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12. Recombinant DNA Technology
Recombinant DNA is made by splicing a foreign DNA fragment into a small replicating molecule (such as a bacterial plasmid), which will then amplify that fragment along with itself and result in a molecular clone of the inserted DNA. Restriction enzymes cut DNA at specific target sites, resulting in defined fragments with sticky ends suitable for insertion into a vector that has been cut open by the same enzyme. A collection of DNA clones that encompasses the entire genome of an organism is called a genomic library. An individual DNA clone can be selected from a library by using a specific probe for the DNA or its protein product or by its ability to transform a null mutant. DNA fragments of different sizes produced by restriction-enzyme digestion can be fractionated because they migrate to different positions on an electrophoretic gel. RNA or restriction-enzyme-cut DNA molecules that have been fractionated by size in an electrophoretic gel can be probed to detect a specific molecule. Restriction-enzyme target sites can be mapped, providing useful markers for DNA manipulation. A gene can be found by testing overlapping clones radiating outward from a linked marker. After a gene has been cloned, its nucleotide sequence can be determined, and the sequence can be used to study gene function and evolution. A pair of replication primers spanning a DNA sequence can be used to amplify that sequence for isolation.
Injection of foreign DNA into an animal cell. The microneedle used for injection is shown at right and a cell-holding pipette at left. (© M. Baret/Rapho/Photo Researchers, Inc.)
Recombinant DNA Technology The goal of genetics is to study the structure and function of genes and genomes. Since Mendel's time, genes have been identified by observing standard phenotypic ratios in controlled crosses. Clues about gene function first came from correlating specific mutations with enzyme and other protein deficiencies. The correlation of mutant sites within a gene with amino acid substitutions in the appropriate protein led to a better understanding of gene structure and function. To these ideas were added discoveries about the nature of DNA and the genetic code, leading to a fairly comprehensive understanding of the basic nature of the gene. However, all were indirect inferences about genes; no gene had ever been isolated and its DNA sequence examined directly. Indeed, it seemed impossible to isolate an individual gene from the genome. Although it is relatively easy to isolate DNA from living tissue, DNA in a test tube looks like a glob of mucus. How could it be possible to isolate a single gene from this tangled mass of DNA threads? Recombinant DNA technology provides the techniques for doing just that, and today individual genes and other parts of genomes are isolated routinely. Why is gene isolation so important? First, isolation of a gene enables the determination of its nucleotide sequence. From this information, the internal landmarks of the gene can be determined—for example, intron number and position. A comparison of DNA sequences between genes also can lead to insights in gene evolution. Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to comparisons with the protein products of known genes; and, from this knowledge, the function of the gene can often be inferred. Function can also be studied by direct modification of part of the gene's DNA sequence followed by the reintroduction of the gene into the genome. Furthermore, a gene can be moved from one organism to another. An organism containing a foreign gene is called transgenic. Transgenic organisms can be used either for basic research or for specialized commercial applications. One application has been to make valuable human gene products such as insulin in transgenic bacteria carrying the appropriate human gene. From this brief overview, we see that gene isolation has become an indispensable tool of modern genetic analysis. What are some examples of interesting genes that could be isolated? The answer depends very much on which biological process is being studied. Let's look at a few cases. A fungal geneticist studying the cellular pathway for synthesizing tryptophan would be interested in the genes that, when mutated, confer an auxotrophic requirement for tryptophan, because each gene would represent a step in the synthetic pathway (see Chapter 10). These genes can be identified through mutation, segregation, and mapping analysis. They would be named trp1, trp2, trp3, and so forth. This geneticist would be very interested in isolating and characterizing one or more of these genes. Likewise, human genes that have mutant alleles conferring some type of functional disorder are interesting for medical and biological reasons. We have seen that these genes are identified by pedigree analysis. Two examples covered 免疫学信息网 http://www.immuneweb.com
Recombinant DNA Technology in Chapters 2 and 9 are the recessive autosomal conditions albinism and alkaptonuria. In these cases, the general nature of the defect has been understood for some time (both are enzyme defects), but it would be very useful to isolate the genes themselves. Other human genes are known from pedigree analysis, but no biochemical function is known for them. Isolating such genes would be particularly useful because the characterization of gene structure might lead to a determination of gene function and the nature of the disease. A good example is cystic fibrosis, a disease known from pedigree analysis to be caused by an autosomal recessive allele of a gene for which no function was known until the gene was isolated and sequenced. Cases such as these would be raised in all the organisms used in genetic research. In our consideration of gene isolation, we shall first examine the nature of recombinant DNA and the principle whereby recombinant DNA technology can be used to isolate a gene. Next, we shall examine the methods for isolating specific genes such as those just discussed.
Making recombinant DNA
How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:
the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses. Some geneticists prefer the alternative name chimeric DNA. after the mythological Greek monster Chimera.Recombinant DNA Technology MESSAGE Cloning allows the amplification and recovery of a specific DNA segment from a large. Therefore. usually from different organisms. a combination of parts of different animals. Likewise. The procedure used for obtaining vector DNA depends on the nature of the vector. the Chimera has stood as the symbol of an impossible biological union. The term recombinant DNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and prokaryotes. Isolating DNA The first step in making recombinant DNA is to isolate donor and vector DNA.com . 免疫学信息网 http://www. Recombinant DNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources. When this clone has been obtained. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology.immuneweb. Figure 12-1 gives a general outline of the approach. Inasmuch as the donor DNA was cut into many different fragments. a different cloned insert). Through the ages. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells. With the use of such methods. these types are generally the ones required for analysis. which we shall consider later in the chapter. and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated as chemical reagents. complex DNA sample such as a genome. the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. recombinant DNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinant DNA technology. most colonies will carry a different recombinant DNA (that is. the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes.
Another protocol relies on the observation that. Production of these sticky ends is another feature of restriction enzymes that http://www. bacterial genomic DNA denatures but plasmids do not. Cutting DNA The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. and they would not be cut in vivo. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 12-2.immuneweb. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. Importantly. rather. Restriction enzymes are produced by bacteria as a defense mechanism against phages. but plasmids stay in solution. Any DNA molecule. Let's look at an example: the restriction enzyme EcoRI (from E. Subsequent neutralization precipitates the genomic DNA. restriction enzymes do not cut randomly.Recombinant DNA Technology Bacterial plasmids are commonly used vectors. from viral to human.” The ends are called sticky because they can hydrogen bond (stick) to a complementary sequence. This staggered cut leaves a pair of identical single-stranded “sticky ends.com 免疫学信息网 . The enzymes act like scissors. Many different restriction enzymes recognize and cut specific palindromes. at a specific alkaline pH. Restriction sites are not relevant to the function of the organism. cutting up the DNA of the phage and thereby inactivating it. and the linear molecule formed has two sticky ends. Figure 12-3 shows EcoRI making a single cut in a circular DNA molecule such as a plasmid: the cut opens up the circle. and these plasmids must be purified away from the bacterial genomic DNA. coli) recognizes the following six-nucleotide-pair sequence in the DNA of any organism: This type of segment is called a DNA palindrome. which is one of the key features that make them suitable for DNA manipulation. The enzyme EcoRI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides. The plasmid band is collected by punching a hole in the plastic centrifuge tube. because most organisms do not have restriction enzymes. which means that both strands have the same nucleotide sequence but in antiparallel orientation. they cut at specific DNA target sequences. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate. contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of a size suitable for cloning.
Even flush cuts. DNA can also be cut by mechanical shearing. At this stage. MESSAGE Restriction enzymes have two properties useful in recombinant DNA technology. First. and many different EcoRI fragments of donor DNA. The principle is simply that. whereas others make flush cuts. making it possible for DNA chimeras to form. Certain ligases are even capable of joining DNA fragments with blunt-cut ends. For example. the backbones can be sealed by the addition of the enzyme DNA ligase. There are many opened-up vector molecules in the solution. However. When the two populations are mixed. Joining DNA Most commonly. because double helices form between their sticky ends. which create phosphodiester bonds at the junctions (Figure 12-4b). some of which are shown in Table 12-1. DNA fragments from the two sources can unite. if both vector DNA and donor DNA are cut with EcoRI. like EcoRI. Dozens of restriction enzymes with different sequence specificities have now been identified. Figure 12-4a shows a plasmid vector that carries a single EcoRI restriction site.com . both will produce fragments with the same complementary sticky ends. Therefore a diverse array of vectors carrying different donor inserts will be produced. agitating DNA in a blender will break up the long chromosome-sized molecules into flush-ended clonable segments. can be used for making recombinant DNA.Recombinant DNA Technology makes them suitable for recombinant DNA technology. many restriction enzymes make staggered cuts that create single-stranded sticky ends conducive to the formation of recombinant DNA. so digestion with the restriction enzyme EcoRI converts the circular DNA into a linear molecule with sticky ends. Amplifying recombinant DNA 免疫学信息网 http://www. both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed. Donor DNA from any other source (say. they cut DNA into fragments of a size suitable for cloning. which lack sticky ends. the sugar-phosphate backbones are still not complete at two positions at each junction. Hence. but. Drosophila) also is treated with the EcoRI enzyme to produce a population of fragments carrying the same sticky ends.immuneweb. if two different DNA molecules are cut with the same restriction enzyme. although sticky ends have united to generate a population of chimeric molecules. Second. You will notice that all the target sequences are palindromes. some enzymes make staggered cuts.
Subsequently. and 免疫学信息网 http://www. Figure 12-1. Recombinant DNA technology enables individual fragments of DNA from any genome to be inserted into vector DNA molecules. the donor DNA is automatically replicated along with the vector. many cycles of cell division will take place. However. This set of amplified copies of the single donor DNA fragment is the DNA clone (Figure 12-5). Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell.com . After it is in the host cell. The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. such as plasmids.immuneweb. the plasmid vector is able to replicate because plasmids normally have a replication origin.Recombinant DNA Technology The ligated recombinant DNA enters a bacterial cell by transformation. and the recombinant vectors will undergo more rounds of replication. now that the donor DNA insert is part of the vector's length.
com . Each amplified fragment is called a DNA clone.immuneweb.” Copyright © 1975 by Scientific American. Figure 12-2. Plasmids such as those carrying genes for resistance to the antibiotic tetracycline (top left) can be separated from the bacterial chromosomal DNA. Cohen. They can then be introduced into bacterial cells by transformation (right). “The Manipulation of Genes. Inc. Because differential binding of ethidium bromide by the two DNA species makes the circular plasmid DNA denser than the chromosomal DNA. N.Recombinant DNA Technology individually amplified in bacteria. (After S. the plasmids form a distinct band on centrifugation in a cesium chloride gradient and can be separated (bottom left). 免疫学信息网 http://www.
