(a) 'Insertional inactivation' is a method to detect recombinant DNA. Explain the method. OR (b) Explain how r

Question 67 of 78

Q. (a) 'Insertional inactivation' is a method to detect recombinant DNA. Explain the method.

(OR)

(b) Explain how recombinant DNA technology is used to detect a disease even before any clinical symptom appears.

CBSECBSE Class XII Board 2023Subjective· 2mImportance★★★★★

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Part (a)Concept understanding — Cloning Vectors and Selectable Markers (pBR322)

Cloning Vectors and Selectable Markers (pBR322)

A cloning vector is a DNA molecule that can carry a foreign DNA fragment into a host cell and replicate there. Plasmids such as pBR322, an early and widely used E. coli cloning vector, are drawn as circular DNA maps and have several essential features that a good vector must possess.

Origin of replication (ori): the sequence at which replication starts; it controls copy number (how many copies of the plasmid are made per cell). In pBR322 the rop gene near the ori helps regulate this copy number.
Selectable markers: genes that allow cells carrying the vector to be distinguished from those that do not, usually by giving antibiotic resistance. pBR322 carries two such genes — ampicillin resistance ( $amp^R$ ) and tetracycline resistance ( $tet^R$ ). …

Part (b)Concept understanding — Recombinant DNA Technology

Imagine you have a cookbook with recipes from all over the world. Normally, you can only cook what's in one book at a time. But what if you could cut out the best recipe from one book and paste it into another, so your new book has a dish that no single cuisine ever had before? That is the basic idea behind Recombinant DNA Technology.

At its simplest, this technology is a way to take a piece of DNA (the genetic instruction manual) from one organism and join it with the DNA of a completely different organism. The result is a new, "recombinant" DNA molecule — a hybrid that never existed in nature. Think of it as genetic tailoring: you cut a gene from a human, a bacterium, or a plant, and stitch it into the DNA of another organism, often a bacterium or yeast. That host organism then reads the new instructions and starts producing the protein the inserted gene codes for.

Why does this matter? Because it lets us manufacture things that living organisms naturally make, but in a controlled, large-scale way. For example, the human insulin gene can be inserted into E. coli bacteria. These bacteria then become tiny factories, churning out human insulin that can be purified and given to diabetic patients. Before this technology, insulin had to be extracted from the pancreases of cows and pigs — a slow, expensive, and sometimes allergenic process.

The NCERT textbook (Class 12 Biology, Chapter 11) defines it precisely: Recombinant DNA Technology is the technique of joining DNA from two different species and inserting it into a host organism to produce a new genetic combination. The textbook highlights three key tools that make this possible:

Restriction Enzymes – These are the "molecular scissors" that cut DNA at specific, predictable points. They allow scientists to cut out a desired gene cleanly.
Vectors – These are the "delivery vehicles," usually plasmids (small circular DNA in bacteria) or viruses, that carry the foreign DNA into the host cell.
Host Organisms – The living factory (like bacteria, yeast, or plant cells) that will replicate the recombinant DNA and produce the desired protein.

Important

The core principle is genetic recombination — creating a DNA molecule that contains sequences from two or more different sources. This is not the same as natural reproduction or mutation; it is a deliberate, laboratory-made hybrid.

The process itself follows a clear sequence:

Isolation of the desired gene (say, the human insulin gene) from the donor organism's DNA.
Cutting both the gene and the vector DNA with the same restriction enzyme, creating matching "sticky ends."
Ligation — using an enzyme called DNA ligase to permanently join the gene and the vector, forming the recombinant DNA.
Transformation — inserting this recombinant DNA into a host cell (like a bacterium).
Selection — identifying and growing only those host cells that successfully took up the recombinant DNA.
Expression — getting the host cells to produce the desired protein in large quantities.

Note

A common confusion is thinking this technology creates "new life." It does not. It creates a new genetic combination inside an existing living cell. The host organism remains the same species, but it now carries an extra instruction — like a factory that gets a new blueprint for a product it never made before. …

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Part (a): Insertional inactivation clones foreign DNA into a marker gene so the insertion destroys that gene's function, letting recombinants be selected as Amp-resistant/Tet-sensitive (or white lacZ colonies).

Part (b): PCR amplifies the suspect sequence and a labelled complementary DNA probe hybridises to it, revealing a mutation or low-level pathogen before symptoms appear.

Part (a)

Concept-first idea: After ligation we need to tell recombinant plasmids (carrying insert) from non-recombinant ones. Insertional inactivation does this by making a successful insertion destroy a detectable marker.

Method.

