Protocol for genome-wide analysis of palindrome formation

Earlier work from our laboratory focused on identifying regions of the genome susceptible to DNA palindrome formation, a rate-limiting step in gene amplification. We described a method to obtain a genome-wide analysis of palindrome formation (GAPF) based on the efficient intrastrand base pairing in large palindromic sequences (1). Palindromic sequences can rapidly anneal intramolecularly to form ‘snap-back’ DNA under conditions that do not favor intermolecular annealing. This snap-back property is used to enrich for palindromic sequences in total genomic DNA by denaturing the DNA at 100˚C, rapidly renaturing it by cooling, and then digesting the mixture with the single-strand specific nuclease S1. Snap-back DNA formed from palindromes is double-stranded and resistant to S1, whereas the remainder of genomic DNA is single-stranded and thus is sensitive to S1 digestion. Using GAPF, we have shown that de novo palindromes can form in cancers, and direct molecular analysis validated that a subset of these GAPF-positive signals represent cancer-specific palindromes located at the boundary of gene amplicons (1,2). We recently discovered that the original GAPF protocol also enriches for differentially methylated DNA (3). We now describe a modification of GAPF to increase the stringency of denaturation using 50% formamide to make the assay specific for DNA palindromes.