Gene Cloning
Short Questions and Answers
One mark Questions with Answers
1. Which of the following is a plasmid?
(a) pBR322
(b) Bam HI
(c) Sal I
(d) Eco RI
Answer: (a) pBR322
2. Which of the following are used in gene cloning?
(a) lomasomes
(b) mesosomes
(c) plasmids
(d) nucleoids
Answer: (c) plasmids
3. The technique used for separating molecules by passing electric current is
(a) fractionation
(b) centrifugation
(c) electrophoresis
(d) ELISA
Answer: (c) electrophoresis
4. Lysozyme that is present in perspiration, saliva and tears get destroyed in
(a) fractionation
(b) centrifugation
(c) electrophoresis
(d) ELISA
Answer: (b) centrifugation
5. The linking of antibiotic resistance gene with the plasmid vector is possible because of
(a) DNA polymerases
(b) exonucleases
(c) DNA ligase
(d) endonucleases
Answer: (c) DNA ligase
6. Which one among the following is just the cloning plasmid not an expression plasmid?
(a) pBAD-18-cam
(b) pBCSK
(c) pUC-18
(d) pET
Answer: (c) pUC-18
7. Bacteria protect themselves from viruses by fragmenting viral DNA upon entry with
(a) exonuclease
(b) gyrase
(c) endonuclease
(d) ligase
Answer: (c) endonuclease
8. Which of the following enzymes is used to join bits of DNA?
(a) ligase
(b) primase
(c) DNA polymerase
(d) endonuclease
Answer: (a) ligase
9. Construction of recombinant DNA involves
(a) cleaving DNA segments with ligase and rejoining them with endonuclease
(b) cleaving and rejoining DNA segments with ligase alone
(c) cleaving and rejoining DNA segments with ennuclease alone
(d) cleaving DNA segments with endonuclease and rejoining them with ligase
Answer: (d) cleaving DNA segments with endonuclease and rejoining them with ligase
10. The sites of DNA where restriction enzymes act are generally
(a) pallindromic
(b) tandem repeats
(c) CG rich region
(d) TATA boxes
Answer: (a) pallindromic
11. Which of the following is necessary to make accurate copies of vector DNA?
(a) RNA polymerase
(b) DNA polymerase
(c) DNA ligase
(d) Endonucleases
Answer: (b) DNA polymerase
12. What is the name of the site where foreign DNA can be inserted in plasmid of Agrobacterium?
(a) t-RNA
(b) c-DNA
(c) T-DNA
(d) B-DNA
Answer: (c) T-DNA
13. A method used to insert DNA molecules into the cells by using short electrical impulses is known as
(a) biolistics
(b) microinjection
(c) liposomes
(d) electroporation
Answer: (d) electroporation
14. In genetic engineering, the antibiotics are used
(a) as selectable markers
(b) to select healthy vectors
(c) as sequences from where replication starts
(d) to keep the cultures free of infection
Answer: (a) as selectable markers
Two marks questions with answers
1. What is Recombinant DNA Technology?
Answer: Recombinant DNA technology is the process of removing the desired piece of DNA from one organism and incorporating it into chromosomal DNA of another organism.It is also termed as genetic engineering. The first successful attempt of joining different pieces of DNA was made by Paul Berg.
2. What are the tools of Recombinant DNA Technology?
Answer: The tools include enzymes termed as restriction endonucleases and also DNA polymerases and DNA ligase. The procedure also uses vectors as vehicles to carry foreign DNA into a host cell or living system. All these form tools for recombinant technology.
3. What are blunt ends and sticky ends?
Answer: The cut produced by restriction endonuclease is important. The resultant DNA sequence may either have blunt ends or sticky ends. Blunt ends or flush ends are produced by many restriction enzymes which leave both strands of DNA exactly at the same nucleotide position, in the centre of recognition site. It cuts both DNA strands producing blunt ends.
Sticky or cohesive ends are produced when restriction enzymes do not cut DNA at the same nucleotide position but cut the recognition sequence unequally. This produces short single stranded overhangs at each end. These are termed as sticky ends. The stickiness helps enzyme ligase to make the DNA pieces to join.
4. Mention the function of DNA ligase.
Answer: This enzyme is useful in joining the bits of DNA. DNA ligase forms phosphodiester bonds between adjacent nucleotides and joins two fragments of double stranded DNA. The enzyme forms phosphodiester bond between hydroxyl group at 3’ carbon of one nucleotide with phosphate group at 5’ carbon of another nucleotide.
5. Yeasts have also been widely used in r-DNA technology. Give reasons.
Answer: Yeasts are very useful because of the following reasons:
(1) these are simplest eukaryotic organisms
(2) yeasts are unicellular and hence easy to handle
(3) the yeasts can be cultured on both small and large scales
Three marks questions with answers
1.Who is the competent host for transformation and recombinant DNA?
Answer: The use of tools finally results in the formation of recombinant DNA. Recombinant DNA thus formed needs to be multiplied inside living system or a host. Many types of host cells are available for this purpose. The choice of host, however, depends upon the purpose of cloning E coli, yeast, animal and plant cells have been variously used. E. coli is the most commonly used organism in recombinant DNA technology. It has many advantages, such as:
(1) It is easy to manipulate and grow in vitro
(2) It can accept a wide range of vectors
(3) It doubles the number of cells every 20 minutes and
(4) It reproduces r-DNA molecule as it does for its own DNA.
2. What is selectable marker gene? Mention its importance in gene cloning.
Answer: Vector should also include a selectable marker which is a gene permitting identification of transformants from non transformants. Common selectable markers in E coli include genes encoding antibiotic resistance such as ampicillin resistance or enzymes such as beta galactosidase (product of z gene of lac operon). A colour reaction is helpful in the identification of these genes.
The process of selection of transformant or recombinant due to antibiotic inactivation is very complex and involves time consuming procedures. A simple and alternative method, therefore, is necessary. A technique termed as insertional inactivation was helpful in overcoming this difficulty. Insertional inactivation is a technique used in recombinant DNA engineering where a plasmid such as pBR322 is useful to disable expression of a gene.
3. What are cloning sites? How important are the cloning site in r-DNA technology?
Answer: Vector should possess a unique recognition site that would allow particular enzyme to cut only once. The restriction enzyme recognizes this site. If is there are more than one recognition sites in the vector, several fragments would be produced. Generally the vectors used possess unique recognition sites for several restriction enzymes in a small region of DNA. This is known as polylinker or multiple cloning sites (MCS). Such a cloning site offers choice of restriction enzyme.
Unique restriction endonuclease recognition site enables insertion of foreign DNA into the vector for production of recombinant DNA. The foreign DNA is inserted and made to join or ligate at a specific restriction site generally in antibiotic resistance gene.
4. What are restriction enzymes?
Answer: Restriction enzymes occur in bacteria where restriction enzyme or restriction endonuclease forms a major part of bacterial defence system by restricting the growth of potentially harmful genetic elements (i.e., DNA) of other bacteria, viruses ( bacteriophages) etc.
Nuclease is an enzyme which breaks phosphodiester bonds between nucleotide subunits of nucleic acids. These are of two types exonucleases and endonucleases. Endonuclease cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain at specific locations. Restriction endonuclease recognises a specific DNA sequence and degrades any DNA containing that sequence. These enzymes differ with different bacterial species. There are three types of restriction enzymes, Type I, Type II and Type III. Restriction enzymes Type II are useful in Recombinant DNA technology. These enzymes are now variously termed as ‘molecular scalpels’, ‘molecular scissors’, ‘chemical scalpels’ etc.