,420
b) 5,120
c) 20,480
d) 10,240
View Answer
Explanation: The number of dsDNA molecules that would be obtained after 10 cycles is 10,240.
Formula: A=2n x N°
Where n=number of cycles of amplification,
N°=Initial number of DNA molecules ,
A=number of DNA molecules after n cycles.
A=210 x 10
A=10,240.
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