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Uneven PCR, Rapid method to walk unknown sequence, http://128.84.203.244

Gerald L. McLaughlin, Ph.D gmclaugh at IUPUI.EDU
Mon Feb 23 09:51:52 EST 1998


I believe that I described something very similar, naming it "plateau PCR",
but this was published in a relatively obscure book chapter (Parasitology
for the 21st Century, eds MAli Azcel and M. Ziya Alkan, Chapter 25,
McLaughlin et al, 261-78, kWallingford Oxon OX 10 8DE, UK.  Essentially we
started at a higher hybridization temperature than the predicted Tm for 15
cycles, then 21 cycles at 55 C, for the primers we chose for various phyla
of pathogens.  We got much more specific target amplification, than whe we
tried similar strategies without this step (McLaughlin et al, 1993, Memoria
Osvaldo Cruz Foundation 87, Suppl III, 57-68.  As you are more likely to
know, about 2 years ago in Biotechniques, someone else's paper was published
describing "Touchdown" PCR, which starts at a still higher temperature and
more incrementally drops the temperature with fewer steps at each
temperature, eventually finishing with a cycle time for many cycles, much
lower than the calculted or empiracal Tm.  We have had good results with
this strategy also.

Please send a reprint when you get one!

Thanks,

Jerry

At 02:45 AM 2/20/98 +0000, Xiongfong Chen wrote:
>Uneven PCR, Rapid method to walk unknown sequence, http://128.84.203.244
>
>This paper describes a new polymerase chain reaction (PCR) cycling condition 
>to generate specific unknown fragments or genes directly from total DNA 
>instead of cloning genes from libraries.  
>
>For example, if you have a cDNA clone but missing the 5'end, or if you have a
>gene coding sequence but you want to get the promoter region fast, or you  
>find that you can not get the damn Inversion PCR to work in your hands.
>Before you decide to buy a kit to do these kind of work. Please try this 
>method first.    
>
>The essence of the method is to use two different annealing temperatures in 
>consecutive PCR cycles to 
>effectively amplify the target products while inhibiting the synthesis of non
>specific products.  We name this method "Uneven PCR".  Under favorable 
>conditions, a desired DNA fragment or gene can be obtained and ready to clone 
>in just a few hours.  The advantages of this method are simplicity, precision, 
>sensitivity, and speed.  We believe that it will have a broad spectrum of 
>application in molecular biology.
>
>This paper has been published in GENE 185(1997)195-197.
>
>A similar method called TAIL PCR has also been published.
>
>
Gerald McLaughlin, Ph.D.
Dept Pathology and Lab Med
635  Barnhill Dr., MS A128
Indianapolis, IN 46202-5120
317-274-2651; gmclaugh at iupui.edu




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