Isothermal nucleic acid amplification methods have been successfully developed and applied for diagnostic purpose, especially for detection of pathogens. However, amplicon size of such methods is relatively short (< 500 bp) to limit their application for long amplicon production that can be used for various downstream applications including genomic surveillance of pathogens. To fill the gap, we developed a method for specific amplification of kilobases-long target sequence from RNA templates. This method, named CREA, utilizes sequence specific recombination of Cre recombinase to generate circular intermediate template for subsequent RCA reaction. CREA with SARS-CoV-2 spike template could amplify ~ 2.9 kb target and up to ~ 1.9 kb amplicon was able to produce in sufficient amount for general cloning. Each step of CREA procedure was thoroughly analyzed to provide directions for further optimizations. Furthermore, we evaluated a variation of CREA which utilized DNA ligase.
【저자키워드】 Biological techniques, RNA, DNA, 【초록키워드】 Variation, diagnostic, amplification, Surveillance, Recombination, Pathogens, nucleic acid amplification, genomic, SARS-CoV-2 spike, sequence, CREA, downstream, RNA templates, limit, RCA, Cre recombinase, analyzed, subsequent, evaluated, generate, applied, can be used, 【제목키워드】 amplification, isothermal, linear,