This set of Basic Vector Biology Questions and Answers focuses on “Restriction Endonucleases – 2”.
1. EcoR1 has which of the following hexanucleotide recognition sequence?
Explanation: E.coli isolated from the gram-negative bacterium Escherichia coli. A recognition sequence is the one where an enzyme cleaves.
2. Which of the following is an example of flush end cutter?
Explanation: Many restriction endonucleases like PvuII, AluI, HaeIII simply cleave the double stranded DNA producing bunt or flush ends.
3. Which of the following does not produce cohesive ends upon cleavage on their recognition sequences?
Explanation: PvuII produces blunt ends whereas all others produce sticky ends. Some endonucleases cleave the DNA strand in a staggered way usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end.
4. What is dithiothreitol?
a) Reducing agent
b) Restriction endonuclease
Explanation: Most endonuclease function at pH 7.4, but different enzymes vary in their requirements for ionic strength and concentration. It is advisable to add a reducing agent like DTT which stabilizes the enzyme and prevents its deactivation. Before adding the enzyme, solution containing DNA must be adjusted to provide the correct conditions for maximum enzyme activity.
5. Restriction digests with which enzyme needs to be kept at a higher temperature than the usual 37 degree temperature?
Explanation: Most endonucleases work best at 37 degrees, TaqI enzyme from Thermus Aquaticus has a high working temperature like Taq DNA polymerase.
6. How can it be determined that a DNA molecule is cleaved by the action of endonuclease?
a) Checking the viscosity of the solution
b) Temperature check
c) Color indication
d) pH check
Explanation: Whether or not a DNA molecule is cut can be determined by viscosity examination. Larger DNA molecules result in more viscous solutions, DNA cleavage is associated with a decrease in viscosity.
7. How can the sizes of individual cleavage products be determined?
a) Temperature check
b) Molecular weight check
d) Gel electrophoresis
Explanation: Gel electrophoresis is a technique by which the sizes of fragmented DNA molecules can be measured against a molecular weight ladder of standard weights.
8. The rate of migration of a DNA molecule in an electrophoresis depends on which two factors?
a) Temperature, length
b) Length, source
c) Shape, charge-to-mass ratio
d) Shape, viscosity
Explanation: Shape and charge-to-mass ratio of nearly all DNA molecules is same and therefore to separate them gel electrophoresis is done in place of conventional electrophoresis.
9. How many basepairs long DNA molecules can be distinguished by the polyacrylamide (40%) gel electrophoresis?
a) 1-100 kbp
b) 1-300 bp
c) 100-200 kbp
d) 300-600 bp
Explanation: A very thin (0.3mm) polyacrylamide gel with extremely small pores can be used to distinguish small DNA molecules, can distinguish between molecules differing in length by just a single nucleotide.
10. What are double digestions?
a) Cutting with an enzyme twice on the same site
b) Cutting with two different enzymes on the same site
c) Cutting with an enzyme twice on different sites
d) Cutting with two different enzymes on the different sites
Explanation: Cutting with two different enzymes and at two different sites is done to create a restriction map of the genome of an organism.
11. What will happen if the incubation period, post restriction digestion is shortened?
b) Partial digestion
c) Double digestion
d) Complete digestion
Explanation: To perform partial digestion either incubation period is shortened or incubation is done at a low temperature at around 4 degrees which limits the activity of the enzyme.
12. What does a complex pattern of bands in a gel electrophoresis signify?
a) Partial digestion
b) Double digestion
c) Complete digestion
d) Contaminated sample
Explanation: As well as the standard fragments of total digestion, partially digested fragments are also seen as additional bands giving overall complexity.
13. What is the full form of OFAGE?
a) Orthogonal field alternation gel electrophoresis
b) Orthogonal field attraction gel electrophoresis
c) Oligonucleotide field alternation gel electrophoresis
d) Oligonucleotide field attraction gel electrophoresis
Explanation: OFAGE is an advanced gel electrophoresis technique used for the separation of DNA molecules of over 50 kb in length.
14. Which of the following techniques cannot be used to separate the genome of lower eukaryote yeast?
d) Gel electrophoresis
Explanation: The eukaryotic genome is large and simple techniques like gel electrophoresis cannot distinguish the DNA molecules ranging in several thousand basepairs.
15. FIGE is the modification of which technique?
b) Agarose gel electrophoresis
d) Acrylamide gel electrophoresis
Explanation: Field Inversion Gel Electrophoresis is a modification of OFAGE in which parallel alternating pairs of electrodes are placed.
Sanfoundry Global Education & Learning Series – Vector Biology & Gene Manipulation.
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