title: Novel 5'/3'RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates.
abstract: To acquire the full-length sequences and to determine the 5'/3'ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols-via cDNA or mRNA templates-are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5'/3'ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5'/3'RACE approaches.
authors: Regional Laboratory of Virology, National Reference Laboratory of Gastroenteric Viruses, ÁNTSZ Regional Institute of State Public Health Service, Szabadság út 7, Pecs, 7623, Hungary.
authors: Péter
authors: P
authors: Pankovics
authors: Regional Laboratory of Virology, National Reference Laboratory of Gastroenteric Viruses, ÁNTSZ Regional Institute of State Public Health Service, Szabadság út 7, Pecs, 7623, Hungary.
authors: Ákos
authors: Á
authors: Boros
authors: Regional Laboratory of Virology, National Reference Laboratory of Gastroenteric Viruses, ÁNTSZ Regional Institute of State Public Health Service, Szabadság út 7, Pecs, 7623, Hungary. reuter.gabor@gmail.com.
authors: Gábor
authors: G
authors: Reuter
mesh_terms: D000818:Animals; D003370:Coturnix / Q000821:virology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009835:Oligochaeta / Q000235:genetics*; D010849:Picornaviridae / Q000235:genetics; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase
keywords: 5′/3′RACE; PCR; RNA genome; mRNA; ssRNA