title: Genome walking of large fragments: an improved method.
abstract: The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.
authors.affiliation: Biotechnology Initiative, Forestry and Forest Products Division, Center for Applied Research and Technology Development (CARTD), Natural Resources Research Institute, University of Minnesota-Duluth, Duluth, MN 55811, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Rishi
authors.forename: Neil D
authors.initials: ND
authors.lastname: Nelson
authors.forename: Arun
authors.initials: A
authors.lastname: Goyal
mesh_terms: D016386:Chromosome Walking / Q000379:methods*; D053938:DNA Fragmentation / Q000235:genetics*; D018745:Genome, Plant; D016133:Polymerase Chain Reaction / Q000379:methods*; D032107:Populus / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D011933:Reagent Kits, Diagnostic