Further Reading 353 Luger, C., et al., 1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389:251–260. Rhodes, D., 1997. The nucleosome core all wrapped up. Nature 389: 231–233. Chromosome Structure Pienta, K. J., and Coffey, D. S., 1984. A structural analysis of the role of the nuclear matrix and DNA loops in the organization of the nucleus and chromosomes. In Cook, P. R., and Laskey, R. A., eds., Higher order structure in the nucleus. Journal of Cell Science Supplement 1:123–135. Sumner, A. T., 2003. Chromosomes: Organization and Function. Malden, MA: Blackwell Science. Tremethick, D. J., 2007. Higher-order structures of chromatin: The elu- sive 30 nm fiber. Cell 128:651-654. Telomeres Axelrod, N., 1996. Of telomeres and tumors. Nature Medicine 2:158–159. Feng, J., Funk, W. D., Wang, S-S., Weinrich, S. L., et al., 1995. The RNA component of human telomerase. Science 269:1236–1241. Chemical Synthesis of Genes Ferretti, L., Karnik, S. S., Khorana, H. G., Nassal, M., and Oprian, D. D., 1986. Total synthesis of a gene for bovine rhodopsin. Proceedings of the National Academy of Sciences U.S.A. 83:599–603. Higher-Order RNA Structure Ban, N., et al., 2000. The complete atomic structure of the large riboso- mal subunit at 2.4 Å resolution. Science 289:905–920. Gray, M. W., and Cedergren, R., eds., 1993. The new age of RNA. The FASEB Journal 7:4–239. A collection of articles emphasizing the new appreciation for RNA in protein synthesis, in evolution, and as a catalyst. Holbrook, S. R., 2005. RNA structure: The long and the short of it. Cur- rent Opinion in Structural Biology 15:302–308. Klosterman, P. S., et al., 2005. Three-dimensional motifs from the SCOR, structural classification of RNA database: Extruded strands, base triples, tetraloops, and U-turns. Nucleic Acids Research 32:2342–2352. Nilsen, T. W., 2007. RNA 1997–2007: A remarkable decade of discovery. Molecular Cell 28:715–720. Scala/Art Resource, NY 12 Recombinant DNA: Cloning and Creation of Chimeric Genes In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules com- posed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, 1 opened exciting new avenues of investi- gation in molecular biology and genetics, and a new field was born—recombinant DNA technology. Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplifi- cation of any particular DNA segment, regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became feasible. 12.1 What Does It Mean “To Clone”? In classical biology, a clone is a population of identical organisms derived from a sin- gle parental organism. For example, the members of a colony of bacterial cells that arise from a single cell on a petri plate are a clone. Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original mol- ecule or cell. So, if a single bacterial cell harboring a recombinant DNA molecule in the form of a plasmid grows and multiplies on a petri plate to form a colony, the plasmids within the millions of cells in the bacterial colony represent a clone of the original DNA molecule, and these molecules can be isolated and studied. Further- more, if the cloned DNA molecule is a gene (or part of a gene)—that is, it encodes a functional product—a new avenue to isolating and studying this product has opened. Recombinant DNA methodology offers exciting new vistas in biochemistry. Plasmids Are Very Useful in Cloning Genes Plasmids are naturally occurring, circular, extrachromosomal DNA molecules (see Chapter 11). Natural strains of the common colon bacterium Escherichia coli isolated from various sources contain diverse plasmids. Often these plasmids carry genes specifying novel metabolic activities that are advantageous to the host bacterium. These activities range from catabolism of unusual organic substances to metabolic functions that endow the host cells with resistance to antibiotics, heavy metals, or bacteriophages. Plasmids that are able to perpetuate themselves in E. coli, the bac- terium favored by bacterial geneticists and molecular biologists, are the workhorses The Chimera of Arezzo, of Etruscan origin and proba- bly from the fifth century B.C., was found near Arezzo, Italy, in 1553. Chimeric animals existed only in the imagination of the ancients. But the ability to create chimeric DNA molecules is a very real technology that has opened up a whole new field of scientific investigation. …how many vain chimeras have you created?… Go and take your place with the seekers after gold. Leonardo da Vinci The Notebooks (1508–1518), Volume II, Chapter 25 KEY QUESTIONS 12.1 What Does It Mean “To Clone”? 12.2 What Is a DNA Library? 