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| Tools |
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| The recombinant DNA technology requires the use of many biological tools. They are as follows: |
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| Several kinds of specific enzymes are employed in genetic engineering. These include lysing enzymes, cleaving enzymes, synthesizing enzymes, joining enzymes and alkaline phosphates. |
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| Lysing Enzymes |
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| These are enzymes used to extract DNA from a cell for genetic experiments. Lysozyme is commonly used to dissolve the bacterial cell wall. |
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| Cleaving Enzymes |
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| These are used to break DNA molecule into fragments. They are further of three kinds, |
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 Exonucleases, which release nucleotides from 5 or 3 ends of a DNA molecule; |
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 Endonucleases, which cleave DNA helix at any point except the at the terminal ends and |
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 Restriction endonucleases, which cleave DNA molecule at specific points in such a way that single-stranded free ends project from each fragment of the DNA molecule. These single-stranded free ends are called “sticky ends” because they join with similar complementary ends of DNA fragment from some other source. Restriction endonuclease was discovered by Arber in 1962 in bacteria. Restriction endonuclease 1 (ECOR-1) is found in the colon bacterium Escherichia coli. It recognizes the base sequence GAATTC in DNA duplex and cleaves its strands between G and A as shown below. The arrows indicate the sites where the enzyme cleaves the sequence. |
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| fig. 25.1 Action of DNA Cleaving Enzymes |
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| Synthesising Enzymes |
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| These enzymes play a role in the synthesis of DNA strands on suitable templates. They are of two types, |
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 Reverse transcriptases, which help in the synthesis of complementary DNA strands on RNA templates, |
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 DNA polymerase, which aid in the synthesis of complementary DNA strands on DNA templates. |
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| Joining Enzymes |
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| These enzymes, also called as ligases, help in sealing gaps in DNA fragments joined by complementary base pairing. e.g. Tuligases. |
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| Alkaline Phosphotase |
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| These enzymes release phosphate group from the 5 end of linearised circular DNA to check its recircularization. |
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| The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle DNA. Two types of DNA are used as vehicles: plasmid DNA and bacteriophage DNA. |
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| Plasmids |
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| The plasmids are small, circular DNA molecules present in bacterial cells in addition to the chromosomal DNA. Genes from one organism may be added to the plasmids of a bacterium and transfer them to another. They retain their characters after combining with the DNA of another organisms. Not being a part of the main genome, they can be easily isolated and transferred. These features make the plasmids most suitable for use as a vehicle DNA. A plasmid having DNA of another organism integrated with it is known as cosmid, also recombinant plasmid or hybrid plasmid. Cosmids are picked up by or can be introduced into plasmid free bacteria. |
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| Bacteriophage DNA |
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| The bacteriophages (or phages), are viruses that infect and kill bacteria. Their circular DNA is also suitable for use as a vehicle DNA. |
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| It is the DNA that is transferred from one organism into another by combining it with the vehicle DNA. Three types of DNA are used as passengers: complimentary, synthetic and random |
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| Complementary DNA (cDNA) |
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| It is the DNA synthesised on RNA template with the help of the enzyme reverse transcriptase and necessary nucleotides. The DNA strand is isolated from the hybrid RNA-DNA complex by using alkaline phosphatase enzyme. A complementary DNA strand is then synthesized on the isolated single-stranded DNA template with the help of DNA polymerase. The cDNA double helix so obtained can be joined with the vehicle DNA for introduction into a new host cell. |
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| Synthetic DNA (sDNA) |
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| It is the DNA synthesised with the help of DNA polymerases on the DNA template or from free deoxybironucleotides without a template. |
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