October 13, 2012

Restriction endonucleases are a key tool in mapping DNA

  • Restriction endonucleases recognize specific short sequences of DNA and cleave the duplex (sometimes at target site, sometimes elsewhere, depending on type).
  • A restriction map is a linear array of sites on DNA cleaved by various restriction enzymes.
  • A kilobase (kb) is a measure of length and may be used to refer to DNA (1000 base pairs) or to RNA (1000 bases).
  • A megabase (Mb) is 1 million base pairs of DNA.
  • Restriction endonucleases can be used to cleave DNA into defined fragments.
  • A map can be generated by using the overlaps between the fragments generated by different restriction enzymes. 

The characterization of eukaryotic genes was made possible by the development of techniques for physically mapping DNA. The techniques can be extended to (single-stranded) RNA by making a (double-stranded) DNA copy of the RNA. A physical map of any DNA molecule can be obtained by breaking it at defined points whose distance apart can be accurately determined. Specific breaks are made possible by the ability of restriction endonucleases to recognize rather short sequences of double-stranded DNA as targets for cleavage.
Each restriction enzyme has a particular target in duplex DNA, usually a specific sequence of 4-6 base pairs. The enzyme cuts the DNA at every point at which its target sequence occurs. Different restriction enzymes have different target sequences, and a large range of these activities (obtained from a wide variety of bacteria) now is available.
A restriction map represents a linear sequence of the sites at which particular restriction enzymes find their targets. Distance along such maps is measured directly in base pairs (abbreviated bp) for short distances; longer distances are given in kb, corresponding to kilobase (103) pairs in DNA or to kilobases in RNA. At the level of the chromosome, a map is described in megabase pairs (1 Mb = 106 bp).
When a DNA molecule is cut with a suitable restriction enzyme, it is cleaved into distinct fragments. These fragments can be separated on the basis of their size by gel electrophoresis, as shown in Figure 2.3. The cleaved DNA is placed on top of a gel made of agarose or polyacrylamide. When an electric current is passed through the gel, each fragment moves down at a rate that is inversely related to the log of its molecular weight. This movement produces a series of bands. Each band corresponds to a fragment of particular size, decreasing down the gel.

By analyzing the restriction fragments of DNA, we can generate a map of the original molecule in the form shown in Figure 2.4. The method is explained in detail in 32.11 Restriction mapping. The map shows the positions at which particular restriction enzymes cut DNA; the distances between the sites of cutting are measured in base pairs. So the DNA is divided into a series of regions of defined lengths that lie between sites recognized by the restriction enzymes. An important feature is that a restriction map can be obtained for any sequence of DNA, irrespective of whether mutations have been identified in it, or, indeed, whether we have any knowledge of its function (Danna, Sack, and Nathans, 1973) (for review see Nathans and Smith, 1975; Wu, 1978).

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