Write the role of 'ori' and restriction site in the cloning vector pBR322.

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Q. Write the role of 'ori' and restriction site in the cloning vector pBR322.

CBSECBSE Class XII Board 2024Subjective· 2mImportance★★★★★

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Concept understanding — Biotechnology Core Concepts

Biotechnology Core Concepts — A First Look

You already know biotechnology better than you think. When you eat yoghurt, that's biotechnology at work. When bread rises, that's biotechnology. When your grandmother used curd to ferment buttermilk, she was practising an ancient form of it. The core idea is simple: using living organisms (or parts of them) to make or modify products for human benefit.

The NCERT textbook defines biotechnology as "the use of living systems and organisms to develop or make useful products." That's the formal version of what you just read.

The Two Big Ideas That Hold Everything Together

Biotechnology rests on two fundamental capabilities that nature gave us, and that scientists learned to harness:

1. Genetic Engineering — the ability to change an organism's DNA directly. Think of it as editing the instruction manual of a living thing. Instead of waiting for nature to produce a trait through slow breeding, scientists can now take a specific gene from one organism and put it into another. A bacterium can be made to produce human insulin because the human insulin gene has been inserted into it.

2. Maintenance of Sterile Conditions — the ability to grow large numbers of cells or microorganisms in a controlled, contamination-free environment. This is called aseptic technique. Without it, the wrong microbes would spoil the process, and you'd get garbage instead of medicine.

Important

These two — genetic engineering and sterile maintenance — are the twin pillars of modern biotechnology. The NCERT explicitly states that biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products useful to humans. The modern era of biotechnology began when we could manipulate DNA directly.

Why This Matters in Everyday Life

You don't need a lab coat to see biotechnology's impact. Consider these examples:

Medicine: Insulin for diabetes, vaccines, and gene therapy all come from biotechnology. Before genetic engineering, insulin was extracted from the pancreases of pigs and cows — expensive and sometimes caused allergic reactions. Now, bacteria make human insulin for us.
Agriculture: Crops that resist pests or tolerate drought. Bt cotton, for instance, has a bacterial gene that makes it produce a protein toxic to certain insects, reducing the need for chemical pesticides.
Environment: Microbes engineered to clean up oil spills or break down plastic waste.
Food: Cheese, beer, wine, and even the citric acid in your soft drinks are products of microbial biotechnology.

Note

The NCERT classifies biotechnology into two eras: Traditional biotechnology (fermentation, breeding) which humans have used for thousands of years, and Modern biotechnology (genetic engineering, cell culture) which began in the 1970s with the discovery of tools to cut and join DNA.

The Core Tools (What Makes It Possible)

Modern biotechnology relies on a few essential techniques. You don't need to memorise details, but understand what each does:

Recombinant DNA technology: Cutting a gene from one organism and pasting it into another. This is how we make human insulin in bacteria.
Gene cloning: Making many identical copies of a gene. If you have one copy of a useful gene, you can make millions.
Tissue culture: Growing plant or animal cells in a lab dish. A whole plant can be regenerated from a single cell.
Fermentation technology: Using microbes in large tanks (bioreactors) to produce substances like antibiotics, enzymes, or alcohol.

A Common Misunderstanding to Avoid …

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The 'ori' sequence in pBR322 initiates DNA replication and controls the vector's copy number, while restriction sites provide specific locations for restriction enzymes to cut and insert foreign DNA, often within selectable marker genes for screening.

Cloning vectors are fundamental tools in genetic engineering, serving as vehicles to carry and replicate foreign DNA within a host organism. Among the most widely used and well-characterized artificial cloning vectors is pBR322, a plasmid constructed in the laboratory. Its effectiveness stems from several key features, two of the most critical being the 'ori' sequence and its multiple restriction sites. Understanding their roles is central to comprehending how recombinant DNA technology works.

The Role of 'ori' (Origin of Replication)

The 'ori' sequence, short for origin of replication, is a specific DNA sequence present in the pBR322 plasmid that dictates where replication will begin. It is a crucial control element for the vector's propagation within a host cell, typically E. coli.

Initiation of Replication: The 'ori' sequence is the recognition site for the host cell's DNA replication machinery. Without an 'ori', the plasmid would not be able to replicate independently within the host cell. Once the host cell's enzymes bind to the 'ori', they initiate the synthesis of new DNA strands, effectively making copies of the entire plasmid, including any foreign DNA inserted into it.
Control of Copy Number: The 'ori' sequence also plays a vital role in determining the number of copies of the plasmid that will exist per host cell. Different 'ori' sequences can lead to high or low copy numbers. For pBR322, its 'ori' allows for a relatively high copy number, meaning many copies of the plasmid (and thus the cloned gene) can be present in each bacterial cell. This is highly advantageous for applications where a large amount of the cloned gene product or DNA is desired.

Important

The 'ori' sequence ensures that the cloning vector, along with the foreign DNA it carries, is replicated and stably inherited by daughter cells during cell division, making it a self-replicating unit within the host.

The Role of Restriction Sites

Restriction sites are specific DNA sequences that are recognized and cut by restriction enzymes. These sites are strategically placed within the pBR322 vector to facilitate the insertion of foreign DNA.

Targeted DNA Insertion: Each restriction site is a unique recognition sequence for a particular restriction endonuclease. For example, pBR322 contains restriction sites for enzymes like PstI, PvuI, EcoRI, BamHI, SalI, and HindIII. When a specific restriction enzyme is used, it cuts the pBR322 plasmid at its corresponding restriction site, creating an opening in the circular DNA. The same restriction enzyme is then used to cut the foreign DNA to be cloned, ensuring that both the vector and the foreign DNA have compatible "sticky ends" or blunt ends, allowing them to be joined together by DNA ligase.
Facilitating Selection of Recombinants (Insertional Inactivation): Many of the restriction sites in pBR322 are located within genes that confer antibiotic resistance, known as selectable markers. pBR322, for instance, possesses genes for resistance to ampicillin (ampR) and tetracycline (tetR).
- The ampR gene contains restriction sites for enzymes like PstI and PvuI.
- The tetR gene contains restriction sites for enzymes like BamHI and SalI. …

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Same / Similar Concept — real previous-year questions on the same or a closely similar concept, not this exact question.

Showing the 12 most recent of 36 on this concept.

CBSE 2026Set 57/3/11 markMCQ
$Q. Match Column I with Column II and select the correct option : Column I a. Primers b. Insertional inactivation c. Bioreactor d. Downstream processing Column II i. β-galactosidase ii. To separate and purify products iii. Chemically synthesized oligonucleotides iv. Large scale production of specific product of activity of microbes Options : (A) a-iii, b-i, c-iv, d-ii (B) a-iii, b-i, c-ii, d-iv (C) a-i, b-iii, c-iv, d-ii (D) a-i, b-iv, c-ii, d-iii$
›Reveal solutionSolution

Primers are chemically synthesized oligonucleotides; insertional inactivation uses β-galactosidase; bioreactors enable large-scale microbial production; downstream processing separates and purifies products.

The question tests your understanding of four fundamental tools and processes in biotechnology, each playing a distinct role in genetic engineering and industrial production. Let's work through what each term means and why it matches its partner.

Primers are short stretches of nucleotides—typically 10 to 20 bases long—that are chemically synthesized in the laboratory. They are oligonucleotides, meaning "few nucleotides." In PCR (polymerase chain reaction), primers bind to specific sequences on the DNA template and provide the starting point for DNA polymerase to begin synthesis. Without primers, the enzyme cannot initiate replication. The NCERT textbook explicitly describes primers as chemically synthesized oligonucleotides used in amplification techniques. So a matches iii.

Insertional inactivation is a clever selection technique used to identify recombinant DNA. The principle relies on disrupting a functional gene when foreign DNA is inserted into a plasmid vector. The classic example involves the lacZ gene, which codes for the enzyme β-galactosidase. When a foreign DNA fragment is successfully inserted into the cloning site within lacZ, the gene is inactivated—the enzyme is no longer produced. Colonies containing recombinant plasmids remain white when grown on medium with a chromogenic substrate (like X-gal), while non-recombinant colonies turn blue because their intact β-galactosidase cleaves the substrate. This visual distinction makes screening straightforward. Thus b matches i.

Note

The blue-white screening method is one of the most widely used techniques in molecular cloning labs because it provides immediate visual confirmation of successful insertion.

Bioreactors are large vessels designed for the controlled, large-scale cultivation of microorganisms, plant cells, or animal cells. They maintain optimal conditions—temperature, pH, oxygen supply, nutrient availability—to maximize the production of desired products like enzymes, antibiotics, vaccines, or recombinant proteins. The NCERT textbook describes bioreactors as systems that provide the ideal environment for growing cultures in volumes ranging from 100 to 1000 liters or more. The entire purpose is large-scale production of a specific product through microbial or cellular activity. Therefore c matches iv. …
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CBSE 2026Set ANNUAL1 markMCQ
$Q. Which is not a step of PCR cycle?$
(a) Denaturation
(b) Primers Annealing
(c) Extension of primers
(d) Identification of DNA with genes
›Reveal solutionSolution
A PCR cycle has three steps: denaturation, annealing and extension; identifying DNA is not one of them.

