title: Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.
abstract: We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Kelli
authors.initials: K
authors.lastname: Sumner
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Swensen
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Melinda
authors.initials: M
authors.lastname: Procter
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Jama
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Whitney
authors.initials: W
authors.lastname: Wooderchak-Donahue
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Tracey
authors.initials: T
authors.lastname: Lewis
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Michael
authors.initials: M
authors.lastname: Fong
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Lindsey
authors.initials: L
authors.lastname: Hubley
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Monica
authors.initials: M
authors.lastname: Schwarz
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Youna
authors.initials: Y
authors.lastname: Ha
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Eleri
authors.initials: E
authors.lastname: Paul
authors.affiliation: ARUP Laboratories, Salt Lake City, Utah.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Brulotte
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Pinar
authors.initials: P
authors.lastname: Bayrak-Toydemir
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Rong
authors.initials: R
authors.lastname: Mao
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah.
authors.forename: Genevieve
authors.initials: G
authors.lastname: Pont-Kingdon
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Department of Pathology, University of Utah, Salt Lake City, Utah. Electronic address: hunter.best@aruplab.com.
authors.forename: D Hunter
authors.initials: DH
authors.lastname: Best
mesh_terms: D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers