Step 1: DNA extracted from an organism, with the gene of interest, is cut into gene-size pieces with restriction enzymes. These enzymes read the nucleotide sequence of the DNA and recognize specific sequences. The enzymes then cut the DNA sequence by breaking the bonds between nucleotides in a DNA strand. Step 2: Bacterial plasmids are cut with 3- Restriction endonuclease (natural scissors) cut the insulin gene fragment by recognizing particular stretches of nucleotides. 1- DNA strand with the specific nucleotide sequence for Insulin chain A and chain B 2- Unraveling strand of the DNA of chromosome 11, with the exposed nucleotides coding for the A & B chain of Insulin Gene cloning involves removing a gene of interest from an organism and inserting that gene into a vector DNA molecule ( figure 12.2 ). The resulting recombinant DNA molecule is then introduced into a host for maintenance. The host can be a bacterium such as Escherichia coli, the yeast Saccharomyces cerevisiae, or in some cases, a virus. Overview of DNA cloning. DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. Molecular cloning is an essential technique to create DNA-based experimental tools for expression in bacterial or mammalian cells. Examples of such DNA constructs include a promoter element fused to a reporter gene or a cDNA sequence under the control of a ubiquitous promoter. Molecular cloning entails the preparation of the vector and insert The basic gene cloning steps are: 1. Cutting and Pasting DNA: A restriction enzyme that recognises a specific target sequence of DNA cuts it into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair with each other. 9. Fig. 10.2 Revealing the patterns of DNA with electrophoresis 1 2 34. 10. Analysis of DNA. 2 . 2 # /. 11. Analysis of DNA. 2 ' 2 1 . DNA Cloning takes place in the following steps: Cutting and Pasting DNA Two types of enzymes are used in this method: Restriction enzymes DNA ligase The restriction enzymes cut the DNA at specific target sequences. The target gene is inserted into the cut site and is ligated by DNA ligase. This is known as a recombinant plasmid. Top 13 Methods of Gene Transfer (With Diagram) Article shared by : The following points highlight the top thirteen methods of gene transfer. Some of the methods are: 1. Calcium Chloride (CaCl2) Mediated DNA Transfer 2. Rubidium Chloride Mediated DNA Transfer 3. Electroporation 4. Liposome Encapsulation (Lipofection) 5. Microinjection Gene Transfer: 2. Introduction • Gene cloning is a common practice in molecular biology labs • It is used to create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein. Gene cloning is the process in which a gene of interest is located and copied (cloned) out of all the DNA extracted from an organism. The basic steps in gene cloning are: DNA extracted from an organism known to have the gene of interest is cut into gene-size pieces with restriction enzymes. Bacterial plasmids are cut wi