” Copyright © 1975 by Scientific American.com . resulting in a linear molecule with single-stranded sticky ends. Figure 12-4. All rights reserved. N. Method for generating a chimeric DNA plasmid containing genes derived from foreign DNA. Cohen. The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target sequence.Recombinant DNA Technology All rights reserved. “The Manipulation of Genes. Inc.) 免疫学信息网 http://www.) Figure 12-3. (From S.immuneweb.
How amplification works. Restriction-enzyme treatment of donor DNA 免疫学信息网 http://www.immuneweb.com .Recombinant DNA Technology Figure 12-5.
and Modification Sites of Various Restriction Enzymes 免疫学信息网 http://www.immuneweb. Recognition.Recombinant DNA Technology and vector allows insertion of single fragments into vectors.com . Making a Library of Wild-Type Yeast DNA. A single vector enters a bacterial host. Cleavage. Table 12-1. where replication and cell division result in a large number of copies of the donor fragment.
a geneticist is interested in isolating and characterizing some particular gene of interest.com . so the procedures must be tailored to isolate a specific recombinant 免疫学信息网 http://www.Recombinant DNA Technology Cloning a specific gene The foregoing descriptions are generic approaches to creating recombinant DNA.immuneweb. However.
The details of the process differ from organism to organism and from gene to gene. but also for vectors containing recombinant DNA. only those that prove to be tetracycline sensitive have inserts. but both have been genetically modified for convenient use as recombinant DNA vectors. Plasmids are also an efficient means of amplifying cloned DNA because there are many copies per cell. some cells have the plasmid. These vectors are derived from natural plasmids.com . The drug-resistance genes are useful because the drug-resistant phenotype can be used to select not only for cells transformed by plasmids. tetR and ampR. as we shall see in Chapter 14. Another important requirement is to have convenient restriction sites that can be used for insertion of the DNA to be cloned. As described earlier. the cloning procedure is to mix the samples of cut plasmid and donor DNA. In Chapter 7. The distribution of any one plasmid within a species is generally sporadic. It must be capable of prolific replication in a living cell. as many as several hundred for some plasmids. but also additional to it. it has two drugresistance genes. and the choice between them often depends on the size of the DNA segment that needs to be cloned and on the intended application for the cloned gene. transform bacteria. thereby enabling the amplification of the inserted donor fragment. which must have been successfully transformed by a plasmid molecule. Many different types of plasmids have been found in bacteria. whereas others do not. and select first for ampicillinresistant colonies. However. For example. which confers certain types of conjugative behavior to cells of E. and the cell will be sensitive to that drug. Choosing a cloning vector The ideal vector is a small molecule. coli. Two plasmid vectors that have been extensively used in genetics are shown in Figure 12-6. which will then no longer confer tetracycline resistance. Plasmid pBR322 is simpler in structure. Both genes contain unique restriction target sites that are useful in cloning. we encountered the F plasmid. facilitating manipulation. Therefore. bacterial plasmids are small circular DNA molecules that are not only distinct from the main bacterial chromosome. Of the AmpR colonies. Plasmids. Unique sites are most useful because then the insert can be targeted to one site in the vector. Numerous cloning vectors are in current use. the plasmids that are routinely used as vectors are those that carry genes for drug resistance. It is also important to have a method for easily identifying and recovering the recombinant molecule.immuneweb. We shall consider several commonly used types. They replicate their DNA independently of the bacterial chromosome. in 免疫学信息网 http://www. An important initial factor is the choice of an appropriate vector for the job at hand. A successful insertion will split and inactivate the tetR gene.Recombinant DNA Technology DNA clone that will contain that particular gene. The F plasmid can be used as a vector for carrying large donor DNA inserts. donor DNA could be inserted into the tetR gene.
com 免疫学信息网 . such as the bacterial M13-based vectors. Some examples of viral vectors used in bacteria are described next. whose structure allows direct visual selection of colonies containing vectors with donor DNA inserts. the AmpR TetS colonies are the ones that contain recombinant DNA. the cloned gene can be introduced into cells at a significantly higher frequency than by simple transformation. Some viral vectors are specialized for producing high levels of proteins encoded by the cloned genes. Vectors derived from retroviruses can effect the stable integration into mammalian chromosomes of cloned DNA. these plasmids are not useful for cloning DNA fragments larger than 20 kb. which colors the colony blue. the enzyme fragment borne on the vector is disrupted. in which the gene or genes of interest are incorporated into the genome of a virus. this property can be used to select for λ molecules with inserts of donor DNA. λ phage heads will selectively package a chromosome about 50 kb in length. as will be seen. Into this region has been inserted a piece of DNA called a polylinker or multiple cloning site. Hence. selection for white AmpR colonies selects directly for vectors bearing inserts. and. The polylinker is in frame translationally with the β-galactosidase fragment and does not interfere with its translation. therefore. coli. as exemplified by the use of insect baculovirus to express foreign proteins in a eukaryotic cell system. so the central part can be cut out by using http://www. An unusual type of complementation takes place in which the partial proteins encoded by the two fragments unite to form a functional β-galactosidase. The central part of the phage genome is not required for replication or packaging of λ DNA molecules in E. offer many advantages for cloning and the subsequent applications of cloned genes. If donor DNA is inserted into the polylinker. Viral vectors. which is detailed in Chapter 13. Other viral vectors. allowing continued expression of the gene.immuneweb. which contains many unique restriction target sites useful for inserting donor fragments. no complete β-galactosidase protein is formed. Phage lambda. Small plasmids that contain large inserts of foreign DNA tend to spontaneously lose the insert. Because viruses infect cells with high efficiency. Viral vectors. The transformation protocol uses recipient cells that contain a β-galactosidase gene lacking the fragment present on the plasmid. and the functional enzyme converts this substrate into a blue dye. coli β-galactosidase gene.Recombinant DNA Technology other words. A colorless substrate for β-galactosidase called X-Gal is added to the medium. Further experiments are needed to find the clones with the specific insert required. First. are designed to facilitate sequencing and the generation of mutations in cloned genes. The key element is a small part of the E. Phage λ is a convenient cloning vector for several reasons. The pUC plasmid is a more advanced vector. and such colonies are isolated for further study. Viral vectors are also the vehicles of choice for gene-therapy strategies. and the colony is white.
and their DNA can replicate in the cell like that of a plasmid or be packaged like that of a phage. The advantage of using these phages as cloning vectors is that single-stranded DNA is the very substrate required for the Sanger DNA-sequencing technique currently in widespread use (page 387). The chimeric molecules can be either introduced into E. because this size of insert substitutes for the deleted central part of the phage genome and brings the total molecule size to 50 kb. Some expression vectors have been designed with restriction sites located just next to a lac regulatory region.com . In the in vitro system. Expression vectors. Phage M13 is the one most widely used for this purpose.to 15-kb inserts are the ones that will be most effectively packaged into phage heads. coli directly by transformation or packaged into phage heads in vitro. and infective λ phages form spontaneously. The two “arms” are ligated to restriction-enzyme-cut donor DNA. recombinant molecules with 10.Recombinant DNA Technology restriction enzymes and discarded. cosmids can carry DNA inserts about three times as large as those carried by λ itself (as large as about 45 kb). it is necessary to be able to express the gene in bacteria. Making a DNA library 免疫学信息网 http://www. Cloning by cosmids is illustrated in Figure 12-8. Cosmids are vectors that are hybrids of λ phages and plasmids.immuneweb. This modified structure enables phage heads to be stuffed with almost all donor DNA. However. Most cloning vectors do not permit expression of cloned genes. Therefore. These restriction sites permit foreign DNA to be spliced into the vector for expression under the control of the lac regulatory system. that is. Some phages contain only single-stranded DNA molecules. but such expression is possible if special vectors are used. DNA and phage-head components are mixed together. The key is that most of the λ phage structure has been deleted. Single-stranded phages. which can be isolated and used for cloning. Therefore the presence of a phage plaque on the bacterial lawn automatically signals the presence of recombinant phage bearing an insert (Figure 12-7). but the signal sequences that promote phage-head stuffing (cos sites) remain. in these cases. A second useful property of a phage vector is that recombinant molecules are automatically packaged into infective phage particles. which can be conveniently stored and handled experimentally. Cosmids. One way of detecting a specific cloned gene is by detecting its protein product expressed in the bacterial cell. However. The cloned gene is inserted next to appropriate bacterial transcription and translation start signals. the single infecting strand is converted into a double-stranded replicative form. In either method. to transcribe it and translate the mRNA into protein. On infection of bacteria. Cosmid DNA can be packaged into phage particles by using the in vitro system. because bacteria cannot process introns. the cloned sequences must be stripped of introns.
or complementary DNA. Different cloning vectors carry different amounts of DNA. This library should be enriched for the gene in question. The collection of clones is called a DNA library. Generally. then a genomic library is necessary at some stage. then it makes sense to use that tissue to prepare mRNA to be converted into cDNA and then make a cDNA library from that sample. and other vectors such as YACs and BACs (see Chapters 13 and 14) carry the largest amounts of all. Ease of manipulation is another important factor in choosing a vector. If a specific gene that is active in a specific type of tissue in a plant or animal is being sought. cDNA. second. according to the source of DNA. A phage library is a suspension of phages. is synthetic DNA made from mRNA with the use of a special enzyme called reverse transcriptase originally isolated from retroviruses. so the choice of vector for library construction depends on the size of the genome (or other DNA sample) being made into the library. The second important decision is whether to make a genomic library or a cDNA library.Recombinant DNA Technology We have learned that the most important goal of recombinant DNA technology is to clone a particular gene or other genomic fragment of interest to the researcher. Therefore cDNA from eukaryotes can be translated into functional protein in bacteria—an important feature when expressing eukaryotic genes in bacterial hosts. reverse transcriptase synthesizes a single-stranded DNA molecule that can then be used as a template for double-stranded DNA synthesis (Figure 12-9). they do have the benefit of containing genes in their native form.com . including introns and regulatory sequences. Although genomic libraries are bigger. so it will inevitably be smaller than a complete genomic library. If the purpose of constructing the library is a prelude to cloning an entire genome.immuneweb. A plasmid or a cosmid library is a suspension of bacteria or a set of defined bacterial cultures stored in culture tubes or microtiter dishes. This step is sometimes referred to as “shotgun” cloning because the experimenter clones a large sample of fragments and hopes that one of the clones will contain a “hit”—the desired gene. Because it is made from mRNA. The approach used to clone a specific gene depends to a large degree on the gene in question and on what is known about it. The next step is to obtain a large collection of clones made from this original DNA sample. A cDNA library is based on the regions of the genome transcribed. There are different types of libraries. first. which should contain all of the genome. cDNA is devoid of both upstream and downstream regulatory sequences and of introns. the procedures start with a sample of DNA such as eukaryotic genomic DNA. according to which vector is used and. The choice between genomic DNA and cDNA depends on the situation. categorized. With the use of an mRNA as a template. 免疫学信息网 http://www. Plasmid and phage vectors carry small amounts of DNA. Cosmids carry larger amounts of DNA. so these vectors are suitable for cloning genes from organisms with small genomes. The task then is to find that particular clone.
