Biotechnology | Part -2

Types of Endonucleases

Endonucleases are enzymes that cut DNA internally at specific sequences. These enzymes are essential tools in molecular biology and genetic modification. They are generally classified according to their source, how they recognize DNA sequences, and their mode of action:

• Type I Endonucleases: These enzymes have both restriction and modification activities. They cut DNA at sites distant from the recognition sequence and require ATP for activity.
• Type II Endonucleases: The most commonly used in biotechnology, these enzymes cleave DNA at or near their recognition sites. They do not require ATP and are highly specific.
• Type III Endonucleases: These recognize specific sequences but cut DNA a short distance away. They need ATP but do not hydrolyze it.

Basic Steps in Genetic Cloning

Genetic cloning refers to the technique used to produce identical copies of a particular gene or DNA fragment. It involves several precise steps:

1. Isolation of DNA: The gene of interest is identified and extracted from the source organism.
2. Insertion into a Vector: The isolated gene is inserted into a plasmid or other vector using restriction enzymes and ligase.
3. Transformation: The recombinant vector is introduced into a host cell, commonly a bacterium like E. coli.
4. Selection and Screening: Only successfully transformed cells are selected, often using antibiotic resistance markers.
5. Cloning and Expression: The cloned gene is replicated and may be expressed to produce the desired protein or trait.

Target of Polymerase Chain Reaction (PCR)

The primary target of PCR is a specific segment of DNA that needs to be amplified. This technique enables researchers to create millions of copies of a particular DNA region in a short amount of time. The key components of PCR include:

• Template DNA: The DNA containing the target sequence.
• Primers: Brief DNA strands that match the beginning and end portions of the specific region to be amplified.
• DNA Polymerase: Usually Taq polymerase, which synthesizes new DNA strands.
• Thermal Cycling: Repeated cycles of denaturation, annealing, and extension to amplify the DNA.

This makes PCR a powerful tool in diagnostics, forensic analysis, gene mapping, and genetic disease research.