Unless the vector and source DNAs are cut, fragments separated and joined, the desired recombinant vector molecules cannot be created. 1. How are the desired DNA sequences cut? 2. Explain the technique used to separate the cut fragments. 3. How are the resultant fragments joined to the vector molecule?

1. * The purified DNA molecule containing the desired gene is incubated with the specific restriction endonuclease, at optimal conditions of pH, temperature, etc. of that enzyme. * Similarly, the vector DNA is also incubated with the same restriction endonuclease under optimal conditions. * This results into cutting of the DNA into fragments. 2. * Gel electrophoresis is used to separate the DNA fragments produced by restriction digestion. * The DNA fragments resolve according to their sizes, through the sieving effect provided bythe agarose gel. 3. The resultant fragments are joined to the vector by use of DNA-ligases.

Unless the vector and source DNAs are cut, fragments separated an…

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Question

Unless the vector and source DNAs are cut, fragments separated and joined, the desired recombinant vector molecules cannot be created.

How are the desired DNA sequences cut?
Explain the technique used to separate the cut fragments.
How are the resultant fragments joined to the vector molecule?

✓

Answer

The purified DNA molecule containing the desired gene is incubated with the specific restriction endonuclease, at optimal conditions of pH, temperature, etc. of that enzyme.
Similarly, the vector DNA is also incubated with the same restriction endonuclease under optimal conditions.
This results into cutting of the DNA into fragments.

Gel electrophoresis is used to separate the DNA fragments produced by restriction digestion.
The DNA fragments resolve according to their sizes, through the sieving effect provided bythe agarose gel.