Use a vector such as pBR322 carrying two antibiotic-resistance genes — e.g., ampicillin resistance (amp^R) and tetracycline resistance (tet^R).
Insert foreign DNA at a restriction site located within one marker (say inside tet^R).
The insertion interrupts and inactivates tet^R, so the recombinant cell can no longer make functional tetracycline-resistance protein, but amp^R stays intact.
Select transformants on ampicillin (only cells with a plasmid survive), then replica-plate onto tetracycline:
- Non-recombinants (empty vector) grow on both antibiotics.
- Recombinants grow on ampicillin but not tetracycline.
Colonies that are Amp-resistant, Tet-sensitive carry the recombinant DNA. …

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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 43 on this concept.

CBSE 2026Set 57/1/11 markMCQ
$Q. Which of the following statements about plasmids is incorrect ? (A) Plasmids have the ability to replicate within the bacterial cell. (B) Their replication is controlled by chromosomal DNA. (C) They are autonomously replicating circular extra-chromosomal DNA. (D) They often carry antibiotic resistant genes.$
›Reveal solutionSolution
The incorrect statement about plasmids is that their replication is controlled by chromosomal DNA; plasmids replicate autonomously.

Plasmids are fascinating and crucial components in the world of microbiology and biotechnology. They are essentially small, circular, extra-chromosomal DNA molecules found predominantly in bacteria, but also in some eukaryotes like yeast. Think of them as accessory genetic units that carry non-essential but often beneficial genes for the host cell.

Let's break down the given statements to understand which one is incorrect:

Statement (A): Plasmids have the ability to replicate within the bacterial cell.

This statement is correct. A defining feature of plasmids is their ability to self-replicate. They possess their own origin of replication (ori) sequence, which allows them to initiate DNA synthesis independently of the main bacterial chromosome. This autonomous replication is what makes them incredibly useful as vectors in genetic engineering, as they can multiply along with the host cell, carrying the inserted foreign DNA.

Statement (B): Their replication is controlled by chromosomal DNA.

This statement is incorrect. As mentioned above, plasmids replicate autonomously. This means their replication is independent of the bacterial cell's main chromosomal DNA replication. While the host cell's machinery (enzymes, nucleotides) is used for plasmid replication, the initiation and control of plasmid replication are governed by sequences within the plasmid itself, not by the bacterial chromosome. This independence is a key distinction between plasmids and the main chromosome.

Important

The autonomous replication of plasmids, independent of the host cell's chromosomal DNA, is a fundamental characteristic that distinguishes them and makes them invaluable tools in molecular biology.

Statement (C): They are autonomously replicating circular extra-chromosomal DNA.

This statement is correct. This is a precise definition of a plasmid.

Autonomously replicating: They have their own origin of replication and can replicate independently.

Circular: Most plasmids are found in a closed circular form. …
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CBSE 2026Set 57/2/11 markMCQ
$Q. Assertion (A) : DNA Ligase is used to join DNA fragments. Reason (R) : It catalyses the formation of glycosidic bonds between nucleotides. (A) Both Assertion (A) and Reason (R) are true and Reason (R) is the correct explanation for Assertion (A). (B) Both Assertion (A) and Reason (R) are true, but Reason (R) is not correct explanation for Assertion (A). (C) Assertion (A) is true, Reason (R) is false. (D) Assertion (A) is false, Reason (R) is true.$
›Reveal solutionSolution
DNA Ligase joins DNA fragments by catalysing phosphodiester bonds, not glycosidic bonds. So Assertion is true, Reason is false — option (C).

The question tests a very specific piece of molecular biology: what bond does DNA Ligase actually form? Many students memorise "DNA Ligase joins fragments" but forget the exact chemistry. Let's break it down cleanly.

Why the Assertion is correct

DNA Ligase is the enzyme used in DNA replication, repair, and genetic engineering to seal nicks in the DNA backbone. It joins two DNA fragments — for example, an Okazaki fragment to the growing lagging strand, or an insert into a plasmid vector. This is a standard fact in biotechnology and molecular biology.

Why the Reason is wrong

The Reason claims DNA Ligase catalyses the formation of glycosidic bonds between nucleotides. That is a critical error.

A glycosidic bond links a sugar (deoxyribose) to a nitrogenous base (A, T, G, C) — that bond is formed during nucleotide synthesis, not by DNA Ligase.

DNA Ligase catalyses the formation of a phosphodiester bond between the 3'-hydroxyl group of one nucleotide and the 5'-phosphate group of the next nucleotide. This is the bond that holds the sugar-phosphate backbone together.

Watch out

A common mistake is confusing "joining nucleotides" with "forming the bond between sugar and base." The bond between nucleotides in a DNA strand is always phosphodiester, never glycosidic.

