Uracil or methylation interference assay. End labeled probe is modified at one site per molecule, and allowed to bind protein. Bound and unbound populations are separated, and strands are cleaved at the modified bases. Bases…

The binding of proteins to specific DNA sites is an important mechanism of cellular regulation. Numerous techniques have been developed to analyze the interactions of regulatory proteins with DNA. Three techniques are presented here which…

Primer extension is another technique used to analyze RNA structure and expression. In this method, an oligonucleotide primer is annealed to RNA and extended to a cDNA copy by reverse transcriptase in the presence of…

Ribonuclease protection is a procedure that uses uniformly labeled RNA probes to analyze sample RNA. In this case, probes are chosen to fall entirely within the coding region, so they are only digested if no…

Nuclease S1 will digest only ssDNA or ssRNA. If a duplex of DNA and/or RNA strands have single-stranded overhangs or unhybridized internal loops, these will be digested away. The remaining intact nucleic acid fragments represent…

In studies of transcriptional regulation, it is often necessary to determine the structure and/or amount of a given RNA species. Several techniques have been developed making use of the fact that some nucleases will only…

Differential display is a powerful technique for detecting and quantitating changes in gene expression patterns between differently treated cells. Fragments of those genes which are induced or suppressed can be identified and isolated for further…

Automated sequencing systems make use of fluorescent dye labeling, in combination with laser scanning and computerized data acquisition and processing to carry out the electrophoresis of up to 96 sequencing reactions on a single gel,…

Denaturing PAGE gels for DNA sequencing generally employ 6-8 M urea as their denaturant and TBE as their buffer system. They are poured as described in the section on denaturing PAGE of DNA and RNA.…

In Sanger dideoxy terminator sequencing, the sample DNA is used as a template for a DNA polymerase. Four polymerase reactions are carried out involving enzyme, primer, and sample DNA, along with dNTPs. Each reaction also…