Gene Cloning — NCERT Solutions

From the chapter, PstI is not matched with Pyrococcus stuartii. The chapter gives examples like Taq polymerase from Thermus aquaticus and Pfu polymerase from Pyrococcus furiosus; HindIII is from Haemophilus influenzae. So the incorrect pair is PstI: Pyrococcus stuartii.

Recombinants are screened by comparing the recombinant and non-recombinant cells using differences in their traits.

1. Direct selection of recombinants

In this method, transformed cells are identified by a selectable marker, usually antibiotic resistance. For example, only bacteria carrying a plasmid with an antibiotic-resistance gene survive on medium containing that antibiotic, while non-transformed cells die.

2. Selection by insertional inactivation

This is more efficient. The vector has two markers, such as amp$^{R}$ and tet$^{R}$, or amp$^{R}$ and lacZ.

• If the insert is cloned into one marker gene, that gene becomes inactivated.
• Example with amp$^{R}$ / tet$^{R}$:
• Cells are first grown on tetracycline so only transformed cells survive.
• Then colonies are replica-plated on ampicillin.
• Colonies that were present on the master plate but absent on replica plate are recombinants, because the insert disrupted amp$^{R}$.

3. Blue-white selection

This uses lacZ and X-gal/IPTG.

• lacZ produces β-galactosidase.
• If lacZ is intact, X-gal is cleaved and colonies become blue.
• If the insert disrupts lacZ, β-galactosidase is not formed and colonies remain white.
• White colonies are recombinants.

Thus, screening is done using selectable markers and insertional inactivation methods.

Southern blotting

• Detects a specific DNA sequence.
• DNA is cut with restriction enzymes, separated on agarose gel, transferred to a membrane, and identified using a labelled DNA probe.

Northern blotting

• Detects specific RNA molecules.
• RNA is separated on agarose gel, transferred to a membrane, and detected using a labelled probe complementary to the RNA.

Western blotting

• Detects specific proteins.
• Proteins are separated by SDS-PAGE, transferred to a membrane, and identified using a specific antibody.

Main difference

• Southern = DNA
• Northern = RNA
• Western = protein

Polymerase Chain Reaction (PCR) is a technique used to make millions of copies of DNA from a very small amount of starting DNA.

Principle

It is based on the natural process of DNA replication and requires a heat-stable DNA polymerase, primers, and dNTPs.

Components

• Template DNA
• Two primers: forward and reverse
• Taq polymerase
• dNTPs
• Buffer and Mg$^{2+}$

Steps of PCR

1. Denaturation: DNA is heated to about 94–95°C so the two strands separate.
2. Annealing: Temperature is lowered to about 50–55°C so primers bind to complementary sequences.
3. Extension: Temperature is raised to about 72°C so Taq polymerase adds nucleotides to the 3'-OH end of the primers.

These steps are repeated for 25–30 cycles. Since the number of DNA copies doubles each cycle, the product increases exponentially, producing amplicons.

Types

• Conventional PCR: product analysed by gel electrophoresis.
• RT-PCR: RNA is first converted into cDNA using reverse transcriptase, then amplified.
• Real-time qPCR: product is measured during each cycle using fluorescent markers such as SYBR Green.

Applications

• Amplification of small DNA samples
• Gene expression analysis
• Crime scene and fossil DNA analysis
• Detection of viral nucleic acids such as SARS-CoV-2

Genomic library

• A collection of clones of DNA fragments that together represent the complete genome of an organism.
• Made by isolating genomic DNA, cutting it with restriction enzymes, inserting fragments into vectors, and cloning them in host cells.
• Contains both expressed and unexpressed sequences.
• Useful for genome sequencing, studying evolution, and identifying genes not expressed in a tissue.

cDNA library

• A collection of clones of cDNA made from the mRNA of a particular tissue or cell type.
• Made by isolating mRNA, converting it into cDNA using reverse transcriptase, then making it double-stranded and cloning it into vectors.
• Contains only genes that were expressed in that tissue or cell type.
• Useful for finding active genes in a tissue and for screening genomic libraries.

Comparison

• Source: genomic DNA vs mRNA
• Content: all genes vs expressed genes only
• Introns: present in genomic library, absent in cDNA library
• Use: whole genome studies vs gene expression studies

The chapter states that the haploid human genome contains about 3.2 billion bp, i.e. $3.2 \times 10^9$ base pairs. Among the given options, that is the correct choice.

EcoRI is a Type II restriction enzyme, not Type I. The other matches are correct: nucleases hydrolyse phosphodiester bonds; restriction enzymes cleave DNA at specific sequences; palindromic sequences read the same forward and backward on complementary strands.

The assertion is true because PCR can amplify a very small amount of DNA into many copies. The reason is also true because Taq polymerase is the heat-stable enzyme used in PCR. Since PCR works through repeated cycles using Taq polymerase, the reason correctly explains the assertion.

The assertion is true: foreign genes can be introduced into bacteria by methods such as electroporation. The reason is also true because bacteria have a cell wall and membrane, which must be crossed or temporarily made permeable for DNA entry. This is why such transformation methods are used, so the reason correctly explains the assertion.