cDNA Synthesis,Gene Cloning Experiments,Transcriptase Enzyme,Genomic DNA,Bacterial Expression,cDNA Clones

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	cDNA Synthesis - Methods of analysing RNA are laborious and critical when compared to methods of DNA analysis. Renee it is best to convert the RNA into DNA molecule. DNA molecule synthesized enzymatically by copying an RNA template are called cDNAs or complementary DNA. cDNA can be used as DNA insert in gene cloning experiments. The first step in cDNA synthesis is isolation of the mRNA from the tissue where they are produced in high quantities. These mRNA are placed in tubes containing reverse transcriptase enzyme, a small primer or oligo(dT), and dNTPs.
The oligo(dT) binds to the mRNA at its 3 -OR end, as mRNA (especially eukaryotic) contains a poly(A) tail at its 3 -OR end. The oligo( dT) acts as a primer and provides 3 -OR end for the reverse transcriptase enzyme. Reverse transcriptase enzyme adds dNTPs to new complementary DNA by taking RNA molecule as template strand. In the second step, using RNase H or alkali treatment, RNA molecule is degraded. Then the newly synthesized cDNA are separated and added to a fresh tube containing DNA polymerase and dNTPs. No primer is required at this step because the variable length of hairpin loop is formed transiently at the 3' -OR end of the first strand, which serves as a primer for the second strand DNA synthesis. After the second strand is synthesized, the hairpin structure is removed by using SI nuclease enzyme. This cDNA, which is double stranded, is used as insert in most of the experiments.
cDNAs have their own special uses, which is derived from the fact that they lack the intron sequence that are usually present in the corresponding genomic DNA. cDNA clones find application or use when bacterial expression of the foreign DNA is necessary as a prerequisite for detecting either the clone or the polypeptide product. The second step in gene cloning is the joining together of the vector molecule and the DNA to be cloned. This process is referred to as ligation. The enzyme which catalyses the reaction is DNA ligase. The optimum temperature for ligation of nicked DNA is 37°C, but at this temperature the hydrogen bonding formed between the sticky ends is unstable. Hence, the ligation reaction is carried out at 4°C. Even though the rate of reaction is slow and takes a long time to complete, this temperature is preferred, as the success rate is high. The ligation reaction of vector and inserts generate 3 types of products
1) the insert gets circularized without ligating to vector 2) The vector recircularizes without insert and 3) the vector-insert gets ligated. The last combination is important. To favour the formation of recombinants, various strategies have been devised. Firstly, performing the reaction at a high DNA concentration can increase the population of recombinants, Le., the ratio of vector and insert is kept at 1 : 2 so that there is maximum chance of vector and insert collision. Secondly, by using alkaline phosphatase, the 5 -end of the plasmid DNA is removed, thus the vector cannot religate itself. But the insert can supply 5 -PO4 to the 3 -OH of vector. Thus a phosphodiester bond will form only when the insert and vectors get circularized, but one nick will exist as the 3 ' -OH of insert cannot ligate to vector as it does not have a 5 -end.
	This nick is repaired by host cellular repair mechanism after recombinant molecule enters into the cell. Generation of recombinant molecules or vector insert is very high when the vector and insert have compatible cohesive ends, Le., when vector and insert are subjected to same restriction enzyme, e.g. Eco RI, which generates cohesive ends of same complementary ends. The cohesive end of the complementary sequence can form hydrogen bonds and can hold vector insert DNA together temporarily, so that DNA ligase can form the phosphodiester bond. The efficiency of recombinant molecule formation is a bit less when both the insert and vector or one of them has a blunt end. Inserting DNA segments into vectors is always more efficient if the vector and insert have matching cohesive ends. There are two methods to convert the blunt ended DNA fragments into cohesive ends.