Step-by-step reasoning

Evaluate Assertion (A): "DNA Ligase is used to join DNA fragments." …
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CBSE 2026Set A1 markMCQ
$Q. Which of the following is the sequence from where replication starts in cloning vector?$
(a) tet^R
(b) amp^R
(c) ori
(d) more than one
›Reveal solutionSolution

ori is the origin of replication; the correct option is (c).

A cloning vector must be able to replicate inside the host. The 'ori' (origin of replication) is the specific DNA sequence from which replication begins; any DNA linked to it can be made to replicate within the host cell. The ori also controls the copy number of the linked DNA.

…

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CBSE 2026Set ANNUAL1 markMCQ
$Q. Given below are the steps carried out to construct a recombinant DNA.$
(i) Isolation of genetic material
(ii) Insertion of recombinant DNA in the host cell /organism
(iii) Obtaining the foreign gene product
(iv) Amplification of gene of interest
(v) Downstream processing. Which one of the following gives the correct sequences of these steps?
(a)
(i)
(iii)
(iv)
(ii)
(v)
(b)
(i)
(iv)
(ii)
(iii)
(v)
(c)
(ii)
(i)
(iii)
(iv)
(v)
(d)
(ii)
(iv)
(v)
(iii) (i)
›Reveal solutionSolution
Constructing and using recombinant DNA follows a fixed logical sequence: first isolate and amplify the gene of interest, then insert the recombinant DNA into a host, let the host express the gene product, and finally process that product for use — i.e., (i) → (iv) → (ii) → (iii) → (v).

Recombinant DNA technology (genetic engineering) involves several sequential processes:

(i) Isolation of genetic material (DNA) — the DNA must first be extracted in a pure form from the source cell, free of other macromolecules.

(iv) Amplification of the gene of interest — using restriction enzymes to cut out the desired gene and, typically, PCR to amplify it, then ligating it into a suitable vector to form the recombinant DNA.

(ii) Insertion of the recombinant DNA into the host cell/organism — the recombinant DNA (vector + gene of interest) is introduced into a competent host cell (transformation) and the host is then cultured/multiplied so the gene is expressed. …
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CBSE 2025Set ANNUAL1 markMCQ
$Q. Which one of the following is a genetic vector?$
(a) Phage
(b) Cosmid
(c) Plasmid
(d) Artificial chromosome
›Reveal solutionSolution

Plasmid is the most fundamental cloning/genetic vector; it is engineered with an origin of replication, selectable marker, and cloning site to carry foreign DNA into a host cell.

A cloning/genetic vector is a DNA molecule used as a vehicle to artificially carry a foreign DNA segment into a host cell, where it can be replicated and/or expressed. Plasmids are small, circular, double-stranded, extra-chromosomal DNA molecules found naturally in bacteria that replicate independently of the main bacterial chromosome. Engineered plasmids (e.g. pBR322) are the workhorse cloning vectors — they carry an origin of replication (ori), one or more selectable marker genes (e.g. antibiotic resistance), and unique restriction sites where foreign DNA is inserted.

…

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CBSE 2025Set ANNUAL1 mark
$Q. Why is it essential to have selectable marker in cloning vector?$
›Reveal solutionSolution

A selectable marker (e.g. an antibiotic-resistance gene) lets us identify and select only those host cells that have successfully taken up the vector, since transformation efficiency is naturally low.

When a recombinant DNA vector is introduced into host bacterial cells (transformation), only a small fraction of cells actually take it up — most cells remain untransformed. Since transformed and untransformed cells look identical, we need a way to selectively grow only the cells carrying the vector. This is achieved using a selectable marker gene on the vector — commonly a gene conferring resistance to an antibiotic (e.g. ampicillin resistance, tetracycline resistance) that the host bacterium is not normally resistant to. After transformation, cells are plated on medium containing that antibiotic: only cells that have taken up the vector (and hence the resistance gene) survive and grow, while untransformed cells are k …

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CBSE 2025Set ANNUAL1 markMCQ
$Q. Which one of the following is not required for rDNA technology ?$
(i) DNA polymerase
(ii) DNA ligase
(iii) Restriction endonuclease
(iv) Reverse transcriptase
›Reveal solutionSolution
Basic recombinant DNA technology always needs a cutting enzyme (restriction endonuclease), a joining enzyme (DNA ligase) and DNA-copying capability (DNA polymerase, e.g. for PCR); reverse transcriptase is only needed in the special case of starting from an RNA/mRNA template, so it is the one "not required" in general.

The core enzyme toolkit of rDNA technology includes:

Restriction endonucleases: "molecular scissors" that cut DNA at specific recognition sequences to isolate the gene of interest and to open up the vector for insertion — always required.

DNA ligase: joins ("glues") the cut gene of interest into the vector DNA to form the recombinant DNA molecule — always required.