MESSAGE The task of isolating a clone of a specific gene begins with making a library of genomic DNA or cDNA—if possible. Probes for finding DNA.Recombinant DNA Technology In some cases. which might contain as many as hundreds of thousands of cloned fragments. Broadly speaking. This approach is possible if the experimenter already knows which chromosome contains the gene. This type of procedure separates shorter DNA fragments.immuneweb. this number will be at least doubled. The appropriate chromosomal fraction is then used to make the library. Another technique possible in organisms with small chromosomes is to fractionate whole chromosomes by using pulsed field gel electrophoresis (PFGE). 免疫学信息网 http://www. The appropriate chromosome can be identified on the gel by probing with a chromosome-specific probe (see the next subsection). One technique used in mammalian molecular genetics is to sort the chromosomes with an instrument called a flow cytometer. must be screened to find the recombinant DNA molecule containing the gene of interest. A suspension of chromosomes is passed through the apparatus. Electrophoresis is a general technique that fractionates nucleic acids or proteins according to size on gels under the influence of a strong electric field.com . eluted from the gel. it is possible to narrow the genomic fraction used in library construction to more easily detect the desired gene. enriched for sequences containing the gene in question. but it does provide a rough estimate of the magnitude of the job of library construction. which sorts the chromosomes according to size (this procedure is discussed in more detail in Chapter 14). PFGE is a specialized type of electrophoresis useful for very long DNA molecules. Finding specific clones by using probes The library. Such screening is accomplished by using a specific probe that will find and mark the clone for the researcher to identify. These electric fields enable large DNA molecules such as whole chromosomes to snake through the gel to different positions according to their size. but a rough idea of the general order of magnitude of the library can be obtained simply by taking the total genome size and dividing by the average size of the inserts carried by the vector being used. It uses several oscillating electric fields oriented in several different directions. How can an experimenter determine whether a library is large enough to contain any one unique sequence of interest with a reasonable degree of certainty? There are formulas for calculating the minimum number of clones needed. Generally. and used to make a chromosome-specific library. there are two types of probes: those that recognize DNA and those that recognize protein. Then the desired chromosome can be cut out.
90 percent of the mRNA is known to be transcribed from the β-globin gene. they are often similar enough to promote hybridization. a genomic library would be probed. The method is jokingly called “clone by phone” because. often as a spot on an autoradiogram. colonies or plaques of the library on a petri plate are transferred to an absorbent membrane (often nitrocellulose) by simply laying the membrane on the surface of the medium.com . In this case. The probe DNA must be denatured. the genomic clone must be obtained. For example. then your job of cloning is made relatively easy. Another source of DNA for a probe might be a homologous gene from a related organism. The probe must be labeled either with radioactivity or with a fluorescent dye. The membrane is peeled off. The next step is to bathe the membrane with a solution of a probe that is specific for the DNA being sought. The need for this kind of analysis depends on which questions are to be asked about the gene. Generally. then the cDNA clone itself could provide that information just as well. when denatured (made single stranded by unwinding the two halves of the double helix). This method depends on the evolutionary conservation of DNA sequences through time. if you can telephone a colleague who has a clone of your gene of interest but from a related organism. 免疫学信息网 http://www. and colonies or plaques clinging to the surface are lysed in situ and the DNA denatured. because the mRNA of a gene is abundant. If only the transcribed sequence is of interest. in mammalian reticulocytes. The position of a positive clone will become clear from the position of the concentrated label. the probe is itself a cloned piece of DNA that has a sequence homologous to the desired gene.immuneweb. A probe that is itself DNA. many of the cDNAs made from this tissue and inserted individually into vectors will very likely be for the desired gene. Identification of a specific clone in a library is a two-step procedure (Figure 12-10). will therefore find and bind to other similar denatured DNAs in the library. For example. First. so reticulocytes would be a good source of mRNA for making a cDNA probe to find a genomic globin gene. Where does the DNA to make a probe come from? The DNA can be from one of several sources.Recombinant DNA Technology These probes depend on the natural tendency of a single strand of nucleic acid to find and hybridize to another single strand with a complementary base sequence. The idea is that. One source is cDNA from tissue that expresses the gene of interest. if introns and control regions are needed. it will then bind only to the DNA of the clone being sought. then it is very likely that this gene can be used as a probe to find the homologous gene in the related fungus Podospora. if a certain gene has been cloned in the ascomycete fungus Neurospora. However. Even though the probe DNA and the DNA of the desired clone might not be identical.
However.immuneweb. At the beginning of the chapter. The reaction would make 2 × 3 × 2 × 2 × 2 = 48 oligonucleotide strands at the same time. can be purified by standard biochemical procedures. To make the library. so. can be isolated. and this antibody is used to screen an expression library. By detecting the correct protein. The library was probed with the antibody to tyrosinase.Recombinant DNA Technology Probe DNA can be synthesized if the protein product of the gene of interest is known and an amino acid sequence has been obtained. This “cocktail” of oligonucleotides would be used as a probe. and several positive clones were detected. we asked how it might be possible to find the gene for human albinism. The chemical DNA synthesizing reaction is a step-by-step process. and subsequently an antibody to the enzyme was prepared in rabbits. It is dried and bathed in a solution of the antibody. like any protein. From tyrosinaseproducing cells. cDNA is inserted into the vector in frame with the bacterial protein. The process is described in Figure 12-12. These libraries are made by using expression vectors designed to express high levels of a specific bacterial protein. The correct strand within this cocktail would find the gene of interest. the fact that most amino acids are coded by more than one codon—several possible DNA sequences could have encoded the protein in question. and the cells will make a fusion protein. because of the redundancy of the code—in other words. It was in fact cloned by using an antibody to the enzyme that is known to be defective in this condition. the antibody effectively identifies the clone containing the gene that must have synthesized that protein. the second antibody is labeled by a radioactive isotope or a chemical that will fluoresce or become a colored dye.com . If the protein product of a gene is known and isolated in pure form. wherever in the sequence there are alternative nucleotides. free RNA can be radioactively labeled and used as a probe. showing 2. Synthetic DNA probes are designed on the basis of knowledge of the genetic code. This enzyme. The cDNA in the positive clone was sequenced 免疫学信息网 http://www. a short stretch of amino acids with minimal redundancy is selected. mRNA was isolated and used to make cDNA. and 2 alternatives. This cDNA was used to make an expression-vector library. so an amino acid sequence merely has to be translated backward to obtain the DNA sequence that encoded it. the enzyme tyrosinase. The nucleotide sequence is calculated by using the codon dictionary. Figure 12-11 shows an example in which there are five positions of redundancy. Twenty nucleotides embody enough specificity to find one unique DNA sequence in the library. Positive clones are revealed by making an antibody to the first antibody. respectively. a mixture of those alternative nucleotides is fed into the reaction and all possible DNA strands are synthesized. An antibody to the protein is prepared. 2. Additionally. A membrane is laid over the surface of the medium and removed with an imprint of colonies. such as rRNA or fractionated tRNAs. Probes for finding proteins. To get around this problem. then this protein can be used to detect the clone of the corresponding gene in a library. 2. 3. This use of labeled free RNA as a probe is possible only when a relatively pure population of identical molecules of RNA.
in this process. we will assume that it is an auxotrophic mutation in a fungus. often a eukaryote. The cDNA was used to probe a library of human genomic DNA. We have already considered transformation in prokaryotes (Chapter 7). For the present purpose. The procedure differs among eukaryotes. The reason that this transformation method works is that the transforming fragment functionally complements the deficiency caused by the mutant allele in the recipient. and. but eukaryotes can be transformed. Here the protocol is: This method depends on the ability to transform the donor organism. This procedure is called functional complementation. For example. We shall use DNA from the library to transform the auxotrophic mutant strain and then plate these recipient fungal cells on minimal medium. MESSAGE A cloned gene can be selected from a library by using probes for the gene's DNA sequence or for the gene's protein product. the production of a wild-type phenotype from the union of two mutant genomes. generally the cell walls must be removed enzymatically. the intact tyrosinase gene was found. Let's assume that we have isolated a mutant that is relevant to some biological process that interests us. which acts as the transformation recipient.com . Fungal cells that contain the wild-type allele (from the wild-type culture used to make the library) will transform the auxotroph to prototrophy and allow growth on minimal medium. too. that is. It might seem at first that this view of complementation is not the same as the one developed in Chapter 4. to transform fungi. It proved to have five exons and four introns.Recombinant DNA Technology and found to contain a gene whose exons total 1590 nucleotide pairs.immuneweb. but generally some special treatment of recipient cells is required. Finding specific clones by functional complementation Specific clones in a bacterial or phage library can be detected through their ability to confer a missing function on a mutant line of the donor organism. the transforming vector contributes 免疫学信息网 http://www. However.
By a process of narrowing down the library subsets that successfully transform. In most cases. In this case. and insertion into the chromosome is not essential. in most eukaryotic organisms. However. Subsets of the cosmid library made from wild-type Neurospora DNA were used to transform trp3 mutant cells.com . If a eukaryotic gene is cloned on a prokaryotic vector but a specific eukaryotic sequence is known that can act as an origin of replication. so a type of complementation is accomplished. this sequence can be added to the vector. then the transforming insert can be recovered simply by isolating the plasmid. These types of vectors are called shuttle vectors because they can be moved back and forth between different hosts. Without an origin of replication. Positional cloning 免疫学信息网 http://www. and trp3+ clones were selected by plating transformed cells on medium containing hygromycin but lacking tryptophan. a cosmid library was used. transformants are found to contain the vector carrying the wild-type allele inserted into one of the recipient's chromosomes at a location that is different from the mutant locus in the recipient. and the recipient genome contributes something that the vector lacks (the entire remainder of the genome). the donor DNA must integrate into the eukaryotic chromosome to effect stable transformation. This is called ectopic insertion (Figure 12-14). the bacterial or phage vector cannot replicate and must insert into the genome to achieve stable transformation. the transforming wild-type allele replaces the resident auxotrophic mutation by a double-crossover-like process. This method uses a library in which the clones are laid out as a collection of numbered bacterial cultures in tubes or microtiter dishes.immuneweb. The cosmid must also carry a marker gene that can be used to select for successful transformants of the fungus. Colonies that grow are likely to contain the trp3+ allele and are isolated from the plate. which are normally sensitive to this drug. The process is illustrated in Figure 12-13. MESSAGE Specific cloned donor genes can be selected by using their DNA to transform and complement null alleles in recipient cells of the donor organism. Less commonly. the clone with the wild-type allele can be identified. Then the vector will be able to replicate in both bacterial and eukaryotic cells. DNA is isolated in bulk from all the strains in specific subsets of the library. as we shall see. A gene for hygromycin resistance is commonly used in fungi. using as an example the Neurospora trp3 gene mentioned at the beginning of the chapter. In these cases. and transformation is attempted. the transforming fragment is relatively inaccessible and must be retrieved from the successful clone in the library. If the transformation recipient is an organism in which plasmid vectors replicate autonomously (mainly bacteria and yeasts).Recombinant DNA Technology something that the recipient genome lacks (the wild-type allele being sought).
