Molecular Biology. A Practical Guide - Velikov V.A. 2013

DNA Restriction
Restriction of Eukaryotic Genomic DNA

Compared to plasmid, phage, or Mitochondrial DNA, eukaryotic chromosomal DNA contains an extremely high number of restriction sites. In addition, it is invariably fragmented randomly during isolation, and its preparations are more difficult to free from Proteins, Polysaccharides, and other contaminants. Furthermore, its degree of methylation can vary significantly, which heavily impacts The activity of endonucleases.

Typically, a 3-5-fold excess of the enzyme per 1 μg of DNA is used, and Digestion is carried out for 2-6 h or left overnight. 10 μg of DNA from most higher plants corresponds to 10⁶-10⁷ genomes.

The DNA concentration in a solution can be determined either by comparison with a known standard using agarose gel Electrophoresis, or by measuring the optical density of a diluted aliquot of the DNA solution.

When genomic DNA is digested with restriction Enzymes that recognize six-nucleotide palindromes (EcoRI, BamHI, PstI, and other common Restriction Endonucleases), restriction fragments ranging from tens of NUCLEOTIDES to 20-30 kb are generated. Thus, complete restriction digestion of eukaryotic DNA yields a massive number of restriction fragments—tens or hundreds of thousands. Upon electrophoresis, genomic DNA that has undergone partial or complete digestion produces a smear of fragments of varying sizes. Visualizing a specific fragment is only possible through Southern blotting—transferring the DNA onto a nitrocellulose or nylon filter—followed by Hybridization with a radiolabeled or fluorescently labeled probe.

So-called "frequent-cutter" restriction enzymes, which recognize 4-nucleotide palindromes (such as Sau3AI), will cleave genomic DNA into smaller fragments and in greater quantities. To effectively separate high- or low-molecular-weight DNA fragments, different gels are used (Topic 3).

Materials and Equipment

Genomic DNA from tobacco (corn, petunia) plants of unknown concentration, BamHI restriction enzyme, DNA sample of known concentration, spectrophotometer, incubator, electrophoresis equipment.

Solutions

- 10x BamHI restriction buffer: 100 mM Tris-HCl, pH 8.0; 50 mM MgCl₂; 1000 mM KCl; 0.02% Triton X-100.

- 10x gel loading buffer (Topic 3).

Procedure

1. Determine the DNA concentration using a spectrophotometer.

2. Assess the purity of the preparation by measuring the optical density at wavelengths of 260, 280, and 235 nm, corresponding to the absorption maxima of DNA, proteins, and polysaccharides, respectively. Pure DNA typically exhibits A260/A235 = 2.2-2.5 and A260/A280 = 1.8-1.9.

3. Digest 1 μg of total tobacco DNA in a 50 μl reaction mixture for 6 h ^a, taking a 10 μl aliquot every hour and stopping the reaction by adding 1 μl of loading buffer ^b.

4. Perform electrophoresis of The DNA Restriction fragments. Use an uncut DNA sample as a control. Photograph the gel.

Notes

^a During prolonged incubation, some restriction enzymes may exhibit star activity (relaxed Specificity), meaning they cleave sites with one or more incorrect nucleotides. This must be taken into account if molecular cloning is planned.

^b The biochemical reaction can be temporarily halted (paused) by placing the tube on ice. In this case, if necessary (incomplete digestion, "underdigestion"), it can easily be resumed. However, if the reaction was stopped using EDTA present in the gel loading buffer, magnesium ions must be added back to the reaction mixture in an equimolar amount.