Gene Cloning

Isolation of DNA involves four main steps:

1. Cell disruption — the cells or tissues are broken open to release DNA. This may be done mechanically or enzymatically.
2. Protection from nucleases — the extracted material is protected from degrading enzymes like DNases by using a suitable buffer containing EDTA and mild alkaline conditions.
3. Separation of DNA from other molecules — proteins and other cell components are removed by treating with detergents like SDS, then chloroform-isoamyl alcohol is used for deproteinisation.
4. Precipitation and concentration — DNA in the aqueous phase is precipitated by adding ethanol or isopropanol, then collected by centrifugation.

Finally, RNase A may be added to remove RNA, and the DNA is stored at low temperature.

A biological detergent helps in lysis of the cell membrane or cell wall so that nucleic acids are released into the extraction medium. For example, SDS disrupts the lipid bilayer of membranes, and in plants CTAB also helps in breaking the tough cell wall. In addition, detergents like SDS help denature proteins and prevent nuclease activity, protecting the nucleic acid.

DNA isolation from plant tissue differs from that of bacterial cells in these ways:

- Bacterial cells are easier to lyse because they have little structure beyond the cell wall and cell membrane.
- In bacteria, lysozyme digests the peptidoglycan cell wall, and SDS lyses the membrane.
- Plant cells have a tough cell wall and are mechanically broken, usually by grinding or blending.
- For plant DNA isolation, CTAB is used as detergent.
- Plant tissues contain more polysaccharides and polyphenols, which interfere with DNA isolation; PVP is added to neutralise phenols.

So, plant DNA extraction needs stronger mechanical treatment and special reagents, while bacterial DNA extraction is comparatively simpler.

There are three types of restriction enzymes: Type I, Type II and Type III.

Not all REs can be used in rDNA technology. Only Type II restriction enzymes are commonly used in recombinant DNA technology because they cleave within the recognition site and have only DNA strand cleavage activity, which makes them useful for precise cutting of DNA.

Type I and Type III enzymes need additional cofactors and cut at sites away from the recognition sequence, so they are not as suitable for routine rDNA work.

The main challenges in nucleic acid extraction are:

- Very low availability of DNA and RNA in cells compared with proteins, carbohydrates and lipids.
- Large length of nucleic acids, which makes them easily cleaved by physical stress.
- Nucleic acids are also vulnerable to chemical agents because of the chemical bonds in their structure.
- During extraction, released nucleases can degrade nucleic acids, especially RNases, which are abundant and difficult to remove completely.

So, the major problems are low quantity, fragility, and degradation by nucleases.

The roles are:

- Alkaline phosphatase: removes the terminal phosphate group from the 5' end of DNA strands.
- DNA ligase: joins DNA fragments by forming a phosphodiester bond between the 5'-phosphate and 3'-OH ends.
- Terminal transferase: adds similar nucleotide residues to the 3' end of DNA to form a homopolymer tail, and it does not require a template.

These enzymes are important for modifying, joining and preparing DNA molecules in recombinant DNA technology.

A chelating agent such as EDTA binds divalent cations like **Mg$^{2+}$ and Mn$^{2+}$. This is important because these ions are required by nucleases. By removing them, EDTA prevents nuclease action** and protects DNA from degradation. It also prevents the formation of salts with phosphate groups of nucleic acids.

The modes of DNA transfer into the host are:

1. Transformation — direct uptake of exogenous DNA from the surroundings through the cell membrane; the recipient cells are called transformants.
2. Transduction — transfer of foreign DNA into bacteria by bacteriophages; the recipient cells are called transductants.
3. Conjugation — transfer of DNA from one bacterium to another by direct cell-to-cell contact, usually through an F plasmid and a pilus.

In biotechnology, foreign DNA can also be introduced by methods such as calcium chloride treatment, electroporation, lipofection, microinjection and gene gun.

Blue-white selection is based on the expression of the lacZ gene. When lacZ is intact, colonies are blue; when it is disrupted by insert DNA, colonies are white. So the correct statement is (b).

The correctly matched pair is Western blot: Detect specific proteins. Southern blot detects DNA, Northern blot detects RNA, and Eastern blot is for certain post-translational modifications of proteins, not RNA transcriptional modifications. So the correct option is (c).