The Polymerase Chain Reaction (PCR) amplifies a specific DNA segment through repeated cycles, each with three steps:

Denaturation: heating (about 94-95 C) separates the double-stranded DNA into single strands.

Annealing: cooling allows two primers to bind (anneal) to their complementary sequences flanking the target. …
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CBSE 2026Set ANNUAL1 mark
$Q. Write answer in one word/sentence: Which type of charge found on DNA fragments?$
›Reveal solutionSolution

DNA fragments carry a negative charge because of their phosphate backbone.

The backbone of a DNA molecule is made of alternating sugar and phosphate groups, and the phosphate groups carry negatively charged oxygen atoms. As a result, DNA fragments are negatively charged. This is the basis of gel electrophoresis: when an electric field is applied, the negati …

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CBSE 2026Set ANNUAL1 markMCQ
$Q. The enzyme required for isolating DNA from fungi is -$
(a) Cellulase
(b) Chitinase
(c) Lysozyme
(d) Endonuclease
›Reveal solutionSolution
Fungal cell walls are made of chitin, so the enzyme chitinase is used to break them open and release the DNA.

The first step in isolating genetic material (DNA) is to break open the cell and its wall so that the DNA is released along with other macromolecules. Because different organisms have different cell-wall chemistry, different enzymes are used:

Bacteria → lysozyme (digests peptidoglycan) …
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CBSE 2026Set ANNUAL1 markMCQ
$Q. Assertion : Ligase enzyme is used to join a foreign gene with a cloning vector. Reason : Ligase forms phosphodiester bonds between complementary base pairs.$
(a) If both Assertion and Reason are true and Reason is a correct explanation of the Assertion.
(b) If both Assertion and Reason are true but Reason is not a correct explanation of the Assertion.
(c) If Assertion is true but Reason is false.
(d) If both Assertion and Reason are false.
›Reveal solutionSolution

Assertion (ligase joins gene to vector) is true, but the Reason is worded wrongly — ligase makes phosphodiester bonds in the backbone between adjacent nucleotides, not between base pairs — so the correct choice is (C).

Evaluating the Assertion: DNA ligase is indeed the enzyme that joins a foreign DNA fragment (gene) to a cloning vector, sealing them into a recombinant DNA molecule. So the Assertion is TRUE.

…

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CBSE 2026Set ANNUAL1 mark
$Q. Give reason for moving DNA fragments towards the anode in gel electrophoresis.$
›Reveal solutionSolution

DNA is negatively charged because of its phosphate backbone, so it migrates towards the positive electrode (anode) during gel electrophoresis.

In gel electrophoresis, DNA fragments are separated according to size by forcing them through an agarose gel in an electric field. Every nucleotide of DNA carries a negatively charged phosphate group in its sugar-phosphate backbone, so the whole DNA molecule is negatively charged.

…

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CBSE 2026Set ANNUAL1 markMCQ
$Q. The enzyme Taq polymerase used in PCR has been isolated from the bacterium :$
(a) Agrobacterium tumefaciens
(b) Thermus aquaticus
(c) Streptomyces albus
(d) Escherichia coli
›Reveal solutionSolution

Taq polymerase, the thermostable DNA polymerase used in PCR, comes from the hot-spring bacterium Thermus aquaticus, so (b) is correct.

In the CBSE/NCERT Biotechnology: Principles and Processes chapter, PCR (Polymerase Chain Reaction) heats DNA to ~94°C to denature it. An ordinary DNA polymerase would be destroyed at that temperature, so PCR uses a thermostable polymerase that survives repeated high-temperature cycles. This enzyme, Taq polymerase, is isolated from *Thermus aquaticus …

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CBSE 2025Set ANNUAL1 markMCQ
$Q.Match the process in column I with their uses in column II and choose the correct option: Column I \tColumn II A) ELISA \ti) Direct introduction of rDNA B) PCR \tii) Gene amplification C) Biolistic \tiii) Antigen-antibody interaction D) Micro-injection \tiv) Gold coated DNA$
(a) A(iv), B(iii), C(ii), D(i)
(b) A(i), B(ii), C(iii), D(iv)
(c) A(ii), B(i), C(iv), D(iii)
(d) A(iii), B(ii), C(iv), D(i)
›Reveal solutionSolution
ELISA uses antigen-antibody interaction, PCR amplifies genes, Biolistic uses gold-coated DNA particles, and Micro-injection directly introduces rDNA — matching gives A(iii), B(ii), C(iv), D(i).

