Phosphorothioate primers in long PCR?
ladasky at leland.Stanford.EDU
Mon Mar 3 21:00:18 EST 1997
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 . 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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