12.3 Can the Cloned Genes in Libraries Be Expressed? 12.4 What Is the Polymerase Chain Reaction (PCR)? 12.5 How Is RNA Interference Used to Reveal the Function of Genes? 12.6 Is It Possible to Make Directed Changes in the Heredity of an Organism? ESSENTIAL QUESTIONS Using techniques for the manipulation of nucleic acids in the laboratory, scientists can join together different DNA segments from different sources. Such manmade products are called recombinant DNA molecules, and the use of such molecules to alter the genetics of organisms is termed genetic engineering. What are the methods that scientists use to create recombinant DNA mole- cules; can scientists create genes from recombinant DNA molecules; and can scientists modify the heredity of an organism using recombinant DNA? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. 1 The advent of molecular biology, like that of most scientific disciplines, generated a jargon all its own. Learning new fields often requires gaining familiarity with a new vocabulary. We will soon see that many words—vector, amplification, and insert are but a few examples—have been bent into new mean- ings to describe the marvels of molecular biology. 12.1 What Does It Mean “To Clone”? 355 of recombinant DNA technology. Because restriction endonuclease digestion of plasmids can generate fragments with overlapping or “sticky” ends, artificial plas- mids can be constructed by ligating different fragments together. Such artificial plasmids were among the earliest recombinant DNA molecules. These recombinant molecules can be autonomously replicated, and hence propagated, in suitable bac- terial host cells, provided they still possess a site signaling where DNA replication can begin (a so-called origin of replication or ori sequence). Plasmids as Cloning Vectors The idea arose that “foreign” DNA sequences could be inserted into artificial plasmids and that these foreign sequences would be car- ried into E. coli and propagated as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes. (The word vector is used here in the sense of “a vehicle or carrier.”) Plasmids useful as cloning vectors possess three common fea- tures: a replicator, a selectable marker, and a cloning site (Figure 12.1). A replicator is an origin of replication, or ori. The selectable marker is typically a gene conferring resistance to an antibiotic. Only cells containing the cloning vector will grow in the presence of the antibiotic. Therefore, growth on antibiotic-containing media “se- lects for” plasmid-containing cells. Typically, the cloning site is a sequence of nu- cleotides representing one or more restriction endonuclease cleavage sites. Cloning sites are located where the insertion of foreign DNA neither disrupts the plasmid’s ability to replicate nor inactivates essential markers. Virtually Any DNA Sequence Can Be Cloned Nuclease cleavage at a restriction site opens, or linearizes, the circular plasmid so that a foreign DNA fragment can be in- serted. The ends of this linearized plasmid are joined to the ends of the fragment so that the circle is closed again, creating a recombinant plasmid (Figure 12.2). Recom- binant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus inserted DNA elements (called inserts). Such hybrid molecules are also called chimeric constructs or chimeric plasmids. (The term chimera is borrowed from mythology and refers to a beast composed of the body and head of a lion, the heads of a goat and a snake, and the wings of a bat.) The presence of foreign DNA sequences does not ad- versely affect replication of the plasmid, so chimeric plasmids can be propagated in bacteria just like the original plasmid. Bacteria often harbor several hundred copies of common cloning vectors per cell. Hence, large amounts of a cloned DNA sequence 4 EcoRI HindIII EcoRV NheI BamHI SalI EagI NruI BspMI BsmI StyI AvaI BspMII PvuII Ndel 3 4 1 2 a m p r t e t r o r i BalI AflIII PpaI PstI PvuI ScaI SspI SphI SalI AatII ClaI pBR322 (4363 bases) FIGURE 12.1 One of the first widely used cloning vec- tors, the plasmid pBR322.This 4363-bp plasmid contains an ori and genes for resistance to the drugs ampicillin (amp r ) and tetracycline (tet r ).The locations of restriction endonuclease cleavage sites are indicated. 