Correct matching of each technique with its principle/use:

A) ELISA (Enzyme-Linked ImmunoSorbent Assay) — based on the specific antigen-antibody interaction (iii); used to detect the presence of an antigen (e.g., a pathogen protein) or antibody (e.g., in disease diagnosis, pregnancy tests) in a sample.

B) PCR (Polymerase Chain Reaction) — used for gene amplification (ii); makes multiple copies of a gene/DNA segment of interest in vitro, using the enzyme Taq polymerase. …
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CBSE 2025Set BOTANY1 markMCQ
$Q. Fill in the blank selecting the appropriate one: A mass of undifferentiated cells that develop from a totipotent cell by tissue culture is ____.$
(a) explant
(b) zygote
(c) callus
(d) plantlet
›Reveal solutionSolution

The undifferentiated proliferating cell mass formed from a totipotent cell in tissue culture is the callus.

Tissue culture exploits totipotency - the capacity of a single plant cell to regenerate a whole plant. A small piece of tissue (the explant) is placed on a sterile nutrient medium with growth hormones (auxin and cytokinin). It first divides to form an unorganised, undifferentiated mass of cells called the callus. On altering the hormone balance the callus differentiates into shoots and roots, giving tiny …

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CBSE 2025Set ZOOLOGY1 markMCQ
$Q. Which enzyme is used for joining the fragments of DNA?$
(i) Ligase
(ii) Polymerase
(iii) Endonuclease
(iv) Transferase
›Reveal solutionSolution

DNA ligase joins DNA fragments by catalysing the formation of phosphodiester bonds between their ends.

In recombinant DNA technology, a restriction endonuclease first cuts DNA at specific sites, producing fragments with sticky/blunt ends. To join the vector DNA with the foreign DNA fragment, the enzyme DNA ligase catalyses the formation of a phosphodiester bond between adjacent nucleotides, sealing the nicks — hence it is call …

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CBSE 2024Set 57/2/11 markMCQ
$Q. Which one of the following is not a feature of plasmids ? (A) Circular (B) Self-replicating (C) Single stranded (D) Extra-chromosomal$
›Reveal solutionSolution
Plasmids are typically circular, self-replicating, double-stranded DNA molecules found outside the main chromosome; therefore, being single-stranded is not a feature.

Plasmids are fascinating genetic elements, primarily found in bacteria, that play a crucial role in their adaptability and evolution. To understand which option is not a feature, we first need to grasp what plasmids are and their fundamental characteristics.

Imagine a bacterium with its main, large chromosome containing all the essential genes for survival. Now, picture smaller, independent loops of DNA floating around in the same cell. These smaller loops are plasmids. They carry non-essential but often beneficial genes, such as those for antibiotic resistance or the ability to degrade unusual compounds. Their unique structure and replication mechanism allow them to be easily transferred between bacteria, contributing to the rapid spread of traits like drug resistance.

Let's examine each option to determine which one does not describe a plasmid.

Analyze Option (A) Circular:

Plasmids are almost universally circular DNA molecules. This closed-loop structure provides stability and protects the DNA from degradation by enzymes that target linear DNA ends. This is a defining characteristic, especially in bacteria.

Analyze Option (B) Self-replicating:

Plasmids possess their own origin of replication (ori) and the necessary genes to initiate their own replication. This means they can replicate independently of the host cell's main chromosome. This self-replicating ability allows them to be maintained and passed on to daughter cells during cell division, often in multiple copies.

Analyze Option (C) Single-stranded: …
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CBSE 2024Set ANNUAL1 markMCQ
$Q. Assertion (A) : Biotechnology started with development of recombinant molecules. Reason (R) : Biotechnology mostly involves in cutting & pasting of desired parts of DNA.$
(a) Assertion (A) and Reason (R) both are true and Reason (R) is the correct explanation of Assertion (A).
(b) Assertion (A) and Reason (R) both are true, but Reason (R) is not the correct explanation of Assertion (A).
(c) Assertion (A) is true, but Reason (R) is false.
(d) Assertion (A) and Reason (R) both are false.
›Reveal solutionSolution

Modern biotechnology's origin is tied to genetic engineering — cutting and joining DNA pieces to make recombinant DNA molecules.

Modern biotechnology, in the genetic-engineering sense, is considered to have begun with the development of techniques to construct recombinant DNA molecules — i.e., DNA that combines genetic material from more than one source. This is achieved fundamentally through 'cutting' DNA at specific sites using restriction enzymes and 'pasting'/joining desired fragments using DNA ligase. This cut-and-paste ability of des …

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Q.Write the role of 'ori' and restriction site in the cloning vector pBR322.

The Role of 'ori' (Origin of Replication)

The Role of Restriction Sites

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