Authors
Takahito Yamasaki, Takehide Murata, Chunyuan Jin, Kohsuke Kato, Michiya Noguchi, Koji Nakade, Jianzhi Pan, Kyousuke Nagata, and Kazunari Yokoyama
Summary
We can use PCR to amplify segments of DNA located between two specified primer-hybridization sites. Single-sided PCR requires specification of only one primer-hybridization site, with the second being defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows the amplification by PCR of any fragment of DNA. Because a defined sequence of discrete-length is added to every fragment, complex populations of DNA, such as sequence ladders, can be amplified intact with retention of single-base resolution. In this method, cleaved DNA is denatured and a gene-specific primer (primer 1) is annealed to the region of interest. During first-strand synthesis, this primer is extended by a processive polymerase (Vent DNA polymerase) to the cleavage site, to create a blunt end. DNA ligase catalyzes the attachment of a unidirectional (staggered) linker to this blunt end. The 3’-end of the longer strand of the linker is ligated to the 5’-end of the genomic DNA. The shorter strand of the linker lacks a 5’-phosphate and, therefore, it is not ligated to the extension product of the gene-specific primer. The DNA is denatured and ligated to the extension product of the gene-specific primer. The DNA is denatured and a second gene-specific primer (primer 2) is annealed to the genomic DNA and extended, by Vent DNA polymerase, through the ligated linker region. The extended product is now a suitable substrate for PCR: at one end, there is a linker sequence to which a linker primer can anneal and, at the other end, there is a genomic sequence to which a gene-specific primer can anneal. Only molecules that have both sequences will be exponentially amplified during subsequent PCR; molecules with only one of the sequences will be linearly, as distinct from exponentially, amplified. For the last extension, a third gene-specific primer (primer 3) that overlaps the second is used to label the DNA indirectly. The end-labeled extension products are visualized on a sequencing gel1. For a detailed introduction to assays of nucleosome assembly and the inhibition of histone acetyltransferase activity, please go here: http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly.phpFurther details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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