356 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes can be recovered from bacterial cultures. The enormous power of recombinant DNA technology stems in part from the fact that virtually any DNA sequence can be selectively cloned and amplified in this manner. DNA sequences that are difficult to clone include in- verted repeats, origins of replication, centromeres, and telomeres. The only practical limitation is the size of the foreign DNA segment: Most plasmids with inserts larger than about 10 kbp are not replicated efficiently. However, bacteriophages such as bacteriophage can be manipulated so that DNA sequences as large as 40 kbp can be inserted into the bacteriophage genome. Such recombinant phage DNA mole- cules lack essential genes and replicate in E. coli as plasmids. Construction of Chimeric Plasmids Creation of chimeric plasmids requires join- ing the ends of the foreign DNA insert to the ends of a linearized plasmid. This ligation is facilitated if the ends of the plasmid and the insert have complementary, single-stranded overhangs. Then these ends can base-pair with one another, an- nealing the two molecules together. One way to generate such ends is to cleave the DNA with restriction enzymes that make staggered cuts; many such restriction endo- nucleases are available (see Table 10.2). For example, if the sequence to be inserted 1 2 3 T T A A C G A A T T G C G C T A T A A T A T C G G C T A T A A T A T C G Cut with EcoRI TTAA C G G C A T A T T A T A C G G C A T A T T A T A C G G C A T A T T A T A C G G CAATT DNA ligase G C A T A T T A T A C G Cut with EcoRI Anneal ends of vector and foreign DNA Seal gaps in chimeric plasmid with DNA ligase ACTIVE FIGURE 12.2 An EcoRI restric- tion fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by (1) cutting the plasmid at this site with EcoRI, (2) annealing the lin- earized plasmid with the EcoRI foreign DNA fragment, and (3) sealing the nicks with DNA ligase. Test yourself on the concepts in this figure at www.cengage.com/ login. Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore the construction of chimeric plasmids. 12.1 What Does It Mean “To Clone”? 357 is an EcoRI fragment and the plasmid is cut with EcoRI, the single-stranded sticky ends of the two DNAs can anneal (Figure 12.2). The interruptions in the sugar–phosphate backbone of DNA can then be sealed with DNA ligase to yield a covalently closed, circular chimeric plasmid. DNA ligase is an enzyme that cova- lently links adjacent 3Ј-OH and 5Ј-PO 4 groups. An inconvenience of this strategy is that any pair of EcoRI sticky ends can anneal with each other. So, plasmid molecules can reanneal with themselves, as can the foreign DNA restriction fragments. These DNAs can be eliminated by selection schemes designed to identify only those bac- teria containing chimeric plasmids. Blunt-end ligation is an alternative method for joining different DNAs. The most widely used DNA ligase, bacteriophage T4 DNA ligase, is an ATP-dependent enzyme that can even ligate two DNA fragments whose ends lack overhangs (blunt-ended DNAs). Many restriction endonucleases cut double-stranded DNA so that blunt ends are formed. A great number of variations on these basic themes have emerged. For example, short synthetic DNA duplexes whose nucleotide sequence consists of little more than a restriction site can be blunt-end ligated onto any DNA. These short DNAs are known as linkers. Cleavage of the ligated DNA with the restriction enzyme then leaves tailor-made sticky ends useful in cloning reactions (Figure 12.3). Similarly, many vectors contain a polylinker cloning site, a short region of DNA sequence bearing numerous restriction sites. Promoters and Directional Cloning Note that the strategies discussed thus far create hybrids in which the orientation of the DNA insert within the chimera is ran- dom. Sometimes it is desirable to insert the DNA in a particular orientation. For ex- ample, an experimenter might wish to insert a particular DNA (a gene) in a vector so that its gene product is synthesized. To do this, the DNA must be placed down- stream from a promoter. A promoter is a nucleotide sequence lying upstream of a gene. The promoter controls expression of the gene. RNA polymerase molecules bind specifically at promoters and initiate transcription of adjacent genes, copying template DNA into RNA products. One way to insert DNA so that it will be properly oriented with respect to the promoter is to create DNA molecules whose ends have different overhangs. Ligation of such molecules into the plasmid vector can only take place in one orientation to give directional cloning (Figure 12.4). (a) P P P P P P Blunt-ended DNA EcoRI linker DNA ligase EcoRI (b) A vector cloning site containing multiple restriction sites, a so-called polylinker. 