Clonal tracking analysis via LAM-PCR and Gene Scan. (A) Outline of LAM-PCR methodology. (1) Linear PCR by repeated primer extension from a biotinylated oligonucleotide and capture of the DNA products with avidin-coated magnetic beads. (2) Double-stranded (ds) DNA synthesis by random hexanucleotide priming. (3) DNA restriction digestion with TasI. (4) Ligation of an oligonucleotide ligation cassette (LC) to the overhanging sequence at the TasI site. (5) Nested PCR amplifications using primer pairs LCI-LTRII, LCII-LTRIII. (B) Final PCR amplification using a fluorescent primer allows separation and precise sizing of LAM-PCR products via comparison to size standards (M) on an automated sequencer and analysis using GeneScan software. (C) Repetitive samples of granulocyte DNA (100 ng) from one animal. The absolute number of independent clones detected by performance of each replicate LAM-PCR procedure is shown. By the time 6 replicates are run, the absolute clone number detected begins to plateau, and by 15 replicates, essentially no additional clones are detected.