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In vivo footprinting requires the precise and sensitive mapping of rucks in highly complex genomic DNA. The Ugation-mediated PCR methodology (LM-PCR (I, 2]) has overcome sensitivity problems due 10 a selective amplification of the DNA fragments under srudy _ 1be currenl procedure (3) employs three genespecific oligooucleotides (pl-P3) to prime three DNA synthesis reactions (Figure I): First, PI is extended on denatured genomic DNA 10 convert all nearby rucks into blunt ends of doublestranded DNA. Tc these ends a small double-stranded linker is ligated. P2 is theo used in conjunction wilh the long linker oligonucleotide for PCR amplification. Finally. radioactivelylabelIed P3 is extended (0 label the set of amplified fragments. We have introduced various modifications 10 the current protocol (3) in order 10 improve the sensitivity, reliability of fragment representation and ease of primer design. First, we exploited the fact that the presence of polyethylene glycol (pEG) during ligations improves the efficiency of blunt end ligation (4). Linker ligation in the presence of 15% PEG resulted in a significant increase in signal and, importantly, improved the equal representation particularly of sma1ler fragments (data submitted but noI shown). Another critica1 aspect in PCR-based genomic sequencing and footprinling experiments is the reliability of the polymerases involved to complete the various primer extension reactions. In a recent improvement of the procedure Garrity and Wold (5) used the thennostable 7hemwcoccus litoralis (Vent) DNA polymerase for the first primer extension and the amplification step and observed an increased sensitivity as weil as a more faithful representation of genomic fragments after amplification. We attributed our initial failure 10 use the Yent polymerase successfully 10 its known 3' 5' exonuclease activity which can ael on doubleand single-stranded DNA. Since the Yent polymerase is not recommended by the supplier for sequencing applications, but rather a mutant enzyme lacking the exonuc1ease activity (Vent Exo) (6), we tested this enzyme for LM-PCR. Figure 2A shows the result of parallel LM-PCR reactions visualising the guanosine-specific cleavage pauem of the genomic Drosophila hsp27 gene. The three DNA synthesis reactions (primer extension on genomic DNA, PCR amp1ification and labelling of amplified DNA) were carried out by all possible combinations of Vent and Yenl Exopolymerases. 100 ng of nicked genomic DNA (purified according 10 ref. 7) and 0.5 pmole Pt (5' AAGCAATIAITAGCCTGTC 3') were incubated in 19 jll Yent buffeT (10 mM KCI. 10 mM (N~hS04' 20 mM

An improved protocol for genomic sequencing and footprinting by ligation-mediated PCR