Restriction Enzymes Resource

2.4 Digestion of High Molecular Weight DNA

1. Isolation of High Molecular Weight DNA
2. Procedure for Embedding Mammalian Cells in Agarose Gel Plugs
3. Digestion of High Molecular Weight DNA Embedded in Agarose Gel Plugs
4. Genome Complexity and Expected Restriction Site Frequency
5. Analysis of Large DNA Fragments
6. References

A. Isolation of High Molecular Weight DNA

High molecular weight genomic DNA can be prepared using several different methods including traditional phenol extraction (1), standard isolation procedures such as the Wizard^® Genomic DNA Purification Kit (Cat.# A1120) (2,3) or by embedding the cells of interest in blocks or beads of agarose and enzymatically digesting the cell membranes and proteins (4). Large DNA is quite susceptible to mechanical shearing and it is difficult to obtain DNA of 50kb or more unless it is embedded in agarose. Regardless of the preparation method, genomic DNA is frequently less pure than plasmid or other smaller DNA that can be treated more harshly during isolation. In addition, genomic DNA, especially that of higher organisms, may contain more modifications such as methylation. The methylation sensitivity (Table 3.5) of potential restriction enzymes may need to be considered for genomic digests. Excess restriction enzyme units and extended incubation times are standard for genomic digestions. For long incubations, especially at elevated temperatures, evaporation of water from the buffer can concentrate components of the reaction and cause star activity. The reaction can be overlaid with mineral oil or the digestion performed in an incubator to avoid evaporation. Addition of spermidine to a final concentration of 1-5mM has also been shown to be helpful for genomic digests (1,5).

For DNA in solution, such as that prepared by phenol extraction or by using the Wizard^® Genomic DNA Purification Kit, viscosity may limit mixing of solutions and diffusion of the enzyme. In general 2-10 units of restriction enzyme per microgram of DNA in a reaction volume of 20µl is recommended. Incubation time is typically 4 hours to overnight.

A general protocol for embedding and digesting mammalian cells in agarose is provided below. Conditions will differ significantly for other cell types.

1. Harvest the cells by centrifugation at 500 x g for 10 minutes. Wash the cells in an isotonic solution and resuspend in the same solution. Count the cells and dilute them to an appropriate density (~5 x 10⁷ cells/ml) in isotonic buffer.

2. Make 1% low melting temperature agarose in isotonic buffer, heat to melt the agarose then cool to 37°C.

3. Warm the cell suspension to 37°C, mix 1:1 with the melted agarose and dispense into appropriate molds. It is best to keep the molds on ice so that the agarose will gel rapidly. This will reduce settling of the cells.

4. After the plugs have set, remove them from the mold and place them in a solution of 1mg/ml pronase in 1% lauryl sarcosine, 0.5M EDTA, and 10mM Tris (pH 9.5).

5. Leave the plugs in this solution at room temperature for 30 minutes to allow diffusion of the pronase and buffer into the plugs.

6. Incubate at 50°C overnight. Replace the buffer/pronase, and incubate at 50°C for another 24 hours.

7. Rinse the plugs in buffer for 2 hours, repeat this rinse once more. Store at 4°C.

C. Digestion of High Molecular Weight DNA Embedded in Agarose Gel Plugs

The conditions required for digestion of agarose-embedded DNA differ from those required for digestion of DNA in solution. In general, much more restriction enzyme is needed. We have tested a number of enzymes for their ability to digest DNA embedded in agarose (Table 2.4). The exact amount of enzyme needed varies depending on the DNA type and preparation. A general protocol for digestion of agarose embedded DNA is provided below.

Protocol

1. Soak the agarose plug in TE buffer (10mM Tris-HCl [pH 7.4], 1mM EDTA) for 30 minutes on ice.

2. Equilibrate the plug in the appropriate restriction enzyme buffer supplemented with 20µg/ml of BSA for 30 minutes on ice.

3. Add the restriction enzyme to each tube (see Table 2.4 for examples of the appropriate amount of enzyme to use). Allow the enzyme to diffuse into the agarose for 30 minutes on ice.

4. Incubate the reaction at the appropriate temperature for 3 hours to overnight.

5. Add EDTA to a final concentration of 60mM to stop the reaction.

6. The digested agarose plug can be stored at 4°C for several days until use.

Table 2.4. Parameters for Digestion of Chromosomal DNA by Promega's Genome Qualified Restriction Enzymes.

*These enzymes are available at high concentration (40-80u/µl).
**Perform 50°C digestions under mineral oil.

