title: Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.
abstract: Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org. zyang@ffame.org.
authors.forename: Zunyi
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org.
authors.forename: Chris
authors.initials: C
authors.lastname: McLendon
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org.|Firebird Biomolecular Sciences LLC, 13709 Progress Blvd, N112, Alachua, FL 32615 (USA).
authors.forename: Daniel
authors.initials: D
authors.lastname: Hutter
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bradley
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hoshika
authors.affiliation: Firebird Biomolecular Sciences LLC, 13709 Progress Blvd, N112, Alachua, FL 32615 (USA).
authors.forename: Carole B
authors.initials: CB
authors.lastname: Frye
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) http://ffame.org. sbenner@ffame.org.|Firebird Biomolecular Sciences LLC, 13709 Progress Blvd, N112, Alachua, FL 32615 (USA). sbenner@ffame.org.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D013696:Temperature
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D004265:DNA Helicases
keywords: helicase-dependent isothermal amplification; nucleic acids; points-of-care diagnostics; polymerase chain reaction; primer dimer