Talk:CH391L/S12/Restriction enzymes and BioBricks assembly standards

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Minor note about endonuclease nomenclature: Endonucleases like I-SceI are not really "restriction endonucleases" because they do not originate from the bacterial restriction system. They are "homing endonucleases" from yeast ("S"accharomyces "ce"revisiae) that help introns move around in yeast genomes. There's lots of other members of this family that have large sites like I-SceI and they are useful, but they aren't "restriction" endonucleases. *Joe Hanson 15:18, 6 February 2012 (EST):

  • Ben Slater 18:35, 12 February 2012 (EST): Duly noted. Thanks!
  • David M. Truong 15:26, 6 February 2012 (EST):You may want to define more clearly what you mean by scar. Simple definition might be the ligation of two different restriction sticky ends, that remove the palindrome, and therefore can no longer be used as a restriction site. Also, you might also discuss the vast commercial sector for restriction enzymes, most importantly, New England Biolabs.
  • Ben Slater 18:35, 12 February 2012 (EST): Done. Thanks!

In order for the Type IIS restriction enzymes to work, would the genes being spliced together have to have complementary code on the ends that are cut and staggered before they anneal together? That would severely limit the application of these enzymes to specific genes that fulfill those 15:37, 6 February 2012 (EST)

  • Ben Slater 19:46, 12 February 2012 (EST): As far as I can tell, that indeed seems to be the case.
  • Yi Kou 14:37, 8 February 2012 (EST):I have a stupid question: have there been any studies on restriction enzyme cutting the single DNA strand, what is the difference, like say, with that of the ds palindromic ones, concerning Km, Kcat?
    • Joe Hanson 16:15, 8 February 2012 (EST): Most common REs, at least Type II's that cut 4-8 bp palindromes operate as dimers. One unit of the dimer cuts one strand and the other monomer cuts the opposite strand. Hence the palindromic nature of recognition sites. A few have been reported to cut ssDNA in vitro, but this probably wouldn't happen in vivo.
    • Jeffrey E. Barrick 10:57, 11 February 2012 (EST): I guess that there are some "nicking" endonucleases that have been discovered and engineered that cut only one strand of a DNA duplex See New England Biolabs article. Some links to NEB from this topic might be helpful - esp. NEBcutter, which is a nice tool for testing a construct you have designed for particular cleavage sites.
  • Yi Kou 17:38, 11 February 2012 (EST): Very helpful. tks!
  • Ben Slater 19:59, 12 February 2012 (EST): Updated with info and links. Thanks!
  • Jeffrey E. Barrick 11:00, 11 February 2012 (EST): It's not quite accurate that Type I RE are not useful "because they cleave the DNA distant from the recognition site". Type IIS also cleave distant from the recognition site. You said the real reason they are not useful in your talk, but it's not there on the page.
  • Jeffrey E. Barrick 11:04, 11 February 2012 (EST):I really like the addition of comments within the reference list.
  • Jeffrey E. Barrick 11:04, 11 February 2012 (EST):In "Zinc finger nucleases", the DNA-binding domain probably can't be replaced to "recognize any sequence" (there are a lot to choose from and specificity might not be achievable in all cases. Probably better to say "recognize new sequences".
  • Ben Slater 19:59, 12 February 2012 (EST): Fixed, thanks.
  • Jeffrey E. Barrick 11:07, 11 February 2012 (EST):I feel like there was at least one more advantage to Bgl bricks that you mentioned in your talk.
  • Ben Slater 20:30, 12 February 2012 (EST): Updated with additional advantages.
  • Jeffrey E. Barrick 11:08, 14 February 2012 (EST):We discussed TAL nucleases during class. They would be an interesting additional topic or way to add to this topic in the future.