(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.

Gujarat GsebCBSE 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 20 on this concept.

GUJCET 2026Set 051 markMCQ
$Q. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as _______. (A) Elution (B) Micro-injection (C) Biolistics (D) Extension$
›Reveal solutionSolution

Extracting separated DNA bands from excised gel pieces = elution.

In gene cloning, the separated DNA bands are cut out from the agarose gel and the DNA is extracted from the gel piece — this step is elution. The eluted DNA fr …
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GSEB Higher Secondary Certificate (HSC) Examination 2026Set ANNUAL1 markMCQ
$Q. If we cut the vactor pBR322 at the place of Bam - HI and make a recombinant DNA from it. Then for which antibitic it remains resistance?$
(a) Ampicillin
(b) Tetracycline
(c) Kanamycin
(d) Chloramphenicol
›Reveal solutionSolution

pBR322 has two antibiotic-resistance genes; BamHI cuts inside the tetR gene, so a recombinant made at that site loses tetracycline resistance but keeps ampicillin resistance (used for selection via insertional inactivation).

The cloning vector pBR322 carries two selectable marker genes: ampR (ampicillin resistance) and tetR (tetracycline resistance), each containing unique restriction sites used for inserting foreign DNA. BamHI's recognition site is located within the tetR gene. If a foreign DNA fragment is inserted at the BamHI site, it disrupts (inactivates) the tetR gene - so recombinant clones …

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GSEB Higher Secondary Certificate (HSC) Examination 2025Set ANNUAL1 markMCQ
$Q. Which scientists isolated the antibiotic-resistance gene and constructed r-DNA (recombinant DNA)?$
(a) Ernest Chain and Howard Florey
(b) Stanley Cohen and Herbert Boyer
(c) James Watson and Francis Crick
(d) François Jacob and Jacques Monod
›Reveal solutionSolution

Cohen and Boyer (1972) constructed the first recombinant DNA by isolating an antibiotic-resistance gene and inserting it into a plasmid vector.

Stanley Cohen (Stanford) and Herbert Boyer (UCSF) isolated a gene that conferred resistance to the antibiotic tetracycline from a Salmonella plasmid and linked it into a plasmid vector (from E. coli) using restriction enzymes and DNA ligase. This recombinant plasmid, when introduced into E. coli, replicat …

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GSEB Higher Secondary Certificate (HSC) Examination 2025Set ANNUAL1 markMCQ
$Q. The ampR gene carries a recognition site for __________.$
(a) Pvu II
(b) Hind III
(c) Bam HI
(d) Pst I
›Reveal solutionSolution

In the cloning vector pBR322, the PstI recognition site lies within the ampicillin-resistance (ampR) gene, allowing insertional inactivation as a screening method.

pBR322 has two antibiotic-resistance genes, ampR and tetR, each containing unique restriction sites that are used for inserting foreign DNA and screening recombinants by insertional inactivation. The ampR gene carries a PstI recognition site — inserting foreign DNA at this PstI site disrupts (inactivates) the ampR gene, so recombinant colonies lose am …

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GSEB Higher Secondary Certificate (HSC) Examination 2025Set ANNUAL1 markMCQ
$Q. The first recombinant DNA was constructed in which microorganism?$
(a) Salmonella typhimurium
(b) Vibrio cholerae
(c) E. coli
(d) Staphylococci
›Reveal solutionSolution

The first recombinant DNA was constructed using the native plasmid of Salmonella typhimurium. The correct option is (a).

The construction of the first recombinant DNA (Stanley Cohen and Herbert Boyer, 1972) came from linking a gene encoding antibiotic resistance with a native plasmid — an autonomously replicating circular extra-chromosomal DNA — of Salmonella typhimurium. The recombinant plasmid so constructed was afterwards introduced into Escherichia coli, where it replicated to make many copies; but E. coli was the propagation host, not …

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GUJCET 2024Set 101 markMCQ
$Q. In which system the specific mRNA becomes silent after complementary to dsRNA molecule? (A) ELISA (B) PCR (C) RNAi (D) DNAi$
›Reveal solutionSolution

Silencing of a specific mRNA by a complementary dsRNA is RNA interference.

