Outline of linear amplification-mediated (LAM)–PCR.

A new combination of linear amplification of target DNA with solid-phase second-strand synthesis, followed by ligation of an oligonucleotide cassette and then nested exponential PCR, was devised for the detection and direct genomic sequencing of unknown retroviral vector integration sites. (A) Linear PCR with a long terminal repeat (LTR)–specific biotinylated primer was performed by repeated primer extension. Subsequently, the amplified fragments of target DNA were enriched by magnetic tag selection of extension primers. (B) A second DNA strand of each enriched target sequence was synthesized by random hexanucleotide priming. (C) Resulting double-stranded DNA was specifically digested with the restriction enzyme Sse9I, which cuts within genomic DNA approximately every 256 bp. The length of each fragment is thus dependent on the distance of the vector insertion site from the next Sse9I recognition sequence. (D) An asymmetric oligonucleotide ligation cassette (LC) was ligated to the end of the Sse9I-digested fragments. (E) Nested exponential PCR amplifications were then performed with LC-specific forward primers (LC 1 followed by LC 2) and LTR-specific reverse primers (LTR II followed by LTR III).