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Cellular and Molecular Biology

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Laboratory Techniques: Restriction Enzyme Cloning

CELLS-CAEOEX

Molecular Cloning has significantly advanced the study of biology by allowing researchers to create recombinant proteins. Recombinant proteins are made by fusing genes (or parts of genes) from various organisms together in order to conduct functional studies using those very same "fusion proteins."

To generate these fusion proteins researchers commonly use restriction enzymes that recognize specific sites to cut (or digest) double stranded DNA and generate overhangs. The overhang generated depends on the enzyme used as well as its recognition site (see image below).

Adapted from http://www.biology-pages.info/R/RestrictionEnzymes.html. "Restriction Enzymes." Restriction Enzymes. N.p., n.d. Web. 03 Apr. 2016.

Once the proper overhangs are generated, what must a researcher do next, and which basic principle of DNA structure makes it possible?

A

The researcher must incubate the DNA fragments with an exonuclease. This enzyme will increase the length of the overhang and allow hybridization of the fragments over a slow cooling step, thereby generating the transcript for the fusion protein.

B

The researcher needs to incubate the DNA fragments with a recombinase. This enzyme will recognize the pallindromic overhangs generated by the restriction enzymes to catalyze cassette exchange, thereby linking the DNA fragments together.

C

The researcher will incubate the DNA fragments with RNA Polymerase II which will transcribe mRNA coding for the fusion protein using the single-stranded overhangs as a starting point. This reaction will take advantage of priming which is necessary to determine the initial binding site of RNA Polymerase II.

D

The researcher must incubate the digested DNA with a ligase. The subsequent ligation reaction takes advantage of the complementarity of nucleic acids by covalently linking the two digested strands of DNA.