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Modern Genetic Analysis Chapter 10. Recombinant DNA Technology
Using Cloned DNA

Cloned DNA can be used in numerous ways dictated by the needs of the experiment. In this section, we consider some of the basic uses that are applicable in a wide range of experimental circumstances.

Cloned DNA Used as a Probe

We have already examined several examples of this type of application, such as using a cloned gene from one organism to select the appropriate clone from another organism. More examples follow.

Probing to Find a Specific Nucleic Acid in a Mixture

In the course of gene and genome manipulation, it is often necessary to detect and isolate specific DNA molecules from a mixture. For example, recall that, in cloning with the use of l phage, it is necessary to separate the two chromosome arms from the unwanted central region. There are several ways of fractionating DNA, but the most extensively used method is electrophoresis (see Figure 6-5 for a drawing of an electrophoretic apparatus). If a mixture of linear DNA molecules is placed in a well cut into an agarose gel and the well is placed near the cathode of an electric field, the molecules will move through the gel to the anode, at speeds dependent on their size (Figure 10-17). Therefore, if there are distinct size classes in the mixture, these classes will form distinct bands on the gel. The bands can be visualized by staining the DNA with ethidium bromide, which causes the DNA to fluoresce in UV light. If bands are well separated, an individual band can be cut from the gel and the purified DNA sample can be removed from the gel matrix. Therefore DNA electrophoresis can be either diagnostic (showing which DNA fragments are present) or preparative (useful in isolating specific DNA fragments).

Restriction-enzyme digestion of genomic DNA results in so many fragments that a stained electrophoretic gel shows a smear of DNA. A probe can identify one fragment in this mixture, with the use of a technique developed by E. M. Southern called Southern blotting. After DNA fragments are fractionated on the gel, an absorbent membrane is laid over the gel and the DNA bands are transferred ("blotted") onto the membrane by capillary action. When transferred to the membrane, the DNA bands stay in the same relative positions as on the gel. The membrane is bathed in a labeled probe, and an autoradiogram is used to reveal the presence of any bands on the gel that are homologous to the probe. If appropriate, those bands can be cut out of the gel and further processed. The gel can be calibrated for DNA fragment size by running a standard "ladder" of fragments of known size on the same gel. Hence the sizes of any interesting fragments in the experimental sample can be inferred. Figure 10-18 on the following page shows the general procedure of Southern blotting. Figure 10-19 on page 323 shows an application in which a cloned fragment of a fungal plasmid was used to detect homologous plasmids in a variety of strains.

The Southern blotting technique can be extended to detect a specific RNA molecule from a mixture of RNAs fractionated on a gel. This technique is called Northern blotting to contrast it with the Southern technique for DNA analysis. The fractionated RNA is blotted onto a membrane and probed in the same way as for Southern blotting. One application of Northern analysis is in determining whether a specific gene is transcribed in a certain tissue or under certain environmental conditions. RNA is extracted from the appropriate cell sample, then electrophoresed, blotted, and probed with the cloned gene in question. A positive signal shows the presence of the transcript.

Hence we see that cloned DNA finds widespread application as a probe, for detecting either a specific clone or a specific DNA fragment or a specific RNA. In all these cases, the ability of nucleic acids with complementary nucelotide sequences to find and bind to each other in solution is being exploited.

A parallel technique called Western blotting has been developed to transfer electrophoretically fractionated protein from a gel and then to visualize specific proteins with antibodies. However, note that the probe here is not DNA but a labeled antibody. These three techniques are very powerful experimental tools in detecting and sizing specific macromolecules in molecular genetics. They are compared in Figure 10-20.

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DNA or RNA fractionated by size in an electrophoretic gel can be blotted onto an absorbent membrane, which in turn can be probed to detect the position of specific fragments.

