Question 1

Describe the importance of host vector system in rDNA technology.

Accepted Answer

The host-vector system is crucial in recombinant DNA technology because it allows the replication and expression of foreign DNA within a host organism. The vector acts as a vehicle to carry the foreign DNA into the host cell, where it can be replicated and sometimes expressed. This system enables the production of multiple copies of the gene of interest, facilitates gene cloning, and allows for the study and manipulation of genes. Without a suitable host-vector system, it would be impossible to propagate recombinant DNA molecules effectively. — The host provides the cellular machinery for replication and expression, while the vector ensures the foreign DNA is maintained and propagated. The combination is essential for cloning, gene expression studies, and genetic engineering.

Question 2

What are the major characteristics of a vector?

Accepted Answer

The major characteristics of a vector include: (1) Ability to replicate independently within the host cell, (2) Presence of a selectable marker gene to identify transformed cells, (3) Presence of multiple cloning sites (MCS) or polylinker for insertion of foreign DNA, (4) Small size to facilitate easy manipulation, (5) Stability within the host, and (6) Ability to accept foreign DNA of desired size. — These characteristics ensure that the vector can efficiently carry and replicate the foreign DNA inside the host, and allow easy selection and identification of transformed cells.

Question 3

What is plasmid and what are its different types?

Accepted Answer

A plasmid is an extrachromosomal, self-replicating circular DNA molecule found in bacteria and some eukaryotes. It carries genes that may provide advantageous traits such as antibiotic resistance. Different types of plasmids include: (1) F-plasmid (fertility plasmid) involved in bacterial conjugation, (2) R-plasmid carrying antibiotic resistance genes, (3) Col-plasmid producing bacteriocins that kill other bacteria, (4) Degradative plasmids enabling the breakdown of unusual substances, and (5) Virulence plasmids carrying genes that make bacteria pathogenic. — Plasmids are important tools in genetic engineering as vectors due to their ability to replicate independently and carry foreign genes.

Question 4

Discuss the strategy applied for the development of (pBR322) plasmid cloning vectors.

Accepted Answer

pBR322 is a widely used plasmid cloning vector developed by Bolivar and Rodriguez. The strategy involved combining antibiotic resistance genes (ampicillin and tetracycline resistance) as selectable markers, and multiple unique restriction sites for insertion of foreign DNA. The plasmid is small (~4361 bp), allowing easy manipulation. The presence of two antibiotic resistance genes allows for screening of recombinant plasmids by antibiotic selection. The vector replicates in E. coli, making it suitable for cloning and propagation of foreign DNA. — The design of pBR322 allows easy insertion of foreign DNA, selection of transformed cells, and replication within host cells, making it an ideal cloning vector.

Question 5

Briefly describe the structure of lambda bacteriophage and also discuss the role of lambda phage based vectors.

Accepted Answer

Lambda bacteriophage is a virus that infects E. coli. Its structure includes a head (capsid) containing double-stranded DNA (~48.5 kb), a tail, and tail fibers. The lambda phage genome has cohesive ends (cos sites) that allow circularization inside the host. Lambda phage based vectors utilize the phage's ability to package DNA and infect bacteria, allowing cloning of larger DNA fragments (up to ~20 kb). These vectors can integrate into the host genome or exist as plasmids, facilitating gene cloning and expression. — Lambda phage vectors are useful for cloning larger DNA fragments than plasmids and have high efficiency of infection and packaging, making them valuable tools in recombinant DNA technology.

Question 6

Discuss the M13 based vectors and its application.

Accepted Answer

M13 is a filamentous bacteriophage whose genome is single-stranded DNA. M13 based vectors are used for cloning single-stranded DNA, which is useful in DNA sequencing and site-directed mutagenesis. The vector can infect E. coli without killing it, allowing continuous production of single-stranded DNA. Applications include generation of single-stranded templates for sequencing and mutagenesis studies. — M13 vectors are valuable for molecular biology techniques requiring single-stranded DNA, such as sequencing and mutagenesis.

Question 7

Differentiate between cosmids and phagemids.

Accepted Answer

Cosmids are hybrid vectors containing plasmid and lambda phage cos sites, allowing packaging of large DNA fragments (~35-45 kb) into phage particles for infection. Phagemids are plasmids containing an origin of replication from filamentous phage (like M13), allowing them to be packaged into phage particles and produce single-stranded DNA. Cosmids are used for cloning large DNA fragments, while phagemids are used for generating single-stranded DNA for sequencing and mutagenesis. — The key difference lies in their origin and application: cosmids combine plasmid and phage features for large insert cloning; phagemids combine plasmid and filamentous phage features for single-stranded DNA production.

