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.

Question

Quizplus · Accepted Answer

BamHI is preferred for cloning because it creates sticky ends, which facilitate more efficient and specific ligation into a plasmid that has been cut with the same enzyme. Sticky ends have overhanging sequences that can easily pair with complementary sequences on another DNA molecule cut with the same enzyme.

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