PCR和磁珠介导的靶标捕获法分离鱼类基因组的短散布元件.doc

  PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes# Liu Dong, Zhu Guoli, Tang Wenqiao, Yang Jinquan, Guo Hongyi* 5 10 15 20 25 30 35 40 45 (College of Fisheries and Life Science, Shanghai Ocean University, ShangHai 201306) Abstract: Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180–360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is first report of a tRNALeu-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs. Keywords: Transposable element; SINE; tRNA; Coilia nasus 0 Introduction Short Interspersed Nucleotide Elements (SINEs) are a class of retrotransposons widely distributed in eukaryotic genomes. They integrate into a new genomic locus through RNA reverse transcription [1]. Most SINEs are 80–400 base-pair (bp) long, with more than