Positional cloning has been particularly useful for cloning human genes. the gene of interest. The linked marker acts as the departure point in a process. Often both probing and complementation are part of positional cloning. a− 免疫学信息网 http://www. called chromosome walking. Figure 12-15 summarizes the procedure of chromosome walking.com . One type of tag is transforming DNA. For any case of chromosome walking. The human gene for cystic fibrosis. that will terminate at the target gene.immuneweb. The specific sequence is then used as a tag to recover the gene. Hence among the x+ transformants. Many of the x+ transformants will be mutant for the genes into which the transforming DNA has inserted ectopically. Positional cloning is a term that can be applied to any method that makes use of such information. The approach is summarized in Figure 12-16. The availability of a large number of neutral DNA markers (restriction fragment length polymorphisms) dispersed throughout most genomes has provided many useful start points. there must be some type of criterion to assess each step of the walk for the gene of interest. it can integrate into the genome and become part of the chromosome. and will be of phenotype a−. and we shall examine its cloning in more detail in Chapter 14). Each clone can be sequenced or otherwise tested. outward fragments are used for a new round of selection of overlapping clones from the library. mentioned at the beginning of the chapter. End fragments of a clone of the linked marker are used as probes to select other clones from the library. again. Hence the walking process moves outward in two directions from the start site. and. This property can be used to good advantage. was cloned by chromosome walking. the integrating fragment acts as a mutagen. When integration takes place within or near a gene. Sometimes a large insert that is known to contain the linked marker will also luckily contain the sought gene. Ectopic integration is random throughout the genome.Recombinant DNA Technology Information about a gene's position in the genome can be used to circumvent the hard work of assaying an entire library to find the clone of interest. When exogenous DNA is added by transformation or by other methods such as injection. A common starting point is the availability of another cloned gene or other marker known to be closely linked to the gene being sought. and apparently no segment of chromosomal DNA is immune to integration. Suppose that we use a specific cloned gene x+ and transform x− cells of the donor organism into x+. disrupting the function of the gene. These probes will detect clones of DNA regions that overlap with the initial clone. Restriction maps (see discussion on pages 389–392) are made of the DNA of this second set of clones. A subset of such x+ cells will be mutant for the target gene a+. Cloning a gene by tagging Tagging is a cloning method that zeros in on the desired gene directly by inducing a mutation in that gene with the use of a specific piece of DNA as an insertional mutagen. and these criteria depend on the individual gene concerned. and subcloning and transformation will narrow down the appropriate region of the cosmid. depending on the intent of the exploration. many of which have no known biochemical function and cannot be easily selected by functional complementation.
a fragment of the disrupted a gene sequence is used in another round of probing.) In a line containing an active transposon. To recover the intact wild-type a gene. and gene x+ can be used as a probe to recover the clone of the disrupted a gene.com . in a manner similar to that shown in Figure 12-16. The next step is to cross the transformants to determine if the a− phenotype segregates with x+. This mutant line is used to make a library.Recombinant DNA Technology phenotypes are identified. A similar approach uses transposons as tags. (Transposons are described in more detail in Chapter 20. A cloned part of the transposon DNA can then be used as a tag to recover the gene. the mutation is likely to have been caused by the integration of the fragment containing x+.immuneweb. When they move. MESSAGE Mutating a gene by the insertion of transforming DNA or a transposon allows the gene to be tagged as a prelude to its isolation. this time with a wild-type library. 免疫学信息网 http://www. they can create a null mutation. Many of these mutants will have been caused by the insertion of the transposon. The DNA of this mutant line is used to construct a library. If they insert into or near a gene. Transposons are naturally mobile DNA fragments found in many organisms. they can insert anywhere in the genome. If it does. mutants for the desired gene are selected.
Recombinant DNA Technology
Figure 12-6. Two plasmids designed as vectors for DNA cloning, showing general structure and restriction sites. Insertion into pBR322 is detected by inactivation of one drug-resistance gene (tetR), indicated by the TetS (sensitive) phenotype. Insertion into pUC18 is detected by inactivation of the β-galactosidase function of Z′, resulting in an inability to convert the artificial substrate X-Gal into a blue dye.
Recombinant DNA Technology
Figure 12-7. Cloning in phage λ. A nonessential central region of the phage chromosome is discarded and the ends ligated to random 15-kb fragments of donor DNA. A linear multimer (concatenate) forms, which is then stuffed into phage heads one monomer at a time by using an in vitro packaging system. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright © 1992 by Scientific American Books.)
Recombinant DNA Technology
Figure 12-8. Cloning by cosmids. The cosmid is cut at a BglII site next to the cos site. Donor genomic DNA is cut by using Sau3A, which gives sticky ends compatible with BglII. A tandem array of donor and vector DNA results from mixing. Phage is packaged in vitro by cutting at the cos site. The cosmid with insert recircularizes after it is in the bacterial cell. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright © 1992 by Scientific American Books.)
Watson. The loop is then cleaved by S1 nuclease (which acts only on the single-stranded loop) to produce a double-stranded cDNA molecule. The resulting cDNA ends in a hairpin loop. H. A short oligo(dT) chain is hybridized to the poly(A) tail of an mRNA strand.Recombinant DNA Technology Figure 12-9. and D.com . Kurtz. which uses the mRNA as a template for the synthesis of a complementary DNA strand. (From J. Freeman and Company. When the mRNA strand has been degraded by treatment with NaOH. J. The oligo(dT) segment serves as a primer for the action of reverse transcriptase.immuneweb. D. the hairpin loop becomes a primer for DNA polymerase I. Recombinant DNA: A Short Course. T.) 免疫学信息网 http://www. which completes the paired DNA strand. The synthesis of double-stranded cDNA from mRNA. Tooze. Copyright © 1983 by W.
com . each plaque in the lawn is inhabited by a single clone of 免疫学信息网 http://www. (a) A genomic library can be made by cloning genes in λ bacteriophages.immuneweb.Recombinant DNA Technology Figure 12-10. When a lawn of bacteria on a petri plate is infected by a large number of different hybrid phages.
) 免疫学信息网 http://www. P.) Figure 12-11. The probe hybridizes with any recombinant DNA incorporating a matching DNA sequence.” and P. Now the desired clone can be selected from the culture medium and transferred to a fresh bacterial host. which is then denatured so that it will stick to the filter. The problem now is to identify the clone carrying a particular gene of interest (dark blue) by probing the clones with DNA or RNA known to be related to the desired gene. Zipursky. Copyright © 1995 by Scientific American Books. A. “The Genetics of Antibody Diversity. All rights reserved. S.Recombinant DNA Technology phages descended from the original infecting phage. and the position of the clone having the DNA is revealed by autoradiography. and J. (After R. A. Inc. The filter is incubated with a radioactively labeled probe. Each clone carries a different fragment of cellular DNA. (From H. Inc. D. Berk. Molecular Cell Biology. 3d ed. Weinberg. a DNA copy of the messenger RNA representing the desired gene. Darnell. so that a pure gene can be manufactured.immuneweb. “A Molecular Basis of Cancer. that is. Lodish. Baltimore. 1982 by Scientific American. Leder. A short sequence of a protein is used to design a set of redundant oligonucleotides for use as a probe to recover the gene that encoded the protein.” Copyright © 1983.com . L. and the phage protein is dissolved. One of the set of probes will be a perfect match for the gene. Matsudaira. leaving the recombinant DNA. (b) The plaque pattern is transferred to a nitrocellulose filter.
Recombinant DNA Technology Figure 12-12. An expression library 免疫学信息网 http://www. Finding the clone of interest by using antibody.com .immuneweb.
the bound antibodies are visualized through binding of a radioactive secondary antibody. the quest is for the trp3 gene of Neurospora.Recombinant DNA Technology made with phage derivative λgt11 is screened with a protein-specific antibody. (After J.com . Fincham. Witkowski. Copyright © 1992 by Scientific American Books.) 免疫学信息网 http://www. Zoller. Copyright © 1995 by Blackwell. R. J.immuneweb. Finding a cloned gene by using progressively smaller pooled DNA samples in transformation. Watson. M.) Figure 12-13. After the unbound antibodies have been washed off the filter. Gilman. D. 2d ed. S. In this example. Recombinant DNA. and M. (From J. Genetic Analysis.
A donor wild-type allele A+ (cloned in a bacterial vector) transforms an A− recipient by one of three different types of insertion. either prokaryotic or eukaryotic.Recombinant DNA Technology Figure 12-14. 免疫学信息网 http://www. I and II.com . Two recipient chromosomes are shown. The possible fates of transforming DNA.immuneweb. Note: The recipient is generally of the same species as the donor DNA.
D. One recombinant phage obtained from a phage library made by the partial EcoRI digest of a eukaryotic genome can be used to isolate another recombinant phage containing a neighboring segment of eukaryotic DNA.immuneweb. Copyright © 1983 by W. T. Kurtz. J. Watson.com . Chromosome walking. Tooze. Freeman and Company.) 免疫学信息网 http://www. (From J.Recombinant DNA Technology Figure 12-15. as described in the text. Recombinant DNA: A Short Course. and D. H.
The bands can be visualized by staining the DNA with ethidium bromide. Probing to find a specific nucleic acid in a mixture In the course of gene and genome manipulation. Cloned DNA used as a probe We have already examined several examples of this type of application. these classes will form distinct bands on the gel. it is necessary to separate the two chromosome arms from the unwanted central region. it is often necessary to detect and isolate specific DNA molecules from a mixture. if there are distinct size classes in the mixture. Using cloned DNA Cloned DNA can be used in numerous ways dictated by the needs of the experiment. For example. More examples follow.com . In this section. the molecules will move through the gel to the anode at speeds dependent on their size (Figure 12-17). Using DNA insertion as a tag for marking and recovering a gene from the genome.Recombinant DNA Technology Figure 12-16. 免疫学信息网 http://www. in cloning with the use of λ phage. which causes the DNA to fluoresce in ultraviolet (UV) light. The tag DNA can be transforming DNA or an endogenous transposon (movable element). but the most extensively used method is electrophoresis (see Figure 4-5 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. such as using a cloned gene from one organism to select the appropriate clone from another organism.immuneweb. Therefore. we consider some of the basic uses that are applicable in a wide range of experimental circumstances. There are several ways of fractionating DNA. recall that.
