Genomic Footprinting by Ligation Mediated Polymerase Chain Reaction

Chromatin structure analysis at single-nucleotide resolution can be done by genomic sequencing, and, recently, several techniques have been developed (1 ) that give improved specificity and sensitivity over the original method of Church and Gilbert (2 ). The most sensitive method uses ligation-mediated polymerase chain reaction (LM-PCR) to amplify all fragments of the genomic sequence ladder (3 ,4 ). The unique aspect of LM-PCR is the ligation of an oligonucleotide linker onto the 5′ end of each DNA molecule. This provides a common sequence on the 5′ end, and in conjunction with a gene-specific primer, allows conventional, exponential PCR to be used for signal amplification. Thus by taking advantage of the specificity and sensitivity of PCR, one needs only 1 μg of mammalian DNA per lane to obtain good quality DNA sequence ladders, with retention of methylation, DNA structure, and protein footprint information. The LM-PCR procedure is outlined in Fig. 1. Briefly, the first step is cleavage of DNA, generating 5′ phospho-rylated molecules. This is achieved, for example, by chemical DNA sequencing (β-elimination) or by cutting with the enzyme DNase I. Next, primer extension of a gene-specific oligonucleotide (primer 1) generates molecules that have a blunt end on one side. Linkers are ligated to the blunt ends, and then an exponential PCR amplification of the linker-ligated fragments is done using the longer oligonucleotide of the linker (linker-primer) and a second gene-specific primer (primer 2). After 15–20 amplification cycles, the DNA fragments are separated on a sequencing gel, electroblotted onto nylon membranes, and hybridized with a gene-specific probe to visualize the sequence ladder. By rehybridization, several gene-specific ladders can be sequentially visualized from one sequencing gel (4 ).