Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 65
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To illustrate one procedure that is used, let us assume that two bacterialspecies have been grown in media that cause the DNA of one species to contain only the nonradioactive isotope ofphosphorus, 31P, and cause the DNA of the second species to contain the radioactive isotope, 32P. The DNAmolecules of the two species are isolated, broken into many small fragments, and denatured.
The nonradioactivemolecules are immobilized on a nitrocellulose filter, and the radioactive DNA is added. The temperature and saltconcentration are raised to promote renaturation, and then, after a suitable period of time, the filter is washed. Theexistence of common base sequences will be detected by the presence of renatured fragments of radioactive 32PDNA on the filter. The extent of common base sequences determined in this manner in the DNA of two speciesgenerally agrees with the evolutionary relatedness of the species as indicated by direct DNA sequencing of asample of their genes.Restriction Enzymes and Site-Specific DNA CleavageOne of the problems with breaking large DNA molecules into smaller fragments by random shearing is that thefragments containing a particular gene, or part of a gene, will all be of different sizes.
With random shearing,owing to the random length of each fragment, it is not possible to isolate and identify a particular DNA fragment.However, there is an important enzymatic technique, described in this section, that can be used for cleaving DNAmolecules at specific sites.As we saw in Chapter 4, members of a class of enzymes known as restriction endonucleases or restrictionenzymes are able to cleave DNA molecules at the positions at which particular, short sequences of bases arepresent.
For example, the enzyme BamHI recognizes the double-stranded sequence5'-GGATCC-3'3'-CCTAGG-5'and cleaves each strand between the G-bearing nucleotides shown in red. Figure 5.30 shows how the regions thatmake up the active site of BamHI contact the recognition site (blue) just prior to cleavage, and the cleavagereaction is indicated in Figure 5.31.Table 5.3 lists six of the several hundred restriction enzymes that are known.
Most restriction enzymes are isolatedfrom bacteria, and they are named after the species in which they were found. BamHI, for example, was isolatedfrom Bacillus amyloliquefaciens strain H, and it is the first (I) restriction enzyme isolated from this organism. Mostrestriction enzymes recognize only one short base sequence, usuallyFigure 5.30Part of the restriction enzyme BamHI in contactwith its recognition site in the DNA (blue). Thepink and green cylinders represent regions ofthe enzyme in which the amino acid chain istwisted in the form of a right-handed helix.[Courtesy of A.A.
Aggarwal. From M.Newman, T. Strzelecka, L. F. Dorner, I.Schildkraut, and A.A. Aggarwal, 1995. Science269:656.]Page 203Figure 5.31Mechanism of DNA cleavage by the restriction enzyme BamHI. The enzyme makes a single cut in the backbone of each DNAstrand wherever the duplex contains a BamHI restriction site. Each cut creates a new 3' end and a new5' end, separating the upper and lower parts of the duplex. In the case of BamHI the cuts are staggeredcuts, so the resulting ends terminate in single-stranded regions, each four base pairs in length.Page 204four or six nucleotide pairs.
The enzyme binds with the DNA at these sites and makes a break in each strand of theDNA molecule, producing 3'-OH and 5'-P groups at each position. The nucleotide sequence recognized forcleavage by a restriction enzyme is called the restriction site of the enzyme. The restriction enzymes in Table 5.3all cleave their restriction site asymmetrically (at different sites in the two DNA strands), but some restrictionenzymes cleave symmetrically (at the same site in both strands). The former leave sticky ends because each end ofthe cleaved site has a small, single-stranded overhang that is complementary in base sequence to the other end(Figure 5.31).
In contrast, enzymes that have symmetrical cleavage sites yield DNA fragments that have bluntends. In virtually all cases, the restriction site of a restriction enzyme reads the same on both strands, provided thatthe opposite polarity of the strands is taken into account; for example, each strand in the restriction site of BamHIreads 5'-GGATCC-3' (Figure 5.31). A DNA sequence with this type of symmetry is called a palindrome. (Inordinary English, a palindrome is a word or phrase that reads the same forwards and backwards, such as ''madam.")Restriction enzymes have the following important characteristics:• Most restriction enzymes recognize a single restriction site.• The restriction site is recognized without regard to the source of the DNA.• Because most restriction enzymes recognize a unique restriction site sequence, the number of cuts in the DNAfrom a particular organism is determined by the number of restriction sites present.The DNA fragment produced by a pair of adjacent cuts in a DNA molecule is called aTable 5.3 Some restriction endonucleases, their sources, and their cleavage sitesEnzymeMicroorganismEcoRITarget sequence andcleavage sitesEnzymeMicroorganismEscherichia coliHindIIIHaemophilusinfluenzaeBamHIBacillus amyloliquefaciens HPstIProvidencia stuartiiHaeIIHaemophilus aegyptusTaqIThermus aquaticusTarget sequence andcleavage sitesNote: The vertical dashed line indicates the axis of symmetry in each sequence.
