Oligo directed Insertions

usenet news poster usenet at nlm.nih.gov
Wed Sep 26 21:27:20 EST 1990


In article <7013.26fd2d8d at jetson.uh.edu> bchs1b at jetson.uh.edu writes:
>
>
>	I need to make some site directed insertions of about 8-10 bases. 
>I have normally been doing sited directed mutagenesis using Kunkel method 
>(dut ung M13 template) with good success, but have never tried insertions. 
>How many bases on either side of the insertion do I need to make it work well,
>assuming reasonable GC distribution. Any problems to watch out for?
>
>	Second related question, are there any procedures for doing site 
>directed mutagenesis on double stranded plasmids which are as easy and 
>efficient as the Kunkel method? Or is it worthwhile cloning things into 
>M13 everytime (I have reasons not to use filamid vectors like pEMBL and pUC118).
>
>thanks,
>
>mike benedik
>biochemical and biophysical sciences
>university of houston				benedik at uh.edu or benedik at uhou

If you have access to an oligo synthesizer and PCR, there is a very simple
and powerful methods you can use.  Given template and a site XX where you
want to make an inwsertion, make oligos "primer-a1" and "primer-s2" spanning
the region with the desired mutation plus enough extra sequence that
they will amplify, and make primers "s1" and "a2" off the native sequence.


	  primer-s1->           primer-s2->
template  ===========================XX==============================
			        <-pimer-a1-               <-primer-a2

First amplify with "s1" and "a1".  In a separate reaction, amplify with
primers "s2" and "a2".

Gel purify the products of both PCR reactions.

Mix the products and amplify with primers "s1" and "a2".  This reaction
will work because the two previous reaction products are able to
hybridize and act as primers on each other.


	  primer-s1-----------------XX--->
				 <--XX---------------------primer-a2

				 |
				 V

	  primer-s1-----------------XX--->>>>>>>>>>>>>>>>>>>>>>>>>>>
          <<<<<<<<<<<<<<<<<<<<<<<<--XX---------------------primer-a2


The products of this reaction can then be purified, cleaved with
restriction enzymes, and subcloned back into your vector.  Since "s1"
and "a2" may be a kb or more apart, you have a fair amount of flexibility
in finding restriction sites.  Because the product will have gone
through two PCR reactions, you will definitely have to resequence the
entire amplified region.

NOTE: the left and right halves of the starting reaction need not
have come from the same segment.  This is a simple and general way
to join to arbitrary sequences together even if no restriction sites
are available at the junction.

See: AW Lee and A Neinhuis, PNAS within the next couple issues for a more
complete description using this technique to construct mutants of the
CSF-1 receptor.


David States
National Center for Biotechnology Information
National Library of Medicine



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