DNA Sequence Determination

Success in cloning a desired gene is merely the beginning of a second round of analysis in which the aim is to characterize the structure and function of that gene. The gene's nucleotide sequence is the ultimate characterization of its genetic structure. One of the requirements for DNA sequencing is the ability to obtain defined fragments of DNA. Thus, there is a strong interdependence of DNA cloning and DNA sequencing technology, inasmuch as DNA cloning provides amplified samples of defined DNA fragments.

Any method of DNA sequencing starts with a population of a defined fragment of DNA labeled at one end. From this population, sets of molecules are generated that differ in size by one base at the other end. These molecules are then fractionated. Fractionation is by acrylamide or agarose gel electrophoresis of single-stranded DNA molecules. The mobility of a strand is inversely proportional to the logarithm of its length. This technique is so sensitive that fragments differing in length by only a single nucleotide can be separated. The base at the truncated end of each of the fractionated molecules is determined and hence the nucleotide sequence is established.

The sequencing method now most commonly used was developed by Fred Sanger. His method is based on DNA synthesis in the presence of dideoxy nucleotides, which differ from normal deoxynucleotides in that they lack a 3-hydroxyl group (Figure 10-21). The respective dideoxy nucleotide triphosphates (ddNTPs) can be incorporated into a growing chain, but, when incorporated, they terminate synthesis because they lack the 3-hydroxyl group necessary to bond with the next nucleotide triphosphate. Each of four reaction tubes is prepared with a single-stranded DNA template for the sequence of interest, plus DNA polymerase and a section of labeled primer. Each tube receives a small amount of a different ddNTP (ddATP, ddTTP, ddCTP, or ddGTP), together with the four normal deoxynucleotide triphosphates (dNTPs). A dideoxynucleotide will be incorporated randomly, at different sites in different syntheses in the reaction tube. Therefore, in any given tube, various truncated chain lengths will be produced, each corresponding to the point at which the respective ddNTP for that tube was incorporated and terminated chain growth. These lengths, in turn, are a clear indication of where the bases complementary to the ddNTPs are on the template strand. Because incorporation is random, all possible truncated fragments will be produced, corresponding to all the various positions of that particular base. The fragments can be visualized by electrophoresis of the four samples in four lanes on an acrylamide gel, where the fragments form bands. The base sequence can be determined by scanning up the gel, encompassing all four lanes, and recording whichever base occupies the terminus in the next band. The procedure is illustrated in Figures 10-22 and 10-23.

Instead of radioactive labels, the tags attached to oligonucleotide primers can be fluorescent dyes. A different fluorescent color emitter is used for each of the four reactions, and the four mixtures are electrophoresed together. Flourescence detection is used in automated DNA sequencing machines, which can read as many as 1000 bases in one separation. Figure 10-24 illustrates a readout of automated sequencing.

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A cloned DNA fragment can be sequenced by generating a set of labeled single-stranded DNAs differing in length by one nucleotide; when these DNAs are electrophoresed, the nucleotide sequence can be read directly from an autoradiogram of the gel.

The nucleotide sequence of a cloned DNA fragment can be used to find the cloned gene or genes that it contains. The nucleotide sequence is fed into a computer, which then scans all six reading frames (three in each direction) in the search for a gene-sized stretch of DNA beginning with an ATG initiation codon and ending with a stop codon. These stretches are called open reading frames (ORFs). They represent sequences that are candidate genes. Figure 10-25 on the following page shows such an analysis in which two candidate genes have been identified as ORFs. It is interesting to note that ORF detection and Mendelian progeny ratio analysis, although poles apart in their approaches, are both ways of identifying genes.

