By: Dr. Rajesh C. Gene cloning involves the formation of a DNA and its introduction into an acceptable host, resembling E. The host used ought to be without plasmids.
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By: Dr. Rajesh C. Gene cloning involves the formation of a DNA and its introduction into an acceptable host, resembling E. The host used ought to be without plasmids. The walls of host bacterium are created permeable by treatment with calcium chloride or a lysozyme. The DNA is additional to the culture within which such host bacterium are growing. The DNA is concerned by the bacterium together with the nutrients. It replicates whenever the bacterial cell divides. The bacteria E. This may produce an outsized clone of recombinant deoxyribonucleic acid.
In some cases, the bacterial cells additionallytranscribe the DNA molecules into messenger RNA, which is, in turn, translated into peptide chains. Hence, cloning may be utilized not solely to produce large quantities of the deoxyribonucleic acid sequence of an individual gene however additionally large amounts of the genes protein product furthermore.
Introduction Opening up a new horizon of research in genetics, gene cloning or the recombinant DNA rDNA technology, made a sensational beginning during s. With sky being the limit, this technology has the most diversifying applications which include production of food and other supplements, diagnosis of infection and different genetic diseases, prophylaxis and therapy against diseases, biofertilizers, pesticides, metal extractions, pollution control etc.
It is within the realm of gene from any living entity can be transferred into another life forms, be it a bacteria, yeast or fungi or mammal and the gene can be amplified immensely and the gene product harvested in leaps and bounds. Methods of cloning and menipulation The mechanics of gene manipulation dictates identification and isolation of the gene of interest, putting it in a host vector like bacteria or yeasts etc.
These enzymes recognize mostly a six base pair pallindromic DNA sequence which reads same from either ends of complementary strand and cuts within the sequence. The restriction fragments are ligated in a vector DNA which has been restricted earlier with the same restriction endonucleases.
These restrictions leave the DNA with identical protruding ends, which are also known as sticky ends or staggered ends or cohesive ends. The ligated passenger and vector DNA are then passaged into competent host cells by the process called bacterial transformation. Competence of E. It can also be performed by electrical treatment called electroporation, which increases the transformation efficiency by about times. Laser beam is also an effective method of developing competence.
It is possible to transform E. This method besides requiring screening of large number of recombinant clones runs the risks that the gene may be ligated in a reverse orientation with respect to the promoter thus gene expressions is not possible. This ensures proper orientation of the insert DNA upon ligation. This also prevents re-circularisation of vector as well as passenger DNA for want of complementarities.
Looking at the nut bolts levels of genetic engineering it will be seen that the cohesive ends bind specifically with complimentary base pairs produced on the vector DNA cut by the same enzyme. However, there are few enzymes, which make blunt end cut which can be ligated by DNA ligase obtained from bacteriophage T4 under optimum ATP concentrations.
Gene cloning can be performed in E. Ligation of passenger DNA can be done on plasmid upto 10 kilobase inserts or in lamda bacteriophase upto 25 kb and in cosmid upto 45 kb vector.
Wide diversity is available today in all the above kinds of vectors, each having some advantage over the other. Thus a proper vector has to be selected before initiating any cloning experiment. The transformed cells are later cultured in broth or on agar plates so that the inserted gene multiplies several folds. Gene cloning when performed in plasmid vector, a high copy number plasmid is selected for getting more copies of the inserted gene.
On the contrary the plasmid whose replication is dependent with the replication of host chromosome yield few copies 1 or few per cell.
These are called the plasmids with stringent replications or stringent plasmids e. Plasmid amplification involves growing the culture further with vigorous shaking in presence of protein synthesis inhibitor like chloamphenicol, tetracycline or spectinomycin.
The amplified recombinant vector is then isolated and the inserted gene is isolated by restriction with the same restriction endonucleases used earlier.
The gene isolated in abundance can be used for sequencing, hybridization, cell free translation or any other purpose. This is the method of in-vivo gene amplification and was the only method of gene amplification till the development of polymerase chain reaction PCR , which is method of in-vitro DNA amplification, developed in s.
Let us now consider the steps, which involve great intricacies. To begin with it is most important to identify the gene of interest, which has to be cloned. Once the gene has been identified it can be isolated by any of the following methods: Cleavage by restriction endonucleases Mechanical shearing RNA directed synthesis Chemical synthesis For identification of the gene either nucleic acid probe or the protein for use as an antigen or an antibody to the product of gene is required.
The product of the gene, say a protein can be used to generate antibody of the protein antigen. The antibody can be utilized for identifying the translating m-RNA producing growing polypeptide chains and nascent protein antigen molecules on a series of ribosome polyribosomes or polysome.
If the relevant nucleic acid, antigen or antibody is not available, the same from related species or even a distantly related species may be tried. The eukaryotic m-RNA which has a poly-A tail except for histone, interferon and immunoglobin can be isolated by affinity chromatography through a poly dT cellulose matrix. Purification of m-RNA is facilitated considerably as it has a characteristic base composition. In silk fibrin and collagen for instance, the known periodic amino acid sequences predict a special composition of the corresponding m-RNAs.
These m-RNAs are easily separated in calcium chloride density gradient centrifugations by virtue to their high guanine content and are relatively large 32 and 27s, respectively in size.
The other methods of identifying a gene are a direct selection. In this case identifications of the gene is not made before cloning, but selection is made after cloning of all the restriction fragments shotgun cloning on different selective media plates.
There are some genes, which are also directly selected as in some cases only the recombinants grow on the selective plates. This provides direct selection of the gene of interest. The cloning vector could be plasmid extra chromosomal DNA , a bacteriophage lamda, baculo virus, adeno virus, retro virus , cosmid a combination of plasmid and phage , yeast, phage M12, T1 plasmid, phasgemids, plants and fungi.
From Genes to Clones: Introduction to Gene Technology
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From genes to clones