A Graduate Student Wants to Clone a Particular Gene into a Plasmid

question 29

Multiple Choice

A graduate student wants to clone a particular gene into a plasmid. The sequence includes AluI and BamHI sites on both sides of the desired fragment. AluI cuts symmetrically directly between the G and C nucleotides in a palindromic 5' AGCT 3' sequence. BamHI cuts asymmetrically directly between the G and G nucleotides in a palindromic 5' GGATCC 3' sequence. Which of the two restriction endonucleases should the graduate student choose, and why?

• A) BamHI to cut both sides-since this enzyme cuts asymmetrically, it'll leave the sticky, cohesive single-strand DNA ends that will make it easier to ligate into a BamHI-cut plasmid DNA sequence.
• B) AluI to cut both sides-it's always easier to ligate together blunt ends of DNA. She should also use AluI on the plasmid she wants to put the fragment into.
• C) BamHI on the fragment, and AluI on the plasmid-this will give her the matching sequences to anneal/ligate together on the fragment/plasmid combination.
• D) BamHI on one side of the fragment, and AluI on the other side-this would keep the fragment from sticking right back to where it was cut out from in the original DNA.
• E) Neither of these endonucleases will really be useful; she should look for a single enzyme that creates either blunt ends or sticky ends, depending on the plasmid that she is using as a vector.

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