Detecting and Amplifying Sequences by the Polymerase Chain Reaction

If a region of DNA has already been cloned and sequenced, the sequence can be used to retrieve parts of the equivalent region from specific individuals or from other species, all without cloning. The technique uses a procedure called the polymerase chain reaction (PCR). Figure 10-26 illustrates the principle of the technique. A temperature-resistant DNA polymerase, Taq polymerase, from the bacterium Thermus aquaticus is used to catalyze growth from DNA primers. Pairs of primers on opposite strands are extended toward each other, as shown in Figure 10-26. After completion of the replication of the segment between the two primers (one cycle), the two new duplexes are heat denatured to generate single-stranded templates, and a second cycle of replication is carried out by lowering the temperature in the presence of all the components necessary for the polymerization. Repeated cycles of synthesis and denaturation result in an exponential increase in the number of segments replicated. Amplifications by as much as a millionfold can be readily achieved. With the use of PCR, a single-copy gene can be amplified out of a genomic sample, provided primers corresponding to known sequences of the gene can be synthesized. Because of the exponential amplification, PCR is very sensitive and can detect target sequences that are in extremely low copy number in a sample. For example, segments of human DNA can be amplified by using just the few follicle cells surrounding a single pulled-out hair. PCR is generally useful in DNA diagnosticsin other words, in checking for the presence of a gene or the mutational state of a specific gene or, preparatively, in amplifying a defined segment.

Note that, in the PCR technique, no restriction digestion of the substrate DNA is needed, because the primers will home in on the appropriate sequence of native DNA. Furthermore, no lengthy cloning procedures are necessary, because enough DNA is amplified that a clear band on a gel is produced. In addition, only very small amounts of substrate DNA are needed.

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The polymerase chain reaction uses specially designed primers to amplify specific short regions of DNA without cloning.

Locating Genes on Restriction Maps

We have already seen the importance of restriction enzymes in cutting DNA for use in cloning. Another useful feature of restriction enzymes is that the positions of their target sites along a DNA molecule (that is, along a chromosome) can be used as DNA markers. Even though the sites exist by chance, they are generally in the same positions on homologous chromosomes. Therefore, the positions of the target sites can be used much like milestones along a road, which, although not important in themselves, can be used as reference points for locating more significant features along the road. Hence, a map of the positions of restriction sites is a valuable tool in genome analysis generally. Most often, a restriction map is made for a localized chromosomal region that is of particular interest or for a relatively small chromosome such as that of a plasmid or an organelle.

One way of making a restriction map is to compare single-enzyme digests with double digests. Two restriction enzymes are applied in separate digestions, and then the two enzymes are used together. After cutting, the fragment sizes are determined by electrophoresis. The double digest shows whether a fragment produced by one enzyme contains sites for the other; if so, the fragment disappears and is replaced by two or more smaller fragments. Comparison of the sizes of fragments produced in the different digestions allows an approximate localization of the restriction target sites. The procedure is illustrated in Figure 10-27.

Another method for making restriction maps is shown in Figure 10-28. In this method, a piece of DNA is labeled at each 5 end with 32P and cut to generate fragments with the label at one end. The longest fragment is isolated and digested with a second restriction enzyme, but the reaction is not allowed to go to completion; therefore, a population of variously sized labeled fragments is produced, each with one of the various restriction sites at the nonlabeled end. These fragments are separated electrophoretically, and the distance of each restriction site from the labeled end can thus be deduced. An example of a restriction map is shown in Figure 10-29. This map is of the DNA of a linear plasmid from a fungus.

After a restriction map has been made, genes can be placed on the map by using Southern analysis. For example, if a cloned gene hybridizes with a fragment A produced from restriction enzyme 1 and with fragment B produced by restriction enzyme 2, then the gene must be located in the region where fragments A and B overlap.

In this chapter, we have introduced the fundamental techniques that have revolutionized genetics. Genetics in Process 10-1 describes the genetic analysis of the disease alkaptonuria, which incorporates and integrates many of the techniques introduced in this chapter.

Genes are no longer hypothetical entities. Thanks to recombinant DNA technology and related technologies, genes can be isolated in a test tube and characterized as specific nucleotide sequences. But even this is not the end of the story. We shall see in the next chapter that knowledge of the sequence is often the beginning of a fresh round of genetic manipulation of that sequence, giving rise to sophisticated new approaches for altering an organism's phenotype.

© 1999 by W. H. Freeman and Company. All rights reserved.