Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR.
Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3' end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
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D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D005819:Genetic Markers; D006824:Hybridization, Genetic; D008969:Molecular Sequence Data; D009115:Muridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D016324:Sequence Tagged Sites; D013045:Species Specificity
D015342:DNA Probes; D005819:Genetic Markers