DNA polymerase: needed for amplifying DNA (e.g. via PCR) and is also the enzyme the host cell itself uses to replicate the recombinant plasmid — required in essentially every rDNA procedure. …
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CBSE 2024Set 57/3/11 markMCQ
$Q. Which native plasmid did Stanley Cohen and Herbert Boyer use for the construction of the first recombinant DNA ? (A) Salmonella typhimurium (B) Streptococcus pneumoniae (C) Escherichia coli (D) Haemophilus influenzae$
›Reveal solutionSolution
The first recombinant DNA experiment used a plasmid from Salmonella typhimurium — specifically, a small, naturally occurring plasmid called pSC101 — which was cut with a restriction enzyme and spliced with foreign DNA. The correct option is (A).

The key to this question is remembering the historical landmark experiment by Cohen and Boyer in 1973. They weren't just using any E. coli plasmid — they deliberately chose a plasmid from a different bacterial species to demonstrate that recombinant DNA could be constructed across species boundaries.

Why not E. coli?

E. coli does have plasmids (like the famous pBR322, developed later), but Cohen and Boyer wanted to prove that DNA from one organism could be stably inserted into the plasmid of another. Using an E. coli plasmid would have been less convincing — it might have been argued that the foreign DNA was just recombining with native E. coli sequences. So they picked a plasmid from a different bacterium: Salmonella typhimurium.

The actual plasmid: pSC101

This plasmid was isolated from Salmonella typhimurium by Stanley Cohen. It was small (about 9.4 kb), had a single EcoRI restriction site, and carried a tetracycline-resistance gene — perfect for selection. Boyer’s team used EcoRI to cut both pSC101 and a piece of foreign DNA (from a frog or another bacterium), then ligated them together. The resulting recombinant plasmid was introduced into E. coli, where it replicated and expressed the foreign DNA.

Why the other options are wrong …
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CBSE 2024Set E1 markMCQ
$Q. Which of the following is not a cloning vector for bacteria?$
(a) Bacteriophage
(b) Plasmid
(c) pBR322
(d) T-DNA
›Reveal solutionSolution

Plasmids, bacteriophages and pBR322 are cloning vectors used in bacteria; T-DNA is the transferred DNA of the Ti plasmid used to transform plant cells.

Cloning vectors for bacteria include plasmids (option b), bacteriophages (option a) and engineered plasmids such as pBR322 (option c). T-DNA (transfer DNA) is the portion of the Ti (tumour-inducing) plasmid of Agrobacterium tumefaciens that is trans …

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CBSE 2024Set E1 markMCQ
$Q. Which of the following is absent in pBR322?$
(a) Origin of replication
(b) Restriction site
(c) T-DNA
(d) Antibiotic resistant genes
›Reveal solutionSolution

pBR322 carries ori + restriction sites + antibiotic-resistance selectable markers; T-DNA is not part of it.

pBR322 is a well-known bacterial cloning vector. Its essential features are: an origin of replication (ori) that allows it to replicate inside the host (option a), several unique restriction sites where foreign DNA can be inserted (option b), and two antibiotic-resistance genes — am …

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CBSE 2024Set E1 markMCQ
$Q. If a foreign DNA is inserted in tetracycline resistant gene, the recombinant plasmid$
(a) Will lose ampicillin resistance
(b) Will lose tetracycline resistance
(c) Cloning would become easier
(d) Ampicillin resistance will become stronger
›Reveal solutionSolution

Inserting DNA into the tetR gene disrupts it — the recombinant plasmid loses tetracycline resistance (insertional inactivation).

In pBR322, foreign DNA is often inserted at a restriction site (like BamHI) lying inside the tetracycline-resistance (tetR) gene. This insertion disrupts the gene so it can no longer function — a process called insertional inactivation. As a result, cells carrying the recombinant plasmid become sensitive to tetracycline wh …

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CBSE 2024Set D1 mark
$Q. Match the following and write the correct pair. Column 'A':$
(i) Structural genes
(ii) T-Lymphocytes
(iii) Competition
(iv) PBR322
(v) Corona radiata. Column 'B':
(a) bone marrow
(b) vectors
(c) ovum
(d) detrimental interaction
(e) z, y and a
(f) RBCs. Find the correct match for item
(iv) 'PBR322' from Column 'B'.
›Reveal solutionSolution

'PBR322' pairs with option (b) 'vectors' — it is one of the earliest and most widely used cloning vectors.

In recombinant DNA technology, a vector is a DNA molecule (commonly a plasmid or bacteriophage) used as a vehicle to artificially carry a foreign piece of DNA into a host cell, where it can be replicated and/or expressed.

…

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Q.(a) 'Insertional inactivation' is a method to detect recombinant DNA. Explain the method.

Part (a)

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