LINE-1 insertion dimorphisms identification by PCR.

Differentiating between recent LINE-1 (L1) insertion dimorphisms (LIDs) is predominantly useful for studying not only the transposition mechanism but also population genetics or evolution based on polymorphic markers where the ancestral state is known (i.e., absence of the insertion) (1–5). Previous techniques such as L1 display (1) or ATLAS (2) have identified new LIDs from distinct populations. To improve both efficiency and simplicity, we designed a PCR-based approach, LID identification by PCR (LIDSIP), by combining and modifying ligation-mediated PCR (LMPCR) (6) and interspersed repetitive sequence PCR (IRSPCR) (7). This method requires only a small amount of DNA, a limited and specific number of PCRs to provide a genome-wide scan within a PCR range of an appropriate restriction site, conventional molecular genetic techniques such as agarose gel electrophoresis devoid of radioactive label, and in addition, this method yields clear, easily distinguishable, specific results. The aim of applying LIDSIP was to globally map active L1 in the human genome by amplifying the 3′ untranslated region (UTR) end of the L1-Ta subset up to its next specific restriction enzyme recognition sequences. First, 500 ng human genomic DNA were digested with BstYI (New England Biolabs, Beverly, MA, USA) and purified with phenol-chloroform extraction and ethanol precipitation. Second, the DNA was ligated to 20 pmol each of LIDSIP-LINK (5′-AGGTAACGAGTCAGACCACCGACTCGTGGACGT-3′) and BstYI-LINK (5′-GATCACGTCCACGAG-3′) using T4 DNA ligase (New England Biolabs) at 16°C overnight. Finally 50 ng of the purified ligated DNA were subjected to nested PCR. The first PCR (50 μL total volume) contained 200 μM each of the four dNTPs, 1× PCR buffer (contains 1.5 mM MgCl2; Qiagen, Valencia, CA, USA), 0.1 U HotStarTaq® (Qiagen), 0.4 μM of L1-ACA (5′-GAGATCTACCTAATGCTAGATGACACA-3′) (1), and 5′ linker (5′-AGGTAACGAGTCAGACCACCGA-3′). The PCR amplification was performed as follows: initial denaturation at 95°C for 15 min, followed by 20 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, extension at 72°C for 2 min, and a final extension at 72°C for 7 min. To divide the number of product types into subsets distinguishable by electrophoresis, 20 cycles of nested PCR were performed using 32 different chimeric primers. This step helps fractionate the repetitive products similar to MseI 3+ primers in sequencespecific amplification polymorphism (S-SAP), which was developed to display plant retrotransposons (8). Each reaction consisted of 20 amplification cycles containing 1 μL primary PCR product, one of 32 chimeric primers [5′-GACTCGTGGACGTGATC(C/ T)XX-3′, where (C/T) was C or T, and XX was 2 bp of randomly selected sequences; e.g., TGA chimeric is 5′GACTCGTGGACGTGATCTGA-3′], GCNP (1), and the L1-nested-primer, GCGCACCAGCATGGCACA. This is further illustrated by the schematic diagram (Figure 1) and an example of LIDSIP (Figure 2a), respectively. When LIDSIP products were compared among 14 Thai individuals, 37 candidate LIDs could be identified. On average, each nested PCR yielded 10 out of a total of 300 products per sample. Five candidate LIDs were selected, cloned to the pGEM-T easy cloning vector (Promega, Madison, WI, USA), and transformed into Escherichia coli DH5α. DNA sequencing was performed on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA), and results were analyzed by Basic Local Alignment Search Tool (BLAST) program (www.ncbi. nih.gov/BLAST). All clones revealed upstream L1, integration site, single copy, and chimeric primer sequences. Nevertheless, the BLAST results were devoid of L1 upstream of the flanking sequence, and only the preintegration sites were actually present in the human genome database (GenBank® accession nos. AC015547 at 86,101 bp, AC087307 at 13,330 bp, AL392087 at 11,869 bp, AL583842 at 37,981 bp, and AP001996 at 37,981 bp), suggesting recent retrotranspositions. LID PCRs (1,2) were performed, and polymorphisms of all markers were confirmed (Figure 2b). Applying LIDSIP has improved both simplicity and efficacy compared with the two previous PCR techniques. The first, L1 display (1), screens for LIDs by PCR using arbitary primers, Southern blot analysis, and hybridization for identification. Each PCR yields a