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PCR Mutagenesis/Co-Transformation Protocol

Mike Stark M.J.R.STARK at dundee.ac.uk
Wed Oct 16 04:23:18 EST 1996


Stephen Bell wrote:-

>Netters,
>
>	  I am interested if anyone has a protocol for PCR 
>mutagenesis of a gene fragment then transforming yeast with 
>the mutagenized PCR product and a gapped plasmid directly 
>(rather than making a mini-library of mutagenized genes 
>before yeast transformation).  In particular I would like to 
>know how much homology is needed between the gapped plasmid 
>and the PCRed fragment to get good efficiency of 
>recombinants and whether there are problems with religation 
>of the plasmid without recombination. 
>
>Thanks, 
>
>Steve Bell

We have used the protocol below (based on Muhlrad, D., Hunter, R. and Parker, R. 
(1992) A Rapid Method for Localized Mutagenesis of Yeast Genes.  Yeast  8, 79-82.) 
to make Ts- alleles of several essential yeast genes. Regarding the overlap 
necessary beteen the gapped plasmid and PCR ragment we have not rigorously tested 
the minimum requirements, but in one case (GLC7) the overlaps were 46 and 374 bp 
while in another instance (PPH22) they were 148 and 187.  In the latter case the 
primers were  flanking polylinker sequence.  This gave hundreds to thousands of 
transformants in each case and greatly stimulated the transformation frequency 
over that obtained just with the gapped vector.  However, since there was a 
background level of transformation without PCR fragment, either ligation or repair 
(in our case from a second plasmid in our strain carrying the wild-type gene in 
question) clearly could be a problem depending on exactly what you are doing.

Gapped plasmid repair with mutated/non mutated PCR product
----------------------------------------------------------

A. Error-prone PCR for random mutagenesis
-----------------------------------------

Mutagenic and non-mutagenic reactions are set up in parallel. Standard mutagenic 
conditions:-

i)   Ratio of (dTTP/dCTP) : (dATP/dGTP)  =  5 : 1 (could also try 1 : 5)
ii)  Include 0.1-0.5 mM manganese chloride in the reaction 
iii) 2X normal (final) conc. of Taq polymerase in reaction (i.e. 0. 05 U/mu-l). 

For each set of template/primers, optimise the following conditions:

a)  Ratio of MgCl2:MnCl2 in the reaction e.g. for GLC7, used 0.5 mM but for PPH22 
		  used 0.3 mM.  This can greatly affect the frequency of mutations obtained.
b)  The PCR conditions for denaturation/amplification/annealing

1.	Stock solutions

	  50 X stock "mutagenic" dNTP mix (low A+G): 	25mM dTTP, dCTP, 5mM dATP, dGTP
	  50 X stock "normal" dNTP mix: 	25mM each dNTP

2. Reaction for 100 ul:
                       	
                      [Stock]              [Final]	       Vol.(mu-l) 
                      -------              -------        --------                                                                                                                                                           
	Primer 1            200 ng/mu-l           4 ng/mu-l       	2.0		  
 Primer 2	           200 ng/mu-l          	4 ng/mu-l       	2.0
	template (1)	        10 ng/mu-l	       0.1-0.2 ng/mu-l	  1.0-2.0
	Reaction buffer (2)  10X	                  1X	            10.0
	MgCl2 (2)             25 mM               	2 mM           	8.0 
	dNTP mix (3)        see above 	          see above	        2.0
	water	                  -                    -             68-72
 [MnCl2                10 mM	               0.3-0.5 mM	   3.0-5.0]  (5)
	Taq pol.(4)	          5 U/mu-l	            0.05 U/mu-l	    1.0	
                                                                                                                                                            
Footnotes   (1) template = circular plasmid
            (2) Some buffers may already have the MgCl2
            (3) diluted from Pharmacia 100 mM stocks
            (4) We have used Promega or Boehringer Mannheim enzyme successfully
            (5) mutagenic reaction only


3.	Mix, heat at 100 deg. C for 5 min, add the Taq polymerase, mix, add oil (100 
mu-l), pulse spin briefly in a microfuge and put in PCR machine using appropriate 
settings variable because dependent on template/primers etc. and ideally 
pre-optimised) eg:

	94 deg. C  45-60 sec ramp 0 (1 deg.C/sec: on a Techne PHC-3)
	55 deg. C  2.0-2.5 min ramp 30 (0.5 deg.C/min)
	72 deg. C  2.5-5.0 min ramp 0

	After 30 cycles, link to 94 deg.C	1.0 min	(ramp 0), 4 deg.C	15 h

4.	Check for purity on gel after ether extraction of oil and if a clean band of 
the expected size is obtained then it can be used directly in gapped plasmid 
method. Alternatively, gel purify the band e.g. with GeneClean II.

5. In our system the 5' primer was around the ATG of GLC7  and the 3' primer 130 
bp downstream of the stop codon.  Convenient sites were chosen so that the 
homologous overlap at the 5' end was 50 bp (better to be longer) and at the 3' end 
300bp.

B. Gapped plasmid Transformation
--------------------------------

For making Ts- mutations, the host strain is a suitable yeast carrying a wild-type 
copy of the gene in question on a URA3-CEN  plasmid and a gene knockout in the 
chromosome.  The gapped plasmid was prepared by digestion and gel purification 
using Geneclean II and carried a copy of the gene on a CEN  plasmid with a 
different market e.g.TRP1.

1.	Usually added following per aliquot of yeast cells made competent TE-LiAC 
method:

	10 mu-l PCR product mutated or non-mutated (didn't quantify but 10 ul is a good 
intensity band on a gel)
	50-100 ng gapped plasmid
	50 mu-g sheared single-stranded salmon sperm DNA
	50 mu-l competent yeast cells

2.	Mix briefly, then added 300 ul 40% PEG/TE/LiAc.  Shake at 26 deg. C 30-45 min 
and heat shock 42C 15 min.

3.	Spin  3000 rpm for 2 min and resuspend in 100 mu-l TE.

4.	Plate out on selective plates.  Include along side gapped plasmid alone and a 
zero DNA sample as controls.  In our system we then patched out approx. 500 
transformants onto selective plates containing 5-FOA and then when these had grown 
up, repatch onto selective plates at 26 deg. C and 37 deg. C.  At this stage we 
got around 10% Ts- mutants but this will no doubt vary from gene to gene.








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Dr. Michael J. R. Stark,
Department of Biochemistry,
The University,
Dundee
DD1 4HN
Scotland
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