National Key Lab of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.
CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), CAS, Shanghai, China.
Methods Mol Biol. 2021;2250:103-110. doi: 10.1007/978-1-0716-1134-0_9.
Extrachromosomal linear DNA (eclDNA) is the reverse-transcribed cDNA intermediate derived from long terminal repeat (LTR) transposable elements (TEs) (Cho et al., Nat Plants 5:26-33, 2018). Given that the eclDNAs are the final intermediate of LTR-TE life cycle prior to integration to the host chromosomes, their presence is considered a strong indication of active LTR retrotransposons (Cho et al., Nat Plants 5:26-33, 2018; Lanciano et al., PLoS Genet 13:e1006630, 2017). Here, we describe a method of amplification of LTR extrachromosomal DNA followed by sequencing (ALE-seq) which determines the 5' LTR sequences of eclDNAs. Briefly, ALE-seq consists of two steps of amplification, in vitro transcription of adaptor-ligated eclDNAs and subsequent reverse transcription to cDNAs primed at the conserved primer binding site (PBS) (Cho et al., Nat Plants 5:26-33, 2018). ALE-seq allows the high-throughput identification of novel LTR-TEs which are active in plants that could be potentially useful for crop biotechnology.
染色体外线性 DNA (eclDNA) 是由长末端重复 (LTR) 转座元件 (TE) 逆转录而来的 cDNA 中间产物 (Cho 等人，Nat Plants 5:26-33, 2018)。鉴于 eclDNA 是 LTR-TE 生命周期在整合到宿主染色体之前的最终中间产物，它们的存在被认为是活跃的 LTR 反转录转座子的强烈迹象 (Cho 等人，Nat Plants 5:26-33, 2018；Lanciano 等人，PLoS Genet 13:e1006630, 2017)。在这里，我们描述了一种扩增 LTR 染色体外 DNA 随后进行测序 (ALE-seq) 的方法，该方法确定了 eclDNA 的 5'LTR 序列。简而言之，ALE-seq 包括两步扩增，即衔接子连接的 eclDNA 的体外转录，以及随后在保守引物结合位点 (PBS) 处起始的 cDNA 的反转录 (Cho 等人，Nat Plants 5:26-33, 2018)。ALE-seq 允许高通量鉴定在植物中活跃的新的 LTR-TE，这可能对作物生物技术有用。
