a) What is DNA cloning and describe briefly what the main idea behind this proce
ID: 223394 • Letter: A
Question
a) What is DNA cloning and describe briefly what the main idea behind this procedure is For DNA cloning we use: a cloning vector, restriction enzymes (endonuclease), a ligase, transformation, resistance to antibiotics for selection of colonies that have been transformed with our recombinant DNA. Explain briefly each of these terms. b) Recombinant DNA technology provides multiple ways to help us understand protein function. For example, explain briefly how DNA technology can be advantageous for overexpression, purification, and mutagenesis of proteins, in comparison with older approaches when tissues were the main source of protein for biochemical studies.Explanation / Answer
a) Basic steps of DNA Cloning:-
(Genomic DNAs, total RNAs, mRNAs)
(Plasmid, Phage, Phasmid, Cosmid, viral)
Main idea behind these proceudre is to express functional gene by tarnsferring it in to foreign enviorment which may br faouvrable or unfavourable for expression of gene. for exapmle:- Insulin produce by recombinate bacteria with gene of insulin in it.
B) Recoambinate DNA technology is very useful for understand the protien function,expression, asweel as overexpression.
1. To understand the epression of functional gene or to understand th site of protien synthesis we can use rDNA tech effectively by probbing our interest of gene with radioalabell tag.
2. overeperssion of particular gene by designing in such way that production of protien we required is increase many time. such as Insulin hormones that is most required for diabetic patience
Ligase: Ligation is a concentration dependant reaction (like any other enzyme catalysed reaction)The concentration of vector DNA and insert DNA should be equimolar. In a ligation monomolecular reactions and bimolecular reactions take place. For a gene cloning experiment the bimolecular reaction is preferred
Transformation is process by which target gene introduced in to host cell with help vector using appropriate methode of transformation. For example Natural tranfromation was Agrobacterium mediated transformation of Ti plasmid.
Selection: Transformation is screned by Particular selective charatcter show by transformed colony of bacteria or cells. Such as trasnformed cells are resistance to some antibiotic as comared to normal untransformed cells.
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