In shotgun method gene cloning is a general one and not specific. As the genes to be cloned are not identified and isolated before, the chances of inserting a particular gene by this method are very rare.
Another specific method of isolating gene is to first obtain the messenger RNA transcribed by that particular gene and then obtain DNA from mRNA. DNA from mRNA can be obtained by the following two methods:
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1. Hybridization method:
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When DNA transcribes and forms mRNA a complex is formed. The hybridization method depends on this mechanism.
At the first step the total DNA from an organism is isolated and treated with heat or alkali to convert it into a single stranded form.
These single strands are then mixed with the specific mRNA obtained from the gene. Obviously the mRNA chains pair with that section of DNA which has complimentary nucleotides to it thus forming a DNA- RNA hybrid.
This complex can be isolated and the DNA can be separated from the RNA. The single strand DNA can be converted into a double strand DNA by enzymes such as polymerase I. This method is highly useful for isolating ribosomal genes.
2. Reverse transcriptase method:
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In the normal transcription the flow of information is from DNA and RNA proteins. Transcription of RNA from DNA is catalyzed by the enzyme transcriptase also known as DNA dependent RNA polymerase.
Temin and Baltimore independently discovered that occasionally the genetic information may flow in the reverse direction also (from RNA to DNA) in some RNA tumour viruses. They named the enzyme Reverse transcriptase (RNA directed DNA polymerase).
These enzyme catalyses the synthesis of a single strand DNA using an RNA template this discovery has proved to be highly useful for isolating particular genes.
In reverse transcriptase method, purified mRNA segments of the required gene are used as a template to produce a fragment of single stranded DNA under the influence of the enzyme reverse transcriptase.
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As this single strand DNA is complementary to mRNA it is called complimentary DNA or cDNA. Hydrolysis of RNA template will separate a single strand DNA.
This single strand DNA is converted into double strand using the enzyme DNA polymerase. Another enzyme S1 nuclease breaks the covalent linkage between two DNA strands.