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 6 EcoRI BamHI SalI AccI HincII PstI SalI AccI HincII BamHI EcoRI ANIMATED FIGURE 12.3 (a) The use of linkers to create tailor-made ends on cloning fragments. Note that the ligation reaction can add multiple linkers on each end of the blunt-ended DNA. EcoRI digestion removes all but the terminal one, leaving the desired 5Ј-overhangs. (b) Cloning vectors often have polylinkers consisting of a multiple array of restriction sites at their cloning sites, so restriction fragments generated by a variety of endonucleases can be incorporated into the vector. Note that the polylinker is engineered not only to have multiple restriction sites but also to have an uninterrupted sequence of codons, so this region of the vector has the potential for translation into protein (see Figure 12.15).(Adapted from Figure 1.14.2 in Greenwich, D., and Brent, R., 2003. UNIT 1.14 Introduction to Vectors Derived from Filamentous Phages, in Current Protocols in Molecular Biology, Ausubel, F. M.,Brent, R.,Kingston, R.E., Moore, D.D., Seidman, J. G., Smith, J. A., and Struhl, K., eds.New York:John Wiley and Sons.) See this figure animated at www.cengage.com/login. Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore blunt-end ligation. 358 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes Biologically Functional Chimeric Plasmids The first biologically functional chimeric DNA molecules constructed in vitro were assembled from parts of differ- ent plasmids in 1973 by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling. These plasmids were used to transform recipient E. coli cells (transformation means the uptake and replication of exogenous DNA by a recipient cell). To facili- tate transformation, the bacterial cells were rendered somewhat permeable to DNA by Ca 2ϩ treatment and a brief 42°C heat shock. Although less than 0.1% of the Ca 2ϩ -treated bacteria became competent for transformation, transformed bacteria could be selected by their resistance to certain antibiotics (Figure 12.5). Conse- quently, the chimeric plasmids must have been biologically functional in at least two EcoRI SacI KpnI SmaI BamHI XbaI SalI SphI HindIII pUC19 EcoRI SacI KpnI pUC19 PstI P 3' 3' 5' 5' P BamHI HindIII Small fragment discarded Digest with HindIII and BamHI Isolate large fragment by electrophoresis or chromatography HindIII BamHI Target DNA Digest with HindIII and BamHI P P Target DNA anneals with plasmid vector in only one orientation. Seal with T4 DNA ligase. EcoRI SacI KpnI SmaI BamHI HindIII pUC19 Large fragment PstI SphI XbaI SmaI SalI ANIMATED FIGURE 12.4 Directional cloning. DNA molecules whose ends have different over- hangs can be used to form chimeric constructs in which the foreign DNA can enter the plasmid in only one ori- entation.The foreign DNA and the plasmid are digested with the same two enzymes. pUC stands for universal cloning plasmid. See this figure animated at www .cengage.com/login. 12.1 What Does It Mean “To Clone”? 359 aspects: They replicated stably within their hosts, and they expressed the drug re- sistance markers they carried. In general, plasmids used as cloning vectors are engineered to be small (2.5 kbp to about 10 kbp in size) so that the size of the insert DNA can be maximized. These plasmids have only a single origin of replication, so the time necessary for complete replication depends on the size of the plasmid. Under selective pressure in a grow- ing culture of bacteria, overly large plasmids are prone to delete any nonessential “genes,” such as any foreign inserts. Such deletion would thwart the purpose of BamHI 2 4 5 68 7 3 1 EcoRI HindIII EcoRV Sal I a m p r t e t r o r i pBR322 (4363 bases) AvaI Sal I PvuI PstI PvuII a m p r t e t r BamHI restriction fragment of DNA to be cloned is inserted into the BamHI site of tet r . amp r gene remains intact. Chimeric plasmid Suspend 20 ng plasmid DNA + 10 7 E.coli cells in CaCl 2 solution. Plate bacteria on ampicillin media. 42ЊC, 2 min 37ЊC, overnight Ampicillin- containing medium Only ampicillin-resistant (amp r ) bacterial colonies grow. Using velvet-covered disc, bacterial colonies are lifted from surface of agar amp r plate and pressed briefly to surface of plate containing tetracycline media. 