Restriction Mapping of Cloned DNA

Abstract

The procedure of restriction mapping involves cleavage of DNA with a variety of restriction endonucleases, followed by separation of the DNA by gel electrophoresis and determination of the number and molecular weight of the fragments generated. Single and double enzyme digests are usually performed and, by comparing the sizes of the fragments generated, cleavage sites for each restriction endonuclease can be localized with respect to one another and a physical map of the DNA can be constructed. An alternative method involves linearizing the DNA, radiolabelling the ends of the DNA fragments, followed by partial digestion using restriction endonucleases with frequent cleavage sites. The fragments are electrophoresed, detected by autoradiography and the cleavage sites are mapped with respect to the end label.

Keywords: restriction endonuclease; restriction enzyme; DNA digestion; DNA cleavage site; mapping; electrophoresis

Figure 1.

Single and double restriction patterns of cloned DNA pDEMO (a demonstration plasmid vector) with restriction endonucleases A, B and C.

Figure 2.

Physical maps of pDEMO deduced by single enzyme digestion using restriction endonucleases A, B and C. The order of fragments is not known and therefore two maps are possible.

Figure 3.

Physical maps of pDEMO deduced by double enzyme digestion using restriction endonucleases A, B and C. Enzyme A + B. The cleavage site for enzyme A is localized within the 5‐kb restriction fragment of enzyme B and generates two fragments 1.7 and 3.3 kb in size. The order of the fragments cannot be deduced and therefore there are two possible arrangements (map 1 and 2).

Figure 4.

Physical maps of pDEMO deduced by double enzyme digestion using restriction endonucleases A, B and C. Enzyme A + C. The cleavage site for enzyme A is localized within the 2.6‐kb restriction fragment of enzyme B and generates two fragments 2.0 and 0.6 kb in size. There are four possible maps because there are two positions of cleavage within the 2.6‐kb fragment (i.e. 0.6 kb from either end) and two orders of fragments generated by enzyme C.

Figure 5.

Physical maps of pDEMO deduced by double enzyme digestion using restriction endonucleases A, B and C. Enzyme B + C. One cleavage site for enzyme B is localized within the 4.1‐kb fragment produced by enzyme C and generated fragments sized 1.4 and 2.7 kb. The second cleavage site for enzyme B is localized within the 2.6‐fragment produced by enzyme C and generates fragments 0.3 and 2.3 kb in size. Only two maps are possible as these are the only ones which give the correctly sized fragments for enzyme B (3.8 kb and 5 kb).

Figure 6.

Physical maps of pDEMO deduced by double enzyme digestion using restriction endonucleases A, B and C. Enzyme A + B + C. Two possible restriction maps are produced which give the correctly sized DNA fragments for each single and double enzyme digest. They both contain the sites in the same order but in opposite orientations.

Figure 7.

Summary of restriction mapping by end‐labelling and partial enzyme digestion.

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How to Cite close
Woodward, K(Jul 2003) Restriction Mapping of Cloned DNA. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003747]