Mouse DNA fragments isolated by genome subtraction

Abstract

Under the assumption that brain genomic DNA undergoes rearrangement for the expression of cell-specific function, genormic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"), was tried to isolate brain-specific genomic DNA fragments. In order to enrich the "target" sequences that were unique to brain, Acc65I degested mouse brain DNA was subtracted with a large excess of KpnI, an isoschizomer of Acc65I, digested liver DNA. Phenol emulsion reassociation technique with either total DNA or size fractionated DNA yielded two defferent clones II2 and X2, respectively. Southern blot analysis and polymerase chain reaction (PCR), however, failed to prove that these fragments were present only in the genomic DNA prepartaions from brain, suggesting that the whole procedure might not be sensitive enough to identify brain specific DNA fragments. Nucleotide sequence analysis showed that II2 was composed of two repetitive sequences, Mouse Transcript (MT) and LINE-1 (L1), and that X2 was of novel sequence. The reaarranged DNA fragment of II2 was observer during subcloning in E. coli. This rearrangement involved two types of inversion processes.