When transferred to the membrane.Recombinant DNA Technology If bands are well separated.immuneweb. the ability of nucleic acids with complementary nucleotide sequences to find and bind to each other in solution is being exploited. One application of Northern analysis is in determining whether a specific gene is transcribed in a certain tissue or under certain environmental conditions. The Southern blotting technique can be extended to detect a specific RNA molecule from a mixture of RNAs fractionated on a gel. The gel can be calibrated for DNA fragment size by running a standard “ladder” of fragments of known size on the same gel.com . with the use of a technique developed by E. Therefore DNA electrophoresis can be either diagnostic (showing which DNA fragments are present) or preparative (useful in isolating specific DNA fragments). Figure 12-18 shows the general procedure of Southern blotting. Figure 12-19 shows an application in which a cloned fragment of a fungal plasmid was used to detect homologous plasmids in a variety of strains. Restriction-enzyme digestion of genomic DNA results in so many fragments that a stained electrophoretic gel shows a smear of DNA. those bands can be cut out of the gel and further processed. Hence the sizes of any interesting fragments in the experimental sample can be inferred. blotted. were introduced briefly in Chapter 1. The membrane is bathed in a labeled probe. In all these cases. note that the probe here is not DNA but a labeled antibody. an individual band can be cut from the gel and the purified DNA sample can be removed from the gel matrix. If appropriate. RNA is extracted from the appropriate cell sample and then electrophoresed. A positive signal shows the presence of the transcript. and the parallel technique to be detailed below. MESSAGE 免疫学信息网 http://www. 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. (Recall that this technique. an absorbent membrane is laid over the gel and the DNA bands are transferred (“blotted”) onto the membrane by capillary action. A probe can identify one fragment in this mixture. Southern called Southern blotting. However. and probed with the cloned gene in question. the DNA bands stay in the same relative positions as on the gel.) After DNA fragments are fractionated on the gel. M. This technique is called Northern blotting to contrast it with the Southern technique for DNA analysis. 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. These three techniques are very powerful experimental tools in detecting and sizing specific macromolecules in molecular genetics. The fractionated RNA is blotted onto a membrane and probed in the same way as in Southern blotting. They are compared in Figure 12-20. and an autoradiogram is used to reveal the presence of any bands on the gel that are homologous to the probe.
inasmuch as DNA cloning provides amplified samples of defined DNA fragments. From this population. The fragments can be visualized by electrophoresis of the four samples in four lanes on an acrylamide gel. are a clear indication of where the bases complementary to the ddNTPs are on the template strand. His method is based on DNA synthesis in the presence of dideoxy nucleotides. One of the requirements for DNA sequencing is the ability to obtain defined fragments of DNA. These molecules are then fractionated. each corresponding to the point at which the respective ddNTP for that tube was incorporated and terminated chain growth. at different sites in different syntheses in the reaction tube. where the fragments form bands.immuneweb. The respective dideoxynucleotide triphosphates (ddNTPs) can be incorporated into a growing chain. there is a strong interdependence of DNA cloning and DNA sequencing technology. or ddGTP). Thus. Any method of DNA sequencing starts with a population of a defined fragment of DNA labeled at one end. The gene's nucleotide sequence is the ultimate characterization of its genetic structure. corresponding to all the various positions of that particular base. together with the four normal deoxynucleotide triphosphates (dNTPs). all possible truncated fragments will be produced. The base sequence can be determined by scanning up the gel. The mobility of a strand is inversely proportional to the logarithm of its length. Each tube receives a small amount of a different ddNTP (ddATP.Recombinant DNA Technology DNA or RNA fractionated by size in an electrophoretic gel can be blotted onto an absorbent membrane. This technique is so sensitive that fragments differing in length by only a single nucleotide can be separated. ddTTP. plus DNA polymerase and a section of labeled primer.com . they terminate synthesis because they lack the 3′-hydroxyl group necessary to bond with the next nucleotide triphosphate. Fractionation is by acrylamide or agarose gel electrophoresis of single-stranded DNA molecules. when incorporated. and recording 免疫学信息网 http://www. various truncated chain lengths will be produced. which in turn can be probed to detect the position of specific fragments. Each of four reaction tubes is prepared with a single-stranded DNA template for the sequence of interest. encompassing all four lanes. 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. ddCTP. in any given tube. sets of molecules are generated that differ in size by one base at the other end. A dideoxynucleotide will be incorporated randomly. The sequencing method now most commonly used was developed by Fred Sanger. in turn. which differ from normal deoxynucleotides in that they lack a 3′-hydroxyl group (Figure 12-21). These lengths. but. Therefore. Because incorporation is random. The base at the truncated end of each of the fractionated molecules is determined and hence the nucleotide sequence is established.
all without cloning. Figure 12-24 illustrates a readout of automated sequencing. The procedure is illustrated in Figures 12-22 and 12-23. MESSAGE 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. A different fluorescent color emitter is used for each of the four reactions.com . The nucleotide sequence of a cloned DNA fragment can be used to find the cloned gene or genes that it contains. and a second cycle of replication is carried out by lowering the temperature in the presence of all the components necessary for the polymerization. Amplifications by as much as a millionfold can be readily achieved. The nucleotide sequence is fed into a computer. A temperatureresistant DNA polymerase. Pairs of primers on opposite strands are extended toward each other. the tags attached to oligonucleotide primers can be fluorescent dyes. Instead of radioactive labels.immuneweb. After completion of the replication of the segment between the two primers (one cycle). are both ways of identifying genes. the two new duplexes are heat denatured to generate single-stranded templates. the sequence can be used to retrieve parts of the equivalent region from a specific individual organism or from other species. from the bacterium Thermus aquaticus is used to catalyze growth from DNA primers. Detecting and amplifying sequences by the polymerase chain reaction If a region of DNA has already been cloned and sequenced. Repeated cycles of synthesis and denaturation result in an exponential increase in the number of segments replicated. 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. which can read as many as 1000 bases in one separation. as shown in Figure 12-26. With the use of PCR. PCR is very sensitive and can detect target sequences that are in extremely low copy number in a sample.Recombinant DNA Technology whichever base occupies the terminus in the next band. the nucleotide sequence can be read directly from an autoradiogram of the gel. Figure 12-25 shows such an analysis in which two candidate genes have been identified as ORFs. provided primers corresponding to known sequences of the gene can be synthesized. They represent sequences that are candidate genes. segments of human DNA can be amplified by using just the few follicle cells surrounding a 免疫学信息网 http://www. and the four mixtures are electrophoresed together. It is interesting to note that ORF detection and Mendelian progeny ratio analysis. Because of the exponential amplification. a single-copy gene can be amplified out of a genomic sample. although poles apart in their approaches. Fluorescence detection is used in automated DNA sequencing machines. These stretches are called open reading frames (ORFs). Figure 12-26 illustrates the principle of the technique. For example. The technique uses a procedure called the polymerase chain reaction (PCR). Taq polymerase.
The double digest shows whether a fragment produced by one enzyme contains sites for the other. the fragment disappears and is replaced by two or more smaller fragments. One way of making a restriction map is to compare single-enzyme digests with double digests. therefore. along a chromosome) can be used as DNA markers. The longest fragment is isolated and digested with a second restriction enzyme. PCR is generally useful in DNA diagnostics—in other words. Furthermore.com . The procedure is illustrated in Figure 12-27. Locating genes on restriction maps We have already seen the importance of restriction enzymes in cutting DNA for use in cloning. Most often. Even though the sites exist by chance. because enough DNA is amplified that a clear band on a gel is produced. in amplifying a defined segment. only very small amounts of substrate DNA are needed. and the distance of each restriction site from the labeled end can 免疫学信息网 http://www. no restriction digestion of the substance DNA is needed. a piece of DNA is labeled at each 5′ end with 32P and cut to generate fragments with the label at one end. MESSAGE The polymerase chain reaction uses specially designed primers to amplify specific short regions of DNA without cloning. Another method for making restriction maps is shown in Figure 12-28.Recombinant DNA Technology single pulled-out hair. In this method. Therefore. each with one of the various restriction sites at the nonlabeled end. no lengthy cloning procedures are necessary. which. Comparison of the sizes of fragments produced in the different digestions allows an approximate localization of the restriction-enzyme target sites. in the PCR technique. Another useful feature of restriction enzymes is that the positions of their target sites along a DNA molecule (that is. Note that. and then the two enzymes are used together. In addition. they are generally in the same positions on homologous chromosomes.immuneweb. the positions of the target sites can be used much like milestones along a road. can be used as reference points for locating more significant features along the road. the fragment sizes are determined by electrophoresis. a map of the positions of restriction sites is a valuable tool in genome analysis generally. a population of variously sized labeled fragments is produced. but the reaction is not allowed to go to completion. in checking for the presence of a gene or for the mutational state of a specific gene or. After cutting. These fragments are separated electrophoretically. Two restriction enzymes are applied in separate digestions. 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. Hence. if so. although not important in themselves. preparatively. because the primers will home in on the appropriate sequence of native DNA.