D. Genome Complexity and Expected Restriction Site Frequency

It is possible to calculate the expected average fragment size for a given genomic DNA if the percent GC content of the DNA and the recognition sequence of the restriction enzyme are known. For example, in a genome with 50% GC content and no dinucleotide bias, a four-cutter can be expected to cut every 4⁴ bases (256), a six-cutter can be expected to digest every 4⁶ (4,096) bases, and an eight-cutter should digest every 4⁸ (65,536) bases. For sequences with GC contents other than 50% it is still possible to do this calculation by considering the probability of a particular nucleotide appearing at each position in the recognition sequence.

The general form of the equation is:

Expected cutting frequency = (0.5 x GC)^a x (0.5 x AT)^b

"GC" & "AT" are the probability that a given base is (G or C) or (A or T) (the GC or AT content of the target DNA), "a" is the number of G's and C's and "b" is the number of A's and T's in the restriction enzyme's recognition sequence.

For example, for an EcoR I digest (GAATTC) of DNA from an organism with 40% GC and no dinucleotide bias the expected chance of cutting would be:

(0.5 x GC)^a x (0.5 x AT)^b = (0.5 x 0.4)² x (0.5 x 0.6)⁴ = 0.000324

The probability of cutting any given 6 base sequence is 0.000324, or an average of one cut every 3086 bases.

The equation can be refined if there is a known bias in the frequency of dinucleotide and trinucleotide repeats in the DNA being digested (5). For a sequence N₁N₂N₃N₄N₅N₆ (where N₁ through N₆ are the bases in the restriction enzyme recognition sequence), the expected frequency of digestion can be calculated as

p(N₁N₂) p(N₂N₃) p(N₃N₄) p(N₄N₅) p(N₅N₆)/p(N₃) p(N₄) p(N₅)

Where p(N) is the frequency of N in the genome and p(N_aN_b) is the dinucleotide repeat frequency.

or

p(N₁N₂N₃) p(N₂N₃N₄) p(N₃N₄N₅) p(N₄N₅N₆)/p(N₂N₃) p(N₃N₄) p(N₄N₅)

Where p(N_aN_b) is the dinucleotide repeat frequency and p(N_aN_bN_c) is the trinucleotide repeat frequency.

The GC content and dinucleotide frequencies of many organisms have been determined (6). Because the sequences of many organisms have been elucidated it is now possible to generate complete restriction maps of entire genomes. Most of the completed genome sequences are available from the World Wide Web from sites such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) at:  http://www.genome.ad.jp/kegg/java/org_list.html.

Other useful web sites include:

• http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed
• http://www.tigr.org/tdb/index.shtml

E. Analysis of Large DNA Fragments

Standard agarose gel electrophoresis can be used to resolve DNA in the ~10bp (4% gel) to ~50kb (0.3% gel) range. For resolution of larger DNA fragments it is necessary to use pulsed field gel electrophoresis (PFGE). PFGE relies on the observation that the rate of re-orientation of DNA within an electric field is proportional to the size of the DNA fragment. In PFGE, the orientation of the electric field relative to the gel, and thus the DNA, is changed throughout the gel run. Larger DNAs re-orient more slowly and thus have slower net migration rates. Several different types of PFGE including: orthogonal field agarose gel electrophoresis (OFAGE), field inversion gel electrophoresis (FIGE), rotating agarose gel electrophoresis (RAGE), and contour clamped homogenous electric field (CHEF) can be used for this purpose.

F. References

1. Ausubel, F.M. et al. (1993) Current Protocols in Molecular Biology , Vol. 1, Greene Publishing Associates, Inc., and John Wiley and Sons, NY. p2.2.1, p3.1.7.
2. Wizard^® Genomic DNA Isolation System Technical Manual #TM050, Promega Corporation.
3. Protocols and Applications Guide, Third Edition (1996), Promega Corporation.
4. Anand, R., and Southern, E. (1990) In Gel Electrophoresis of Nucleic Acids: A Practical Approach, Second Edition. Rickwood, D., and Hames, B. eds. IRL Press, Oxford, U.K.
5. Perbal, B. (1998) A Practical Guide to Molecular Cloning, John Wiley and Sons, NY, p.327.
6. McClelland, M. et al. (1987) Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucl. Acids Res. 15, 5985.
7. Normore, W.M., Shapiro, H.S. and Setlow, P. (1976) In CRC Handbook of Biochemistry and Molecular Biology. Fastman, G.D. ed., CRC Press.

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