Padmakumar, VC and Varadarajan, Raghavan (2003) A gradient PCR-
based screen for use in site-directed mutagenesis. In:
Analytical Biochemistry, 314 (2). pp. 310-315.
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mutagenesis is widely used to study protein and nucleic acid
structure and function. Despite recent advancements in the
efficiency of procedures for site-directed mutagenesis, the
fraction of site-directed mutants by most procedures rarely
exceeds 50% on a routine basis and is never 100%. Hence it is
typically necessary to sequence two or three clones each time a
site-directed mutant is constructed. We describe a simple and
robust gradient-PCR-based screen for distinguishing site-
directed mutants from the starting, unmutated plasmid. The
procedure can use either purified plasmid DNA or colony PCR,
starting from a single colony. The screen utilizes the primer
used for mutagenesis and a common outside primer that can be
used fo