37ЊC, overnight Only tet r colonies appear; tet s colonies can be recovered from amp r plate by comparing two plates. tet r gene is inactivated by the insertion of DNA fragment. amp r gene remains intact. Tetracycline-containing medium A plasmid with genes for ampicillin resistance (amp r ) and tetracycline resistance (tet r ). A BamHI restriction site is located within the tet r gene. ACTIVE FIGURE 12.5 A typical bacterial transformation experiment. Here the plasmid pBR322 is the cloning vector. (1) Cleavage of pBR322 with BamHI, followed by (2) annealing and ligation of inserts generated by BamHI cleavage of some foreign DNA, (3) creates a chimeric plasmid. (4) The chimeric plasmid is then used to transform Ca 2ϩ -treated heat-shocked E. coli cells, and the bacterial sample is plated on a petri plate. (5) Following incubation of the petri plate overnight at 37°C, (6) colonies of amp r bacteria are evident. (7) Replica plating of these bacteria on plates of tetracycline-containing media (8) reveals which colonies are tet r and which are tetracycline sensitive (tet s ). Only the tet s colonies possess plasmids with foreign DNA inserts. Test yourself on the concepts in this figure at www.cengage.com/login. 360 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes most cloning experiments. The useful upper limit on cloned inserts in plasmids is about 10 kbp. Many eukaryotic genes exceed this size. Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms Shuttle vectors are plasmids capable of propagating and transferring (“shuttling”) genes between two different organisms, one of which is typically a prokaryote (E. coli) and the other a eukaryote (for example, yeast). Shuttle vectors must have unique origins of replication for each cell type as well as different markers for se- lection of transformed host cells harboring the vector (Figure 12.6). Shuttle vectors have the advantage that eukaryotic genes can be cloned in bacterial hosts, yet the expression of these genes can be analyzed in appropriate eukaryotic backgrounds. Artificial Chromosomes Can Be Created from Recombinant DNA DNA molecules 2 megabase pairs in length have been successfully propagated in yeast by creating yeast artificial chromosomes or YACs. Furthermore, such YACs have been transferred into transgenic mice for the analysis of large genes or multi- genic DNA sequences in vivo, that is, within the living animal. For these large DNAs to be replicated in the yeast cell, YAC constructs must include not only an origin of replication (known in yeast terminology as an autonomously replicating sequence or ARS) but also a centromere and telomeres. Recall that centromeres provide the site for attachment of the chromosome to the spindle during mitosis and meiosis, and telomeres are nucleotide sequences defining the ends of chromosomes. Telomeres are essential for proper replication of the chromosome. 12.2 What Is a DNA Library? A DNA library is a set of cloned fragments that collectively represent the genes of a specific organism. Particular genes can be isolated from DNA libraries, much as books can be obtained from conventional libraries. The secret is knowing where and how to look. Insert DNA amp r Polycloning site Yeast LEU2 + Yeast origin of replication Bacterial origin of replication Transform LEU – yeast E.coli Yeast cell Plasmids can be shuttled between E.coli and yeast Shuttle vector Transform E.coli ANIMATED FIGURE 12.6 A typical shuttle vector. LEU2 ϩ is a gene in the yeast pathway for leucine biosynthesis.The recipient yeast cells are LEU2 Ϫ (defective in this gene) and thus require leucine for growth. LEU2 Ϫ yeast cells transformed with this shuttle vector can be selected on medium lacking any leucine supplement. See this figure animated at www.cengage.com/login. 12.2 What Is a DNA Library? 361 Genomic Libraries Are Prepared from the Total DNA in an Organism Any particular gene constitutes only a small part of an organism’s genome. For ex- ample, if the organism is a mammal whose entire genome exceeds 10 6 kbp and the gene is 10 kbp, then the gene represents less than 0.001% of the total nuclear DNA. It is impractical to attempt to recover such rare sequences directly from isolated nu- clear DNA because of the overwhelming amount of extraneous DNA sequences. In- stead, a genomic library is prepared by isolating total DNA from the organism, di- gesting it into fragments of suitable size, and cloning the fragments into an appropriate vector. This approach is called shotgun cloning because the strategy has no way of targeting a particular gene but instead seeks to clone all the genes of the or- ganism at one time. The intent is that at least one recombinant clone will contain at least part of the gene of interest. Usually, the isolated DNA is only partially digested by the chosen restriction endonuclease so that not every restriction site is cleaved in every DNA molecule. Then, even if the gene of interest contains a susceptible re- striction site, some intact genes might still be found in the digest. Genomic libraries have been prepared from