Gel Electrophoresis of DNA and RNA

In the absence of denaturants double stranded DNA retains its double helical structure, which gives it a rodlike form as it migrates through a gel (for non-denaturing electrophoresis of single stranded DNA, see SSCP Analysis). Double stranded DNA of up to 1000 bp can be separated on polyacrylamide gels. DNA over 1kb is generally fractionated…

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 critical for protein binding will not appear as bands in the bound population. Both the methylation and uracil interference assay…

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 analyze the site of protein binding on the DNA (“Footprint” Analysis). A DNA binding protein will attach itself only to…

n Primer Extension, the probe introduced to the mRNA pool will hybridize with the RNA of interest if it is present. Hybrids are then extended by reverse transcriptase. The information gained through this method includes the confirmation of the presence of the RNA of interest, the location of the transcription start site, and if an…

In RNase Protection, an excess of the labeled probe is hybridized into the mRNA pool. Digestion with RNase followed by gel electrophoresis (Probe + mRNA) provides quantitation of the amount of probe complementary mRNA expressed. After digestion in the absence of mRNA (Probe – mRNA) no probe remains. Ribonuclease protection is a procedure that uses…

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 regions of identity between two strands of the duplex. If one of the strands is labeled at one end, the…

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 digest single-stranded nucleic acids (ssDNA or ssRNA). Duplexes, whether DNA: DNA, RNA: RNA, or DNA: RNA, are resistant to digestion.…

The differential display is a PCR-based technique that generates a characteristic set of DNA fragments from the messenger RNA pool within a cell. The use of random hexamers (brown) in combination with oligo-dT primers (blue) allows the amplification of a population of DNA species which changes with the composition of the starting RNA pool. The…

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, and read over 1,000 bases from each reaction. A single run on an automated sequencer can thus produce as much…

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. After a 2-2.5 hour run, a 6% polyacrylamide sequencing gel will give 200-250 bases of readable sequence starting at or…