PCR amplification follows an exponential curve until a saturation point is reached, after which further amplification often serves only to degrade purity. Curve shape depends upon the amount of substrate present initially. PCR reactions produce product in a nonlinear pattern (See figure below). Amplification follows a typical exponential curve until some saturation point is reached.…

The polymerase chain reaction (PCR) is a powerful technique which uses repetitive cycles of primer annealing, primer extension, and product denaturing to produce an exponential increase in the copy number of the target DNA. Two primers are used, which flank the region of interest (See figure below). In the presence of a thermostable polymerase, the…

An exploded view of a gel cassette. Native PAGE gels are prepared by mixing an acrylamide/bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate, and water to achieve the desired gel concentration. TEMED and ammonium persulfate are added to initiate polymerization and the solution is poured into a cassette. The comb is then inserted. The…

Sample preparation for native PAGE is straightforward. Since the DNA does not need to be denatured, one is concerned mainly with buffer content, density, and visibility. The salt content of the sample should be adjusted to be no greater than the 200mM salt concentration in the running buffer. Gross imbalances in salt content between sample…

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…