Red arrows indicate the sites of cutting. The enzymeTaqI yields cohesive ends consisting of two nucleotides, whereas the cohesive ends produced by the other enzymes contain fournucleotides. Pu and Py refer to any purine and pyrimidine, respectively.Page 205Figure 5.32Restriction maps ofλ DNA for the restriction enzymes. (A) Eco RI and (C)BamHI. The vertical bars indicate the sites of cutting.
The blacknumbers indicate the approximate percentage of the total length of λ DNAmeasured from the end of the molecule that is arbitrarily designated theleft end. The numbers within the arrows are the lengths of the fragments, eachexpressed as percentage of the total length. (B) An electrophoresisgel of BamHI and EcoRI enzyme digests of λ DNA. Numbers indicatefragments in order from largest (1) to smallest (6); the circlednumbers on the maps correspond to the numbers beside the gel. The DNAhas not undergone electrophoresis long enough to separate bands 5 and6 of the BamHI digest.restriction fragment. A large DNA molecule will typically be cut into many restriction fragments of differentsizes. For example, an E. coli DNA molecule, which contains 4.7 × 106 base pairs, is cut into several hundred toseveral thousand fragments, and mammalian nuclear DNA is cut into more than a million fragments.
Althoughthese numbers are large, they are actually quite small relative to the number of sugar-phosphate bonds in the DNAof an organism. Restriction fragments are usually short enough that they can be separated by electrophoresis andmanipulated in various ways—for example, using DNA ligase to insert them into self-replicating molecules such asbacteriophage, plasmids, or even small artificial chromosomes. These procedures constitute DNA cloning and arethe basis of one form of genetic engineering, discussed further in Chapter 9.Because of the sequence specificity, a particular restriction enzyme produces a unique set of fragments for aparticular DNA molecule.
Another enzyme will produce a different set of fragments from the same DNA molecule.Figure 5.32A and 5.32C show the sites of cutting of E. coli phage λ DNA by the enzymes EcoRI and BamHI. Amap showing the unique sites of cutting of the DNA of a particular organism by a single enzyme is called arestriction map.
The family of fragments produced by a single enzyme can be detected easily by gelelectrophoresis of enzyme-treated DNA (Figure 5.32B), and particular DNA fragments can be isolated by cuttingout the small region of the gel that contains the fragment and removing the DNA from the gel. Gel electrophoresisfor the separation of DNA fragments is described next.Gel ElectrophoresisThe physical basis for the separation of the DNA fragments in Figure 5.32 is that DNA molecules are negativelycharged and can move in an electric field.
If the terminals of an electrical power source are connected to theopposite ends of a horizontal tube containing a DNA solution, then the molecules will move toward the positiveend of the tube, at a rate that depends on the electric field strength and on the shape and size of the molecules. Themovement of charged molecules in an electric field is called electrophoresis.The type of electrophoresis most commonly used in genetics is gel electrophoresis. An experimental arrangementfor gel electrophoresis of DNA isPage 206Figure 5.33Apparatus for gel electrophoresis capable of handlingseven samples simultaneously. Liquid gel is allowed toharden in place, with an appropriately shaped moldplaced on top of the gel during hardening in order tomake "wells" for the samples (purple). After electrophoresis,the samples, located at various positions in the gel,are made visible by removing the plastic frame and immersingthe gel in a solution containing a reagent that binds toor reacts with the separated molecules.
The separatedcomponents of a sample appear as bands, whichmay be either visibly colored or fluorescent whenilluminated with fluorescent light, depending onthe particular reagent used. The region of a gel in whichthe components of one sample can move is calleda lane. Thus, this gel has seven lanes.shown in Figure 5.33. A thin slab of a gel, usually agarose or acylamide, is prepared containing small slots (calledwells) into which samples are placed. An electric field is applied, and the negatively charged DNA moleculespenetrate and move through the gel.
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