1

FIGURE 20-2 Pulsed-field gel electrophoresis. In this figure, the agarose get is shown from above wifh the head of the gel and a series of sample wells, at the top. A and B represent two sets of electrodes. These ate switched on and off alternately, as described in the text When A is on, the DNA is driven toward the bottom right corner of the gel where the anode of that pair is situated. When A is switched off, and B is switched en, the DMA moves tcvwd the bottom left corner. The arrows thus show the path followed by the OKiA as electrophoresis proceeds. (Source: Adapted from Sambrook J. and Russell D.W. ?0Q 1 Molecular cloning: A laboratory manual, 3rd edition, p 555, hg 5-7 Cdd Spring Harbor Laboratory Press, Cold Spring Harbor, NY)

Restriction Endonucleases Cleave DNA Molecules at Particular Sites

Most naturally occurring DNA molecules are much larger than can readily be managed, or analyzed, in the lab. Thus, as we have seen, chromosomes are extremely long single DNA molecules that can contain thousands of genes (see Chapter 7). if we are to study individual genes and individual sites on DNA, (he large DNA molecules found in cells must be broken into manageable fragments. This is done using restriction endonucleases. These are nucleases that cleave DNA at particular sites by the recognition of specific sequences.

Restriction enzymes used in molecular biology typically recognize short (4-8 bp) target sequences, usually palindromic, and cut at a defined position within those sequences. Thus, consider one widely used restriction enzyme, EcoRI, so named because it was found in certain strains of Escherichia coli, and was the first (I) such enzyme found in that species. This enzyme recognizes and cleaves the sequence 5'-GAATTC-3f. (Because the two strands of UNA are complementary, we need specify only one strand and its polarity to describe a recognition sequence unambiguously.)

This hexameric sequence (like any other) would be expected to occur once in every 4 kilobases on average. (This is because there ere four possible bases that can occur at any given position within a UNA sequence, and so the chances of finding any given specific G bp sequence is 1 in 4li.) So, consider a linear DNA molecule with six copies of the GAATTC sequence: FcoRI would cut it into seven fragments in a range of sizes reflecting the distribution of those sites in the molecule. Suppose we then subject the ¿'roRIcut DNA to electrophoresis through a gel: the seven fragments would separate from each other on the basis of their different sizes (Figure 20-3). Thus, in the experiment shown, £'coR] has dissected the DNA into specific fragments, each corresponding to a particular region of the molecule.

If the same DNA molecule had been cleaved with a different restriction enzyme—for example, H/ndlll, which also recognizes a 6 bp target, but of a different sequence (5'-AAGCTT-3f)-—the molecule would have been cut at different positions and generated fragments of different sizes. Thus, the use of multiple enzymes allows different regions of s DNA molecule to be isolated. It also allows a given molecule to be identified. Thus, a given molecule will generate a characteristic series of patterns when digested with a set of different enzymes.

Other restriction enzymes such as Sau'SAi (which is found in the bacterium Staphylococcus aureus) recognize tetrameric sequences (5'-GATC-3') and so cut UNA more frequently, approximately once every 250 bp. At the other extreme is Notl, which recognizes an octameric sequence (5'-GCGGCCGC-3'j and cuts, on average, only once every 65 kilobases (Table 20-1).

FIGURE 20-3 Digestion of a DNA fragment with endonudease FcoRl. At the top ts shown a PI\SA molecule and the positions within it at which EtoRl cleaves When the molecule, digested with that enzyme, is run on an agarose gel, the pattern of bands shown are observed.

Was this article helpful?

0 0

Post a comment