Steps of DNA Cloning
DNA cloning process is divided into 3 major steps: Cutting and pasting DNA, Bacterial Transformation and Selection, Protein Production.
Cutting and Pasting DNA
- Isolation of DNA: The target DNA sequence, usually a gene of interest, is isolated from the source organism’s genome. This can be done using various methods like PCR (Polymerase Chain Reaction), restriction enzyme digestion, or chemical extraction.
- Selection of Vector: A vector is chosen based on the intended purpose of the cloning experiment. Common vectors include plasmids, bacteriophages, or artificial chromosomes.
- Preparation of Vector: The vector DNA is prepared by treating it with restriction enzymes, which cut the DNA at specific recognition sequences. This creates “sticky ends” that can base pair with complementary ends of the target DNA.
- Ligation: The target DNA and the vector DNA are mixed together with DNA ligase, an enzyme that catalyzes the formation of phosphodiester bonds between adjacent nucleotides. This results in the fusion of the target DNA with the vector DNA to create a recombinant DNA molecule.
Also Read: Ti-Plasmid : Features, Diagram and Uses
Bacterial Transformation and Selection
- Transformation: The recombinant DNA molecule is introduced into a host organism, usually bacteria or yeast, through a process called transformation. The host organism takes up the recombinant DNA and replicates it with its own genome.
- Selection and Screening: Selective pressure, such as antibiotic resistance encoded by the vector, is applied to ensure that only host cells containing the recombinant DNA survive. Screening techniques like PCR, restriction enzyme digestion, or DNA sequencing are then used to identify host cells that have successfully incorporated the target DNA.
Protein Production
- Propagation: The host cells containing the recombinant DNA are cultured under appropriate conditions to allow for the production of multiple copies of the cloned DNA.
- Harvesting: The cloned DNA can be harvested from the host cells using various methods, such as cell lysis and purification kits, depending on the scale and downstream applications.
DNA Cloning – An Overview
DNA cloning is an essential technique in molecular biology that enables researchers to produce multiple copies of a specific DNA sequence. It involves isolating a target DNA fragment and inserting it into a vector (such as a plasmid or a viral genome), which is then introduced into a host organism (usually bacteria or yeast) to produce multiple copies of the inserted DNA. In this article, find the DNA cloning notes and diagram.
Table of Content
- DNA Cloning Definition
- What is DNA Cloning?
- Diagram of DNA Cloning
- Methods of DNA Cloning
- Steps of DNA Cloning
- Application of Gene Cloning
- DNA Cloning Vector
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