of which the most conspicuous is that the urine turns black when exposed to air. then the gene must be located in the region where fragments A and B overlap. There was a 50-year delay before it was shown by others that. activity for the enzyme that normally splits homogentisic acid. In the next section. An example of a restriction map is shown in Figure 12-29. In this chapter. In 1898. In 1992. an English doctor named Archibald Garrod (Figure 12-30) showed that the substance responsible is homogentisic acid. in 1908. which is excreted in abnormally large amounts into the urine of alkaptonuria patients. giving rise to sophisticated new approaches for altering an organism's phenotype.” an enzyme deficiency caused by a defective gene. 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. which incorporates and integrates many of the techniques introduced in this chapter. genes can be isolated in a test tube and characterized as specific nucleotide sequences.Recombinant DNA Technology thus be deduced. we describe the genetic analysis of the disease al-kaptonuria. But even this is not the end of the story. Because of this enzyme deficiency. This map is of the DNA of a linear plasmid from a fungus. he proposed that the disorder was caused by the lack of an enzyme that normally splits the aromatic ring of homogentisic acid to convert it into maleylacetoacetic acid. they used the deduced amino acid sequence of this gene to do a computer search through a large number of 免疫学信息网 http://www.com . After a restriction map has been made. Soon after. and. Genes are no longer hypothetical entities. Jose Fernandez-Canon and colleagues characterized a gene encoding HGO activity in the fungus Aspergillus nidulans. is indeed totally absent. on the basis of pedigree patterns. In 1902. genes can be placed on the map by using Southern analysis. in 1996.2-dioxygenase (HGO). Thanks to recombinant DNA technology and related technologies. the alkaptonuria gene was mapped genetically to band 2 of the long arm of chromosome 3 (band 3q2). if a cloned gene hybridizes with a fragment A produced from restriction enzyme 1 and with fragment B produced by restriction enzyme 2. For example. that alkap-tonuria is inherited as a Mendelian recessive (Figure 12-31). we have introduced the fundamental techniques that have revolutionized genetics. he reasoned. Garrod suggested. Alkaptonuria is a disease with several symptoms. A century of genetic research on alkaptonuria The story of the cloning of the human gene defective in alkaptonuria brings together many of the techniques introduced in this chapter. early in the post-Mendelian era. In 1995. an enzyme called homogentisate 1. in the liver of patients with alkaptonuria. homogentisic acid accumulates.immuneweb. Thus alkaptonuria is among the earliest proposed cases of an “inborn error of metabolism. It seemed likely that the enzyme HGO was normally encoded by the alkaptonuria gene.
which showed 52 percent similarity to the Aspergillus gene. Copyright © 1995 by Scientific American Books.) (From H. (M represents lanes containing standard fragments acting as markers for estimating DNA length. Molecular Cell Biology. Lodish. The cDNA clone was used to recover the full-length gene from a genomic library.immuneweb. The other parent was heterozygous for a valine → glycine substitution at position 300 in exon 12 (mutation V300G). therefore. All three children with alkaptonuria were of the constitution P230S V300G. the probe bound to band 3q2. Inc. P. showing that it was indeed the gene for alkaptonuria. the samples are five recombinant vectors treated with EcoRI.com . The amplified products were sequenced. Berk. Darnell. A. In this case. Matsudaira.Recombinant DNA Technology sequenced fragments of a human cDNA library. The mixtures are applied to wells at the top of the gel. When the human gene was expressed in an E. it showed HGO activity. PCR analysis was used to amplify all the exons individually. number of charges). Furthermore. When the cloned gene was used as a probe on chromosomes in which the DNA had been partly denatured (in situ hybridization—see Chapter 14). as expected if these were the mutant sites inactivating the HGO enzyme. Mixtures of different-size DNA fragments separated electrophoretically on an agarose gel. The gene was found to have 14 exons and spanned a total of 60 kb. 3d ed. The DNA bands have been visualized by staining with ethidium bromide and photographing under UV light. Zipursky. when the gene was used as a probe in a Northern analysis of liver RNA. and fragments move under the influence of an electric field to different positions dependent on size (and. They identified a positive clone that contained a human gene that encodes 445 amino acids. a single RNA of the expected size was hybridized. Baltimore. S. Figure 12-17.) 免疫学信息网 http://www. A family of seven in which three children suffered from alkaptonuria was tested for mutations. and J. D. coli expression vector. L. One parent was heterozygous for a proline → serine substitution at position 230 in exon 10 (mutation P230S).
com .immuneweb.Recombinant DNA Technology 免疫学信息网 http://www.
The lane marked “L” is a calibration ladder composed of DNA fragments of known size. Recombinant DNA. 2d ed. Comparison of these bands with labeled markers reveals the number and size of the fragments in which the targeted sequences are found. Because the radioactive probe has hybridized only with its complementary restriction fragments. The filter is then removed and incubated with a radioactively labeled single-stranded probe that is complementary to the targeted sequence. and M. M. Zoller. The plasmid in strain 11 was cloned and used as a probe in a Southern hybridization.immuneweb. Gilman. and X-ray film is exposed to the filter.5 kb. the film will be exposed only in bands corresponding to those fragments. The various fragments migrate at differing rates according to their respective sizes. up to 12 kb. RNA or DNA restriction fragments are applied to an agarose gel and electrophoresed. which is wicked up by the towels. all homologous to the strain 11 plasmid. The bright bands are linear plasmids. Unbound probe is washed away. strain 7 has a family of three plasmids. An example of a Southern analysis. The autoradiogram shows that several other plasmids have sequences homologous to the strain 11 plasmid. (From J. The fragments are denatured to single strands so that they can stick to the filter.) Using the Cloned GAL Gene as a Probe for GAL mRNA. This information can be used to position the sequences along restriction maps. Indeed. Watson. J. and the positions of some of these plasmids are marked. (From Simon 免疫学信息网 http://www. D. and the gel was stained with ethidium bromide. The gel is placed in buffer and covered by a nitrocellulose filter and a stack of paper towels. and then one additional kilobase for each rung.com . They are carried to the filter by the buffer. Gel electrophoresis and blotting in identifying specific cloned genes. 2 kb. Witkowski. This procedure is termed Southern blotting when DNA is transferred to nitrocellulose and Northern blotting when RNA is transferred.Recombinant DNA Technology Figure 12-18. The sizes of the rungs visible in this figure are (from the bottom up) 1. Copyright © 1992 by Scientific American Books. Figure 12-19. The DNA from the mitochondria of 11 strains of Neurospora from different geographical locations was electrophoresed.
which are employed in the 免疫学信息网 http://www.) Figure 12-21.) Figure 12-20. Northern.com . B and H represent target sites for restriction enzymes B and H.immuneweb. (Note: Positions of bands on Southern blot cannot be compared with positions of bands on Northern and Western gels. Comparison of Southern.3′-dideoxynucleotides. The structure of 2′. and Western analyses of gene X.Recombinant DNA Technology Yang.
Zoller. Witkowski. D. 2d ed. Copyright © 1992 by Scientific American Books. Gilman. The dideoxy sequencing method. Watson. The addition of four different dideoxynucleotides (ddATP is shown here) randomly arrests synthesis. (b) The resulting fragments are separated electrophoretically and subjected to autoradiography.Recombinant DNA Technology Sanger DNA-sequencing method.immuneweb. J. Recombinant DNA. M. (From J. and M.) 免疫学信息网 http://www. Figure 12-22. (a) A labeled primer (designed from the flanking vector sequence) is used to initiate DNA synthesis.com .
Any piece of DNA has six possible reading frames. Here the computer has scanned a 9-kb fungal plasmid sequence in looking for ORFs (potential genes). Two large ORFs. three in each direction. N represents a base that cannot be assigned. Printout from an automatic sequencer that uses fluorescent dyes. Figure 12-25.Recombinant DNA Technology Figure 12-24. are the most likely candidates 免疫学信息网 http://www.immuneweb.com . 1 and 2.
Recombinant DNA Technology as potential genes.immuneweb. 免疫学信息网 http://www.com .
(b) Two chosen or created primers have sequences complementing primer-binding sites at the 3′ ends of the target gene on the two strands. at the other end of the sequence.) The two primers again bind to their respective strands at the 3′ ends of the target region. (From J. These first two strands are of varying length. This is because each new strand begins at the primer-binding site. The strands are separated by heating.com . (f) Each new strand now begins with one primer sequence and ends with the primer-binding sequence for the other primer. Copyright © 1992 by Scientific American Books. and proceeds until it runs out of template. (a) Double-stranded DNA containing the target sequence. 免疫学信息网 http://www. (d) The two duplexes are heated again. (e) Taq polymerase again synthesizes two complementary strands.) (g) The process can be repeated indefinitely. allowing the two primers to anneal to the primer-binding sites. 2d ed. The polymerase chain reaction. (For simplicity.immuneweb. only the two new strands are shown. Together.) Polymerase Chain Reaction. Witkowski. They extend beyond the ends of the target sequence as delineated by the primer-binding sites. the primers again anneal and the strands are extended to the length of the target sequence. J. Zoller. and M. at one end of the target sequence. Recombinant DNA. (c) Taq polymerase then synthesizes the first set of complementary strands in the reaction. the two strands just synthesized from them are precisely the length of the target sequence desired. each time creating two double-stranded DNA molecules identical with the target sequence. M. Gilman. Subsequent to strand separation. D. the primers thus flank the targeted sequence. Although the template strands at this stage are variable in length. because they do not have a common stop signal. exposing four binding sites. (The variable-length strands from part c are also producing target-length strands.Recombinant DNA Technology Figure 12-26. Watson.
which is 17 kb long.and 8-kb segments intact but cleaves the 3-kb fragment. of the three restriction fragments produced by enzyme 1. If the 3-kb section were on the outside of the sequence being studied. In this simplified example. Restriction mapping by comparing electrophoretic separations of single and multiple digests.immuneweb. Because this is not the case. showing that enzyme 2 cuts at a site within this enzyme 1 fragment. That the restriction enzyme 2 (RE2) site lies closer to the 6-kb section than the 8-kb section can be inferred from the 7.and 10-kb lengths of 免疫学信息网 http://www.Recombinant DNA Technology Figure 12-27. Combined digestion by both enzyme 1 and enzyme 2 leaves the 6. digestion with enzyme 1 shows that there are two restriction sites for this enzyme but does not reveal whether the 3-kb segment is in the middle or on the end of the digested sequence.com . the 3-kb fragment must lie in the middle.or 2-kb fragment. digestion by enzyme 2 alone would yield a 1.
The new sets of fragments are separated on gels. and the other is partly digested with HaeIII. which could just as easily be HaeIII. The longest fragment is selected (after testing on a gel to compare lengths) and is divided into two samples. The location of the labeled fragment can be determined by autoradiography of the gels. In this example. the ring of DNA with a single EcoRI site is opened into a linear molecule by cleavage with EcoRI.immuneweb. One sample is partly digested with Alu. the restriction enzyme from Haemophilus aegyptius) to produce fragments of varying lengths. The molecule is labeled on its 5′ end with 32P and then partly digested with Alu (restriction enzyme from Arthrobacter lutens. The sequence of restriction sites can now 免疫学信息网 http://www. Figure 12-28.Recombinant DNA Technology the enzyme 2 digestion.com . Each fragment now carries the radioactive label at only one end. Identification of the sequence of restriction-enzyme sites in a circular DNA molecule.
Figure 12-29. showing sites for six different enzymes.Recombinant DNA Technology be read in order as the two gels are compared. 免疫学信息网 http://www.immuneweb.com . Restriction map of a linear fungal plasmid.
