Chemical mutagenesis of plasmids - Why only hydroxylamine ? (Jul/12/2002 )

Dear all,

I would like to chemicaly mutagenize plasmid DNA (double stranded). I cannot find any protocol for this, except for hydroxylamine mutagenesis.
I have 2 questions :
      1. Is there any special reason, why is hydroxylamine most often used for plasmid DNA mutagenesis ?
      2. If You have plasmid mutagenesis protocol for any other chemicals eg. EMS, please, send it to me.

Thank You in advance

Barbara

Hi,

I assume hydroxylamine is usually used because it is much less dangerous to the user than EMS or MNNG.......however, I don't actually even know if these agents are active in vitro.

Although hydroxylamine is a weak mutagen, by altering the length of incubation you can get as much or as little mutation as you wish; weak mutagens are far more useful for most purposes where you just want single nucleotide substitutions.......if you start using stonger mutagens on plasmids in vitro, the transformation efficiency drops off very quickly.

If you need a different or wider spectrum of mutagenic events I find UV, 2-aminopurine or cloning into mutS coli (Promega) useful, although they are of course all in vivo methods.

Hope this is some use.

Thank You !

For one thing it works. The others are less mutagenic. You can try biologycal mutagenesis e.g. E. coli MutD strain. Yoram