Phosphorothioate primers in long PCR?

John Ladasky ladasky at leland.Stanford.EDU
Mon Mar 3 21:00:18 EST 1997


Greetings,

	I am searching for all possible ways to improve my genomic PCR.
I'm working from mammalian genomic DNA templates, and my products are 5.5
to 7 kB.  Using a mixture of Taq and Pfu polymerases, I get good yield of
my desired products, but they are inevitably accompanied by shorter side
products.  I would prefer to eliminate all of the side products, thus avoid-
ing a band purification step.

	Recently I came across Arne Skerra's article, "Phosphorothioate
primers improve the amplification of DNA sequences by DNA polymerases with
proofreading activity," NAR 20:14, 3551-3554 [1992].  In it he describes 
how a modification of the primer backbone prevents chewing back of the 
primer by Pfu or Vent.  The paper only describes PCR using these polymer-
ases by themselves, not in combination with Taq.  Has anyone used this
primer modification in Taq+Pfu PCR, and did it help you?

	Thanks in advance!

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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