Summary Genetics focuses on the nature of genes. Note that the terms are listed in no particular order. Probes can detect specific DNA or RNA molecules on the gel. which cut DNA into defined fragments. Recombinant DNA technology has allowed individual genes to be isolated in a test tube and then characterized at the molecular level. Common vectors are plasmids. An entire genome can be cloned in a set of clones known as a library.com . Suppose that you want to clone a fungal gene that encodes a certain tRNA. Tagging also is useful for cloning a gene: transforming DNA or a transposon is used to cause a mutation by insertion. Restriction target sites can be mapped and act as DNA landmarks. Cloned DNA can be sequenced by several methods.immuneweb. starting from a marker linked to the desired gene. You have a sample of the purified tRNA and an E. respectively. coli plasmid that contains a single EcoRI cutting site in a tetR (tetracycline-resistance) gene. Concept Map Draw a concept map interrelating as many of the following terms as possible. It is a way of rapidly isolating DNA whose structure is already partly sequenced and of detecting small amounts of one specific type of DNA. as well as a gene for resistance to ampicillin (ampR). phages. Gel electrophoresis separates variously sized DNA or RNA molecules from a mixture. we studied the structure of tRNA molecules. A specific clone can be found in a library by using a probe that specifically binds to the DNA or to the protein of the desired clone. How can you clone the gene of interest? See answer 免疫学信息网 http://www. Chromosome walking provides a way of isolating a gene by sequential isolation of overlapping clones. enabling them to be inserted into a vector capable of replicating in a bacterial cell. and a major goal is to characterize their structure and function. The polymerase chain reaction uses primers to amplify DNA sequences. Restriction fragments often have sticky ends. The analysis of alkaptonuria (black urine disease). in procedures known as Southern and Northern analyses. and cosmids.Recombinant DNA Technology Figure 12-31. including the arrest of DNA chain growth by dideoxynucleotides. and the DNA adjacent to the tag is isolated. Chapter Integration Problem In Chapter 10. Such molecular hybrids are known as recombinant DNA. Specific clones can also be isolated by their ability to transform null mutants. Bacteria amplify a single recombinant DNA molecule to form a DNA clone. The technology is based on restriction enzymes.
On average. See answer Solution Both restriction enzymes recognize a six-base-pair sequence. and the restriction enzyme HpaII cuts DNA at the sequence CCGG. AmpR colonies are selected. determine whether the EcoRI enzyme or the SmaI enzyme would be more useful for cloning. indicating successful transformation. Explain your answer. what is the average spacing between restriction sites?) See answer Solution We need to consider only one strand of DNA. so both would be expected to have approximately the same number of recognition sites per genome. Alternatively. or 1/256. because there are four possibilities at each of the six positions. These TetS colonies will contain vectors with inserts. One method is to digest the genomic DNA with EcoRI and then mix it with the plasmid. which you also have cut with EcoRI. how frequently will each enzyme cut double-stranded DNA? (In other words.25 kb. because both sequences will be present on the opposite strand at the same site owing to the symmetry of the sequences. This region of the gel can be cut out and used as a source of enriched DNA to clone into the plasmid cut with EcoRI. Therefore. From Table 12-1. and a great number of them are needed to make the library.com . whereas 免疫学信息网 http://www. The major difference between the two is that EcoRI leaves staggered ends. The restriction enzyme HindIII cuts DNA at the sequence AAGCTT. Test the library by using the tRNA as the probe. See question 2. See question Solved Problem 1. select the colonies that are TetS. Of these AmpR colonies. The frequency of the six-base-long HindIII sequence is (1/4)6 = 1/4096.immuneweb. the frequency of the four-base-long sequence is (1/4)4. You can examine those colonies hybridizing with the probe further to find out how much of the tRNA sequence they contain. The average spacing between HpaII sites is approximately 0. For HpaII.Recombinant DNA Technology Solution You can use the tRNA itself or a cloned cDNA copy of it to probe for the DNA containing the gene. After transformation of an ampR tetS recipient. the average spacing between HindIII sites is approximately 4 kb. you can subject EcoRI-digested DNA from organism X to gel electrophoresis and then identify the correct band by probing with the tRNA.
what bands do you expect to see on the autoradiogram? Unpacking the Problem 1. which bands do you expect to appear radioactive on an autoradiogram? c.) See question Problems 1. If the same tetR gene cloned in a completely nonhomologous vector is made radioactive and used as a probe in a Southern blot of this gel. The sizes of the bands (in kilobases) are shown alongside. Lavett. If the same gene of interest from a fly closely related to Drosophila has been cloned in a nonhomologous vector and this clone is used as a probe for the same gel. Why is it important that the single site is in a resistance gene? Would it be useful if not? 4. Fruit fly genomic DNA is digested with HindIII.com . What is the importance of the single HindIII restriction site? 3. Clone 15 is studied by restriction analysis with HindIII and another restriction enzyme. b. A circular bacterial plasmid (pBP1) has a single HindIII restriction-enzyme site in the middle of a tetracycline-resistance gene (tetR). EcoRV. showing the sites of the target sequences and the approximate position of the tetR gene.Recombinant DNA Technology SmaI leaves blunt ends.) a.immuneweb. Staggered ends are much easier to manipulate during cloning because of the base-pairing capacity inherent in them. What is the effect of insertion of donor DNA into the resistance gene? Is this effect important for this problem? 免疫学信息网 http://www. Draw restriction maps for plasmid pBP1 with and without the insert. Probing reveals that clone 15 contains a specific Drosophila gene of interest. The ethidium bromide-stained electrophoretic gel shows bands as illustrated in the diagram below (the control was an uninserted plasmid pBP1). (Solution from Diane K. and a library is made in pBP1. (Note: Circular molecules do not give intense bands on this type of gel. Which plasmid described in this chapter seems closest in type to pBP1? 2. so you can assume that all bands represent linear molecules.
What function does ethidium bromide serve in this experiment? 9. If one enzyme produces n fragments and another produces m fragments. Does the gel shown represent a Southern or a Northern blot or neither? 10. at what positions were the DNA samples loaded into the gel? 16.096 kb. on average. how many fragments are produced if both enzymes are used? 15. what is the average spacing between restriction sites?) See answer GTTAAC appears. Is it a coincidence that the 1.and 2-kb fragments together equal the 5-kb fragment in size? 19. Is it a coincidence that the 3. What is a library? What type was used in this experiment and does it matter for this problem? 6. and the enzyme HaeIII cuts DNA at the sequence GGCC. which is 4. why is it stressed that a nonhomologous vector is used? Now attempt to solve the problem. 2. In the diagram. How many fragments are produced if a circular molecule is cut twice? 13. What is the total molecular weight of the fragments in all the lanes? What patterns do you see? 18. what does this signify? 21. What determines whether a probe will hybridize to a DNA blot (denatured)? 22. every 46 bases. Can you write a simple formula relating the number of restriction-enzyme sites in a circular molecule to the number of fragments produced? 14. Genetically.5-kb fragment in size? 20. If a fragment produced by one enzyme disappears when the DNA is treated with that same enzyme plus another enzyme.5. What kind of probing would have shown that clone 15 contains the gene of interest? And is this relevant to the present problem? 7.immuneweb. How many fragments are produced if a circular molecule is cut once? 12. Why are the smaller-molecular-weight fragments at the bottom of the gel? 17. In part c. The restriction enzyme EcoRI cuts DNA at the sequence GTTAAC.com . what types of molecules are visible on the gel? 11.and 1-kb fragments together equal the 2. On average. What is an electrophoretic gel? 8. how frequently will each enzyme cut double-stranded DNA? (In other words.Recombinant DNA Technology 5. 免疫学信息网 http://www.
256 kb. whose amino acid sequence is known? b. You have isolated and cloned a segment of DNA that is known to be a unique sequence in the genome. After digestion with EcoRI. Draw a restriction map of this DNA. partly digesting one with HaeIII and the other with HindII. and 4. you recover four pieces: A.5 kb long. and B yields 23 and 4. B. After Drosophila DNA has been treated with a restriction enzyme. on average. 3.5 kb. You separate the 8. David Hogness recovered every DNA sequence of Drosophila in a library. you obtain the following results: Draw a restriction-enzyme map of the complete 10-kb molecule. 22. coli. 3. After digestion of the entire DNA molecule with HindII.com . Piece C is identical with 22. You obtain two fragments: one is 8. It maps near the tip of the X chromosome and is about 10 kb in length. you obtain four fragments: 1. After digestion of each of these fragments with HindII.Recombinant DNA Technology GGCC is present. the fragments are attached to plasmids and selected as clones in E. a. You label the 5′end with 32P and cleave the molecule with EcoRI. 2. 免疫学信息网 http://www. and D.5-kb fragment into two fractions. which is 0. piece D yields fragments 1 and 31. and 23). How would you identify a clone encoding a specific tRNA? 5. How would you identify the clone that contains DNA encoding the protein actin. When these pieces are treated with EcoRI. you find that fragment 3 yields two subfragments (31 and 32) and that fragment 2 yields three (21. and you wish to map restriction-enzyme sites along its length. every 44 bases. By autoradiography. A yields 32 and 21.immuneweb. the other is 1. C. You then separate each sample on an agarose gel. You have a purified DNA molecule. By using this “shotgun” technique. See answer 4.
immuneweb. 免疫学信息网 http://www. 1989. Freeman and Company. Draw the restriction map. The mixture of fragments produced by the combined enzymes is cleaved with the enzyme EcoRI. F. McPherson.) See answer AluI (1/4)4 = every 256 nucleotide pairs EcoRI (1/4)6 = every 4096 nucleotide pairs AcyI (1/4)4(1/2)2 = every 1024 nucleotide pairs 7. The fragments obtained are: a. Mark the EcoRI cleavage site on the restriction map. Griffiths and J. (Problem 7 courtesy of Joan McPherson.) See answer a. resulting in the loss of the 3-kb fragment (band stained with ethidium bromide on an agarose gel) and the appearance of a band stained with ethidium bromide representing a 1. H.5-kb fragment. W. b. the sequence is: 6.com .Recombinant DNA Technology See answer Read from the top. Calculate the average distances (in nucleotide pairs) between the restriction sites in organism X for the following restriction enzymes: (Note: Py = any pyrimidine. A linear fragment of DNA is cleaved with the individual restriction enzymes HindIII and SmaI and then with a combination of the two enzymes. 100+ Principles of Genetics. From A. Pu = any purine. J.
