Exam 8: Recombinant Dna Technology and Molecular Cloning

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Cloning a DNA fragment into bacteriophage lambda involves several steps. First, the DNA fragment and the bacteriophage lambda DNA are cut with the same restriction enzyme to create compatible ends. The DNA fragment is then ligated into the bacteriophage lambda DNA using DNA ligase. The resulting recombinant DNA is then packaged into lambda phage particles using in vitro packaging extracts. The packaged phage particles are then used to infect E. coli cells, where the recombinant DNA is integrated into the bacterial genome.

To screen for clones that contain an insert, a common method is to use a technique called blue-white screening. In this method, the recombinant DNA is ligated into a plasmid vector that contains a lacZ gene. When the lacZ gene is intact, it produces an enzyme that turns a substrate blue. However, if the lacZ gene is disrupted by the insert, the enzyme is not produced and the colonies remain white. Therefore, by screening for white colonies, one can identify clones that contain an insert. Additionally, PCR or DNA sequencing can also be used to confirm the presence of the insert in the clones.

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Restriction mapping is a technique used to determine the location of restriction sites within a DNA sequence. To determine the orientation of a restriction fragment ligated into a restriction site in a vector, you can use restriction mapping as follows:

1. First, digest the vector with the restriction enzyme used to create the original site where the fragment was ligated. This will result in two fragments, one containing the vector and the ligated fragment, and the other containing the remaining vector sequence.

2. Next, run the digested DNA on an agarose gel to separate the fragments based on size.

3. After the gel electrophoresis, visualize the DNA fragments using a staining method such as ethidium bromide. The gel will show the sizes of the fragments, allowing you to determine the orientation of the ligated fragment in the vector.

4. By comparing the sizes of the fragments with the expected sizes based on the known sequence of the vector and the ligated fragment, you can determine the orientation of the ligated fragment in the vector.

In summary, restriction mapping can be used to determine the orientation of a restriction fragment ligated into a restriction site in a vector by digesting the vector, running the fragments on an agarose gel, and comparing the sizes of the fragments to the expected sizes based on the known sequences.