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).
KeywordsNylon Membrane Xylene Cyanol Sequence Ladder Saran Wrap Cytosine Methylation Pattern
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