In RNAi, a double-stranded RNA complementary to a target mRNA binds it and prevents its translation, effectively silencing that gene. This is used, …
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GSEB Higher Secondary Certificate (HSC) Examination 2024Set ANNUAL1 markMCQ
$Q. The ampR gene contain the recognition sites for which endonucleases in pBR 322 ___________.$
(a) Pvu II and Pvu I
(b) Pst I and Pvu I
(c) Sal I and BamH I
(d) Pst I and Sal I
›Reveal solutionSolution

On the pBR322 cloning vector map, the ampicillin-resistance gene carries unique recognition sites for the restriction enzymes PstI and PvuI, which is exactly why inserting foreign DNA at either site inactivates ampicillin resistance (used for insertional-inactivation screening).

pBR322 is a classic cloning vector engineered with an origin of replication (ori), two selectable antibiotic-resistance genes (ampR and tetR), and multiple unique restriction sites mapped across the plasmid. The ampR gene specifically contains recognition sites for PstI and PvuI; cutting and inserting foreign DNA at one of these sites disrupts (inactivates) the ampR gen …

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GUJCET 2023Set 071 markMCQ
$Q. In extraction of genetic material (DNA), which enzyme is not used? (A) Ribonuclease (B) Cellulose (C) Protease (D) Both Protease and Ribonuclease$
›Reveal solutionSolution
DNA isolation uses protease and ribonuclease; cellulose is not an enzyme at all.

Concept — DNA extraction. To purify DNA, cells are treated with enzymes: protease (digests proteins) and ribonuclease (removes RNA); plant cell walls are broken with cellulase.

Steps.

Ribonuclease — used (removes RNA).

Protease — used (removes protein). …
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GSEB Higher Secondary Certificate (HSC) Examination 2023Set ANNUAL1 markMCQ
$Q. Select the correct option for containing the antibiotic resistance genes in E. coli cloning vector pBR322.$
(a) Pvu II, tet^R
(b) Bam HI, Sal I
(c) amp^R, tet^R
(d) Hind III, amp^R
›Reveal solutionSolution
pBR322 has two selectable-marker genes, ampR (ampicillin resistance) and tetR (tetracycline resistance).

pBR322 is a classic E. coli cloning vector. It contains:

ampR: gene for ampicillin resistance.

tetR: gene for tetracycline resistance.

An origin of replication (ori) and recognition sites for restriction enzymes (e.g. within tetR). …
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GSEB Higher Secondary Certificate (HSC) Examination 2023Set ANNUAL1 markMCQ
$Q. Select correct sequence for the process of VNTR technique. X = Hybridisation using labelled VNTR probe. Y = Digestion of DNA by restriction endonucleases. Z = Transferring (blotting) of separated DNA fragments to synthetic membrane, such as nitrocellulose.$
(a) Y -> X -> Z
(b) Z -> X -> Y
(c) Y -> Z -> X
(d) X -> Z -> Y
›Reveal solutionSolution
DNA fingerprinting by VNTR: restriction digestion (Y) -> Southern blotting/transfer (Z) -> hybridisation with a labelled VNTR probe (X).

Steps:

Y: Isolate DNA and digest it with restriction endonucleases into fragments.

(Electrophoresis separates the fragments by size.)

Z: Transfer (blot) the separated fragments onto a synthetic membrane such as nitrocellulose (Southern blotting). …
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GSEB Higher Secondary Certificate (HSC) Examination 2023Set ANNUAL1 markMCQ
$Q. To cure ADA deficiency, as a first step towards gene therapy, which cells from the blood of the patient are grown in culture outside the body?$
(a) Platelets
(b) WBCs
(c) Lymphocytes
(d) RBCs
›Reveal solutionSolution

In ADA-deficiency gene therapy, the patient's lymphocytes are grown in culture, given a functional ADA gene, and returned to the body.

Adenosine deaminase (ADA) deficiency causes severe immunodeficiency. As a first step of gene therapy, lymphocytes from the patient's blood are grown in culture outside the body; a functional ADA gene (introduced via a retroviral vector) is inserted, and the cells are return …

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GUJCET 2022Set 171 markMCQ
$Q. Statement A - DNA cannot pass through cell membrane Reason R - DNA is a hydrophobic molecule (A) Statement A is correct and Reason R is wrong (B) Statement A and Reason R both are correct because R is not the explanation of A (C) Statement A and Reason R both are correct because R is the explanation of A (D) Statement A is wrong and Reason R is correct$
›Reveal solutionSolution
A is correct; the reason given is wrong.

Concept. DNA is a large, negatively charged (phosphate backbone) hydrophilic molecule, so it cannot passively cross the lipid cell membrane.

Statement A — correct: DNA cannot pass through the cell membrane. …
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Q.(a) 'Insertional inactivation' is a method to detect recombinant DNA. Explain the method.

Part (a)

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