thousands of different species. Many clones must be created to be confident that the genomic library contains the gene of interest. The probability, P, that some number of clones, N, contains a particular fragment representing a fraction, f, of the genome is P ϭ 1 Ϫ (1 Ϫ ƒ) N Thus, N ϭ For example, if the library consists of 10-kbp fragments of the E. coli genome (4640 kbp total), more than 2000 individual clones must be screened to have a 99% probability ln (1 Ϫ P) ᎏᎏ ln (1 Ϫ ƒ) CRITICAL DEVELOPMENTS IN BIOCHEMISTRY Combinatorial Libraries Specific recognition and binding of other molecules is a defining characteristic of any protein or nucleic acid. Often, target ligands of a particular protein are unknown, or in other instances, a unique ligand for a known protein may be sought in the hope of blocking the activity of the protein or otherwise perturbing its func- tion. Or, the hybridization of nucleic acids with each other accord- ing to base-pairing rules, as an act of specific recognition, can be exploited to isolate or identify pairing partners. Combinatorial li- braries are the products of strategies to facilitate the identification and characterization of macromolecules (proteins, DNA, RNA) that interact with small-molecule ligands or with other macromole- cules. Unlike genomic libraries, combinatorial libraries consist of synthetic oligomers. Arrays of synthetic oligonucleotides printed as tiny dots on miniature solid supports are known as DNA chips. (See the section titled “DNA Microarrays (Gene Chips) Are Arrays of Dif- ferent Oligonucleotides Immobilized on a Chip.”) Specifically, combinatorial libraries contain very large numbers of chemically synthesized molecules (such as peptides or oligonu- cleotides) with randomized sequences or structures. Such libraries are designed and constructed with the hope that one molecule among a vast number will be recognized as a ligand by the protein (or nucleic acid) of interest. If so, perhaps that molecule will be useful in a pharmaceutical application. For instance, the synthetic oligomer may serve as a drug to treat a disease involving the pro- tein to which it binds. An example of this strategy is the preparation of a synthetic combinatorial library of hexapeptides. The maximum number of sequence combinations for hexapeptides is 20 6 , or 64,000,000. One approach to simplify preparation and screening possibilities for such a library is to specify the first two amino acids in the hexa- peptide while the next four are randomly chosen. In this approach, 400 libraries (20 2 ) are synthesized, each of which is unique in terms of the amino acids at positions 1 and 2 but random at the other four positions (as in AAXXXX, ACXXXX, ADXXXX, etc.), so each of the 400 libraries contains 20 4 , or 160,000, different sequence combinations. Screening these libraries with the protein of interest reveals which of the 400 libraries contains a ligand with high affin- ity. Then, this library is expanded systematically by specifying the first three amino acids (knowing from the chosen 1-of-400 libraries which amino acids are best as the first two); only 20 synthetic libraries (each containing 20 3 , or 8000, hexapeptides) are made here (one for each third-position possibility, the remaining three positions being randomized). Selection for ligand binding, again with the protein of interest, reveals the best of these 20, and this particular library is then varied systematically at the fourth posi- tion, creating 20 more libraries (each containing 20 2 , or 400, hexapeptides). This cycle of synthesis, screening, and selection is repeated until all six positions in the hexapeptide are optimized to create the best ligand for the protein. A variation on this basic strategy using synthetic oligonucleotides rather than peptides identified a unique 15-mer (sequence GGTTGGTGTGGTTGG) with high affinity (K D ϭ 2.7 nM) toward thrombin, a serine pro- tease in the blood coagulation pathway. Thrombin is a major target for the pharmacological prevention of clot formation in coronary thrombosis. From Cortese, R., 1996. Combinatorial Libraries: Synthesis, Screening and Ap- plication Potential. Berlin: Walter de Gruyter. 