Wayne M. Barnes, Ph.D. Associate Professor Biochemistry and Molecular Biophysics Plant Biology Program Biochemistry Program Molecular Genetics and Genomics Program
Office Phone: 314-362-3351 Lab Phone: 314-362-3350 Other Phone: FAX: 314-754-9556 Box: 8231 Lab Address: 3830 North Building Email: barnes@wustl.edu Website: http://barnes1.wustl.edu Keywords: DNA; genome analysis; plant biology Short Research Description: Improvements to DNA technology, applied to plant genetic engineering.
Research Abstract: PCR is a central technology for all biomolecular research. Having improved PCR by some tenfold in several respects, especially length (from a few kb to 35 kb) and fidelity (from errors at 1/1000 to 1/20,000), I am continuing efforts to improve it further. I have invented several new ways to conduct a hot start for PCR. One is a buffer concoction that precipitates the magnesium until the PCR starts. The other hot start method is mutants of the DNA polymerase (a variant of Taq that we call Klentaq1) that are cold-sensitive; they switch on automatically when warmed up for the PCR. A recent method also allows Klentaq1 to carry out PCR on whole blood added directly to the reaction. I am constructing a large library of mutagenized Klentaq1 in order to select for other possibly interesting mutations in this workhorse enzyme. Plant genetic engineering often requires genes with codon preferences of plants (high GC in the 3d position), so to be in this game often requires artificially constructed genes, not just clones. We have improved gene construction technology by making changes to the gene assembly procedure. These changes are also potentially valuable for reassembly of ancient or museum DNA. My PCR breakthrough occurred while I was amplifying a Bt gene for plant genetic engineering. Transgenic Bt insecticidal protein is becoming an important and safe weapon against insects in agriculture, but resistance is expected to arise. I am attempting to fuse other entomotoxic proteins to Bt in forms that should be stabilized in the insect gut and/or have another domain active against insects, to create new forms that may overcome resistance.
Selected Publications: Kermekchiev MB, Tzekov A, Barnes WM. Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR. Nucleic Acids Res 2003 3:6139-6147. Pergams RW, Barnes WM, Nyberg D. Chicago mice replace their mitochondria DNA. Nature 2003 423:397. Stratman JL, Barnes WM, Simon TC. Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res 2003 12:521-522. Barnes WM, Rowlyk KR. Magnesium precipitate hot start method for PCR. Mole Cell Probes 2002 16:167-171. Barnes WM. PCR amplification of up to 35 kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA 1994 91:2216-2220.