Question 8

Why is a vector required for cloning of a gene?

Accepted Answer

A vector is required for cloning a gene because it acts as a carrier to transfer the foreign DNA into the host cell. It provides the necessary elements for replication, selection, and sometimes expression of the cloned gene within the host. Without a vector, the foreign DNA cannot be maintained or propagated inside the host cells. — Vectors ensure the foreign gene is replicated and can be selected for in host cells, enabling gene cloning and study.

Question 9

A plasmid capable of getting integrated into host chromosome is called:
(a) Col plasmid
(b) Episome
(c) Ti plasmid
(d) R plasmid

Accepted Answer

The correct answer is (b) Episome. An episome is a plasmid that can integrate into the host chromosome and replicate along with it. Other plasmids like Col plasmids produce bacteriocins, Ti plasmids are tumor-inducing plasmids in Agrobacterium, and R plasmids carry antibiotic resistance genes but do not necessarily integrate into the host genome. — Episomes have the ability to exist either as independent plasmids or integrate into the host chromosome, which distinguishes them from other plasmid types.

Question 10

Why the replication of single copy plasmid called stringent replication?

Accepted Answer

Replication of single copy plasmids is called stringent replication because it is tightly regulated to maintain only one or a few copies of the plasmid per cell. The plasmid replication is controlled by the host cell's regulatory mechanisms, ensuring that the plasmid does not replicate excessively, which could be detrimental to the host. — Stringent replication ensures plasmid copy number is kept low and stable, preventing metabolic burden on the host cell.

Question 11

Identify the incorrect match pair from the following:

(i) Multicopy plasmid
(a) Stringent replication
(ii) Col plasmid
(b) Kills bacteria
(iii) pBR322
(c) Plasmid
(iv) Prophage
(d) Phage genome inserted into a host genome

Accepted Answer

The incorrect match is (i) Multicopy plasmid - (a) Stringent replication. Multicopy plasmids replicate via relaxed replication, producing many copies per cell, whereas stringent replication is characteristic of single copy plasmids. The other pairs are correct: (ii) Col plasmid kills bacteria by producing bacteriocins, (iii) pBR322 is a plasmid, and (iv) Prophage is a phage genome inserted into the host genome. — Multicopy plasmids have relaxed replication, not stringent. Stringent replication applies to single copy plasmids. The other matches are correct based on plasmid and phage biology.

Question 12

How can a large size eukaryotic gene insert be cloned?

Accepted Answer

Large size eukaryotic gene inserts can be cloned using vectors capable of carrying large DNA fragments, such as cosmids, bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs). These vectors can accommodate large inserts (up to several hundred kilobases), allowing cloning of entire genes including regulatory regions. — Standard plasmids have limited capacity (~10 kb), so for large eukaryotic genes, vectors with higher capacity and stability are used to clone and maintain large DNA fragments.

Question 13

Assertion: An ideal vector should have selectable marker.

Reason: Selectable markers are required to screen out transformation.

(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.

Accepted Answer

The correct option is (a). Both the assertion and reason are true, and the reason correctly explains the assertion. An ideal vector should have a selectable marker gene so that transformed cells can be easily identified and selected, which is essential for screening successful transformation events. — Selectable markers like antibiotic resistance genes allow only transformed cells to grow in selective media, facilitating identification of recombinant clones.

Question 14

Assertion: Cosmid is a hybrid vector.

Reason: Cosmid has properties of both plasmids and lambda phage vector.

(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false

Accepted Answer

The correct option is (a). Both assertion and reason are true, and the reason correctly explains the assertion. Cosmids are hybrid vectors that combine features of plasmids (such as replication origin and selectable markers) and lambda phage (cos sites for packaging), enabling cloning of large DNA fragments. — Cosmids can be packaged into phage particles due to cos sites and replicate as plasmids in host cells, making them hybrid vectors.

Question 15

In recombinant DNA technology, what are the two key components involved in gene cloning?

Accepted Answer

Host and vector — Recombinant DNA technology involves two key components: a compatible host organism and a vector DNA molecule. The vector carries the insert (target gene) and replicates autonomously, while the host provides the cellular machinery for replication and expression of the recombinant DNA, enabling gene cloning.

Recombinant DNA Technology

Recombinant DNA Technology — Study Notes

Host-Vector System

2.1 TWO KEY COMPONENTS OF RECOMBINANT DNA TECHNOLOGY

2.2 Host

Practice Questions — Recombinant DNA Technology

All 13 Chapters in Biotechnology