362 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes (P ϭ 0.99) of finding a particular fragment. Since ƒ ϭ 10/4640 ϭ 0.0022 and P ϭ 0.99, N ϭ 2093. For a 99% probability of finding a particular sequence within the 3 ϫ 10 6 kbp human genome, N would equal almost 1.4 million if the cloned fragments aver- aged 10 kbp in size. The need for cloning vectors capable of carrying very large DNA inserts becomes obvious from these numbers. Libraries Can Be Screened for the Presence of Specific Genes A common method of screening genomic libraries is to carry out a colony hybridization experiment. In a typical experiment, host bacteria containing a plasmid-based library are plated out on a petri dish and allowed to grow overnight to form colonies (Figure 12.7). A replica of the bacterial colonies is then obtained by overlaying the plate with a flexible, absorbent disc. The disc is removed, treated with alkali to dissociate bound DNA duplexes into single-stranded DNA, dried, and placed in a sealed bag with labeled probe (see the Critical Developments in Bio- chemistry box on page 364). If the probe DNA is duplex DNA, it must be denatured by heating at 70°C. The probe and target DNA complementary sequences must be in a single-stranded form if they are to hybridize with one another. Any DNA se- quences complementary to probe DNA will be revealed by autoradiography of the absorbent disc. Bacterial colonies containing clones bearing target DNA are identi- fied on the film and can be recovered from the master plate. Probes for Southern Hybridization Can Be Prepared in a Variety of Ways Clearly, specific probes are essential reagents if the goal is to identify a particular gene against a background of innumerable DNA sequences. Usually, the probes that are used to screen libraries are nucleotide sequences that are complementary to some part of the target gene. Making useful probes requires some information about the gene’s nucleotide sequence. Sometimes such information is available. Alterna- tively, if the amino acid sequence of the protein encoded by the gene is known, it is possible to work backward through the genetic code to the DNA sequence (Figure 12.8). Because the genetic code is degenerate (that is, several codons may specify the same amino acid; see Chapter 30), probes designed by this approach are usually degenerate oligonucleotides about 17 to 50 residues long (such oligonucleotides are so-called 17- to 50-mers). The oligonucleotides are synthesized so that different bases are incorporated at sites where degeneracies occur in the codons. The final prepa- ration thus consists of a mixture of equal-length oligonucleotides whose sequences vary to accommodate the degeneracies. Presumably, one oligonucleotide sequence in the mixture will hybridize with the target gene. These oligonucleotide probes are at least 17-mers because shorter degenerate oligonucleotides might hybridize with sequences unrelated to the target sequence. A piece of DNA from the corresponding gene in a related organism can also be used as a probe in screening a library for a particular gene. Such probes are termed 1 3 2 4 5 Master plate of bacteria colonies Replicate onto absorbent disc. Denatured DNA bound to absorbent disc Radioactive probe will hybridize with its complementary DNA Autoradiograph film Place disc in sealable plastic bag with solution of labeled DNA probe. Treat with NaOH; neutralize, dry. Wash disc, prepare auto- radiograph, and compare with master plate. Darkening identifies colonies containing the DNA desired. ACTIVE FIGURE 12.7 Screening a genomic library by colony hybridization. Host bacteria transformed with a plasmid-based genomic library are plated on a petri plate and incubated overnight to allow bacterial colonies to form. A replica of the colonies is obtained by overlaying the plate with a flexible disc composed of absorbent material (such as nitrocellulose or nylon) (1). Nitrocellulose strongly binds nucleic acids; single-stranded nucleic acids are bound more tightly.Once the disc has taken up an impression of the bacterial colonies, it is removed and the petri plate is set aside and saved.The disc is treated with 2 M NaOH, neutralized, and dried (2). NaOH both lyses any bacteria (or phage particles) and dissociates the DNA strands. When the disc is dried, the DNA strands become immobilized on the filter.The dried disc is placed in a sealable plastic bag, and a solution containing heat-denatured (single-stranded), labeled probe is added (3). The bag is incubated to allow annealing of the probe DNA to any target DNA sequences that might be present on the disc.The filter is then washed, dried, and placed on a piece of X-ray film to obtain an autoradiogram (4). The position of any spots on the X-ray film reveals where the labeled probe has hybridized with target DNA (5). The location of these spots can be used to recover the genomic clone from the bacteria on the original petri plate. Test yourself on the concepts in this figure at www.cengage.com/login.