Recombinant DNA Technology b. c.) 9. it is hybridized to a specific cDNA previously constructed from viral-gene-V mature mRNA. McPherson. Griffiths and J. First. What is the size of the mature mRNA for gene V? b. The following results suggest that the pathologic nature of the virus is time related (the number of nucleotides is indicated on the bands observed): a. The radiolabeled viral RNA is treated as follows. 100+ Principles of Genetics. Freeman and Company. using as the cloning vector the pBR E. the hybrid is denatured and electrophoresed on a gel. J. 1989.immuneweb. F. The gene for β-tubulin has been cloned from Neurospora and is available. Second. After incubation with 32P-labeled ribonucleotides. List a step-by-step procedure for cloning the same gene from the related fungus Podospora. W. 8. the virus-encoded RNA product is isolated at two timed intervals. Finally. Draw a diagram of each hybrid and indicate what the illustrated bands represent. A viral DNA fragment carrying a specific gene V is introduced into a muscle cell culture by transformation. From A.com . H. the hybrid is treated with RNase. coli plasmid shown here. which is then subjected to autoradiography. Why is protein V not produced until after 2 hours? (Problem 8 courtesy of Joan McPherson. where kan encodes kanamycin and tet encodes tetracycline: 免疫学信息网 http://www.
Explain this result. and 8.5. A fragment of mouse DNA with EcoRI sticky ends carries the gene M. The recombinant plasmid is cut with three different restriction-enzyme treatments. What can be deduced from this result? One recombinant clone was cut with BglII. 11.Recombinant DNA Technology 10. of 2. What can be deduced from this result? Plasmid DNA cut with BglII was mixed and ligated with donor DNA fragments also cut with BglII to make recombinant DNA molecules. f. What can be deduced from this result? The plasmid was cut with EcoRV and electrophoresis produced two bands. is inserted into the bacterial plasmid pBR322 at the EcoRI site.immuneweb. 5. The same clone was treated with EcoRV and fragments of 2. The patterns of ethidium bromide fragments. after electrophoresis on agarose gels. e. Which fragments will hybridize to a probe (32P) of pBR plasmid DNA? 免疫学信息网 http://www.5 were observed.5.5 kb. and 6 kb. a. This DNA fragment. Electrophoresis showed one band of 14 kb.com . A circular bacterial plasmid containing a gene for tetracycline resistance was cut with restriction enzyme BglII.5. What can be deduced from this result? Digestion with both enzymes together resulted in three bands of 2. and fragments of 4 and 14 kb were observed.5 and 11. which is 8 kb long. are shown in this diagram: A Southern blot is prepared from gel iii. Explain these results by showing a restriction map of the recombinant DNA. a. b. d. c. 7. All recombinant clones proved to be tetracycline sensitive.
If gene X encodes a protein X of 400 amino acids. a revertant of the auxotrophic mutation? 13. Gene X is carried on a plasmid consisting of 5300 nucleotide pairs (5300 bp). Cleavage of the plasmid with the restriction enzyme BamHI gives fragments 1. F.com . in fact.immuneweb. From A. replaced the resident gene of the recipient by double crossing-over or single crossing-over? b. a prototrophic cell that has entered the recipient culture as a contaminant? b.) 12. What experimental design would you use to make sure that a colony that you hope is a transformant is not. prototrophy is often the phenotype selected to detect transformants. a. In any particular transformed eukaryotic cell (say. H. (Problem 11 courtesy of Joan McPherson. 1989. then this DNA is cloned and the clones are added to an auxotrophic recipient culture. J. as indicated in the following diagram. how could you tell if the transforming DNA (carried on a circular bacterial vector) a. Successful transformants are identified by plating the recipient culture on minimal medium and looking for colonies.Recombinant DNA Technology b. Griffiths and J. 2. Prototrophic cells are used for donor DNA extraction. was inserted ectopically? 14. 100+ Principles of Genetics. W. In functional complementation in both prokaryotes and eukaryotes. Freeman and Company. In an electrophoretic gel across which is applied a powerful electric alternating 免疫学信息网 http://www.) Tandem copies of gene X are contained within a single BamHI fragment. indicate the approximate positions and orientations of the gene X copies. (B = BamHI restriction site. of Neurospora). McPherson. and 3.
and the label is found associated with the 6. Draw a restriction-enzyme–target-site map of this molecule.4 kb are produced.immuneweb.4.5-. McPherson.9-kb fragments. BamHI cleaves the same molecule into fragments of sizes 6.0. Draw the approximate location of gene X on the restriction map. How would you show which band corresponds to which chromosome? 15.0.0. 1989. showing relative positions and distances apart. J.5. H. A radioactive probe made from a cloned phage gene X is added to Southern blots of single-enzyme digests of phage DNA. the DNA of the haploid fungus Neurospora crassa (n = 7) moves slowly but eventually forms seven bands. a. which represent DNA fractions that are of different sizes and hence have moved at different speeds. See answer 16. and 7. and b. 4. 6.0 fragments. From A. 6.5. and 12.0 and 10. Combined digest with HaeIII and TaqI enzymes Electrophoretic fragments stained with ethidium bromide: 免疫学信息网 http://www. 100+ Principles of Genetics.0. the structural characteristics of the gene and its transcript can be determined: cDNA Map The fragment of cDNA was excised from a plasmid and end labeled with 32P. and 8. When EcoRI and BamHI are used together. The cDNA clone and genomic clone for a phosphatase enzyme have been isolated. and 12.9. The autoradiograms show hybridization associated with the 4. 7. EcoRI produces fragments of sizes 2. A linear phage chromosome is labeled at both ends with 32P and digested with restriction enzymes.2. 10.2 and 8. 6.) a. 3. W.1. 2. F. Griffiths and J. 2.1 fragments. These bands are presumed to be the seven chromosomes. An autoradiogram developed from a Southern blot of this digest shows radioactivity associated with the 6. (Problem 15 courtesy of Joan McPherson.2. fragments of sizes 1. Freeman and Company.9.9. 10.1-. From the following data.Recombinant DNA Technology pulsed field. b.0 kb.com .
com .immuneweb. that is. Determine the cDNA map. the autoradiogram pattern: Digestion with TaqI enzyme alone a. Complete BamII digest 免疫学信息网 http://www. Genomic DNA Map The fragment of genomic DNA was cleaved from a λ phage clone with EcoRI enzyme. and its restriction characteristics were determined.Recombinant DNA Technology Gel from the combined digestion exposed to X-ray film. The fragment was end labeled and digested.
If the phosphatase gene is a single-copy gene.and 2. Which persons are homozygous for the α-interferon gene? b.4-kb genomic fragment. Freeman and Company. will detect only interferon sequences. 1989. What do you think are the causes of the immune deficiencies of persons 3.Recombinant DNA Technology b. Which part of the gene does the 3. and 5 have immune deficiencies. J. blood samples from patients and unaffected people are screened for the α-interferon gene and its expression. which.immuneweb. From A. 100+ Principles of Genetics. J. McPherson.) 免疫学信息网 http://www. 1989. 100+ Principles of Genetics. marking the restriction sites.1-kb TaqI fragment hybridize? d. 4. under the conditions of hybridization. The 1. Persons 1 and 2 are normal for immune capacity. F. F. W.2-kb genomic fragments. McPherson.0-kb genomic fragment represent? (Problem 16 courtesy of Joan McPherson. The following drawing represents autoradiograms for Southern and Northern blots and a Western blot probed by antibody that recognizes α-interferon sequences. c. W. H. Griffiths and J. To determine the cause of unknown immune deficiencies. persons 3.com . and 5? Describe the type of gene mutation for each person. Draw the genomic map. The BamHI restriction fragment containing the complete gene can be identified on a Southern blot by hybridization to a specific interferon cDNA probe (32P). Freeman and Company. to which genomic fragment might the 1.2-kb TaqI fragment hybridized to the 3. (Problem 17 courtesy of Joan McPherson. From A.4. 4. α-Interferon is encoded by a gene that does not contain introns. a.) 17. H. Griffiths and J. A labeled cDNA probe hybridized to the 3.
The 10-kb fragment is partly digested with HpaII and the products are electrophoresed. 32 Southern blots of complete digest 免疫学信息网 http://www. The cDNA for gene P can be cut by HpaII into only two fragments. giving two fragments. and each is used as a P probe to investigate fragments from a complete digest of GP by using HpaII and Bam enzymes. giving the following results: Autoradiogram of HpaII digest of GP fragment a.com .Recombinant DNA Technology 18. The fragment is initially digested with Bam. indicating the Bam site and the HpaII sites. and the ends of the gene fragment (GP) are labeled with 32P.0 kb and 10 kb. Map the genomic fragment. A fragment of human genomic DNA for gene P is excised from a λ vector by using HindIII. Both are labeled.immuneweb. of 1. on Southern blots.
Child 1 For child 2. (Problem 18 courtesy of Joan McPherson. Two children are investigated for the expression of a gene (D) that encodes an important enzyme for muscle development.Recombinant DNA Technology b. From A. Freeman and Company. F. Child 2 免疫学信息网 http://www. the enzyme activity of each stage was very low and could be estimated only at approximately 0.immuneweb. Griffiths and J. J. 1989.com . McPherson. 3. Mark the HpaII site on the cDNA and the orientation of the two Hpa fragments. The results of the studies of the gene and its product follow. Explain the differences. 100+ Principles of Genetics.) 19. Draw a diagram comparing genomic DNA with cDNA. c. Indicate the restriction sites. W. and 4.1 unit at ages 1. H. 2.
W. How might you explain the change in the Southern blot for child 2 compared with that for child 1? d. Freeman and Company. 免疫学信息网 http://www. For both children.) b. From A. If only one mutant gene has been detected in family studies of the two children. J.) 20. (Fully label both axes. F. Griffiths and J. A cloned fragment of DNA was sequenced by using the dideoxy method. A part of the autoradiogram of the sequencing gel is represented here. H. 1989. How can you explain the very low levels of active enzyme for child 2? (Protein degradation is only one possibility.immuneweb. (Problem 19 courtesy of Joan McPherson. McPherson.Recombinant DNA Technology a. draw graphs representing the developmental expression of the gene. define the individual children as either homozygous or heterozygous for gene D. 100+ Principles of Genetics.) c.com .
The protein encoded by the alkaptonuria gene is 445 amino acids long. Deduce the nucleotide sequence of the DNA nucleotide chain used as the template strand. no genomic fragments bound to the probe.immuneweb. How is this possible? 免疫学信息网 http://www. Deduce the nucleotide sequence of the DNA nucleotide chain synthesized from the primer. in this case.com . Albino mice were used in a Southern analysis. Write the nucleotide sequence of the DNA double helix (label the 5′ and 3′ ends). Label the 5′ and 3′ ends. d. Explain these results in relation to the nature of the wild-type and mutant mouse alleles. 22. The cDNA clone for the human gene encoding tyrosinase was used in a Southern analysis of EcoRIdigested genomic DNA of wild-type mice. yet the gene spans 60 kb (see the section “A Century of Genetic Research on Alkaptonuria”). Label the 5′ and 3′ ends. How many of the six reading frames are open as far as you can tell? 21.Recombinant DNA Technology a. Three mouse fragments were found to be radioactive (were bound by the probe). but. c. b.
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