What is cloning? Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. It describes the processes used to create an exact genetic replica of another cell, tissue or organism. The copied material, which has the same genetic makeup as the original, is referred to as a clone. When is cloning done? Cloning is done to amplify the DNA fragments containing the gene of interest and ensures pure sample of gene. When the concentration of the required gene products (proteins) is very low, the production of multiple clones makes it easy to obtain large quantity of the gene products. What all can be cloned? Cloning is a powerful techninque that can be implemented to obtain clones of genes (molecular cloning), cells (cell cloning) and organisms(organism cloning). There are three different types of cloning: Gene cloning, which creates copies of genes or segments of DNA Reproductive cloning, which creates copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. What are the requisites for cloning? In biotechnology, cloning requires a gene of interest, a vehicle (vectors) to carry the gene, a host to provide an environment for multiplication of genes and a medium for growth of the host strain. What is DNA cloning? DNA cloning is the procedures to produce multiple copies of a single gene or segment of DNA. a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA. What are the steps involved in cloning? The major steps involved in cloning a gene are: 1. Preparation of carrier DNA (vector DNA). 2. Isolation of the desired gene. 3. Insertion of the isolated gene into the vector (which results in the rDNA). 4. Transformation of rDNA into a suitable host. 5. Expression of rDNA(cloned gene). CLONING VECTORS- Cloning vectors are self replicating DNA , that is used to carry our gene of interest into a host system where the gene is then expressed. There are many cloning vectors used in cloning procedures. Usually, the following are used : 1) Plasmids. 2) Bacteriophages 3) Bacterial artificial chromosomes 4) Yeast artificial chromosome. Plasmids: Plasmids are self replicating, double stranded, extra chromosomal , covalently linked circular DNA. They range in size from 5000 to 400000 bp. They can be introduced into bacterial cells by a process called transformation. The usual strategy is to use a plasmid that includes a gene that the host cell requires for growth under specific conditions, such as a gene that confers resistance to an antibiotic. Only cells transformed by the recombinant plasmid can grow in the presence of that antibiotic, making any cell that contains the plasmid “selectable” under those growth conditions. We can insert and clone effectively upto 15000 bp sized DNA fragment. Bacteriophage λ: Bacteriophage λ has a very efficient mechanism for delivering its 48,502 bp of DNA into a bacterium, and it can be used as a vector to clone somewhat larger DNA segments . Two key features contribute to its utility: 1) About one-third of the λ genome is nonessential and can be replaced with foreign DNA. 2) DNA is packaged into infectious phage particles only if it is between 40,000 and 53,000 bp long, a constraint that can be used to ensure packaging of recombinant DNA only. Bacteriophage λ vectors permit the cloning of DNA fragments of up to 23,000 bp. Bacterial Artificial Chromosomes (BACs): Bacterial artificial chromosomes are simply plasmids designed for the cloning of very long segments (typically 100,000 to 300,000 bp) of DNA . They include specific marker-like sites such as resistance to the antibiotic chloramphenicol (CmR), as well as a very stable origin of replication (ori) that maintains the plasmid at one or two copies per cell. DNA fragments of several hundred thousand base pairs can be cloned into the BAC vector. Yeast Artificial Chromosomes (YACs): The genome of the most commonly used yeast, Saccharomyces cerevisiae, contains only 14 x 106 bp (less than four times the size of the E. coli chromosome), and its entire sequence is known. It helps us to the study of many aspects of eukaryotic cell biochemistry. YAC vectors contain all the elements needed to maintain a eukaryotic chromosome in the yeast nucleus: a yeast origin of replication, two selectable markers. Those with inserts of more than 150,000 bp are nearly as stable as normal cellular chromosomes, whereas those with inserts of less than 100,000 bp are gradually lost during mitosis. |
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