CHAPTER THREE Recombinant DNA Technology and Genomics chapter, you ® Define recombinant DNA technology and explain how itis used to clone genes and manipulate DNA. ® Compare and contrast different types of cloning vectors and deseribe their Practical features and applications © Understand the importance of DNA libraries as collections of cloned DNA that were historically used to isolate specific genes. ® Describe how agarose gel electrophoresis, polymerase chain reaction (PCR), DNA sequencing, and other common laboratory techniques, including CRISPR-Cas for genome editing, are used to study gene structure, function, and expression for biotechnology applications. ® Describe how whole-genome sequencing and advances in DNA sequencing techniques enable Scientists to rapidly analyze genomes. ® Understand the major goals and findings of the Human Genome Project. © Explain why genomics-rolated “omics” disciplines are rapidly doveloping areas of biotechnology research, ® Provide examples of how bioinformatics can be used to analyze nucleic acid sequences and structures, © Discuss basic concepts of systems biology end synthetic biology as emerging disciplines, and provide examples of potential applications ofeach field 80 —— excitem, "apies th, Since its discovery. "? lent as a genom? an CRISPR-Cas.3.1. Introduction to Recombinant DNA Technology and DNA Cloning 81 s you have learned, biotechnology is not a new science. However, the modern era of biotech- nology began when DNA cloning techniques were developed. Since the 1970s and continuing today, amazing and rapidly developing methods in recombi- nant DNA technology, which enable the joining together of DNA from different sources, have led 10 genetic engineering—genctically modifying cells and organisms in various ways. Genetic engincering appli- cations have changed molecular biology, basic sci and medical research forever. In this chapter, we present an overview of recom- binant DNA technology. We then take a look at an amazing range of techniques that scientists use to clone and manipulate genes and to study gene struc- ture and function. The chapter concludes with an introduction to genomics and bioinformatics, which provide methods for studying genomes. From these introductions you will be well prepared to learn about modern applications of recombinant DNA and genom- ics in the biotechnology world. FORECASTING THE FUTURE There are many exciting potential future directions in recombinant DNA and genomics research. Without question, one area of ongoing and substantial progress is the completion of thousands of genome projects for different plents, animels, bacteria, viruses, and other organisms. Scientists continue to prospect genomes from organisms around the world to look for new genes with potential applications in biotechnology. Following on the success of the Human Genome Project, the use of genomic information for new applications—including novel methods for the detection of disease genes, tar- geted therapies based on genetics, and novel geno- based editing strategies for a range of diseases—holds tremendous promise for alleviating pain and suffering through biotechnology. As the cost of DNA sequencing decreases, the sequencing of individual genomes (per: sonal genomics) will be more common. 3.1 Introduction to Recombinant DNA Technology and DNA Cloning In their landmark paper detailing the double-helical structure of DNA, the scientists James Watson and Fran Ge Crick hinted that DNA’S very structure conveyed @ means for its replication, thus underscoring the potential importance andl impact of this discovery. However, not even these Nobel Prize winners could have imagined the astonishing pace at which molecular biology would advance over the next half century and beyond. In the late 1960s, many scientists were interested in copyin, DNA. or gene cloning. They speculated that it might be possible to clone DNA by cutting and joining together DNA from different sources (recombinant DNA technol ogy). The terms ger cloning, recombinant DNA teclmology, and_genetic engineering describe different, but closely interrelated, processes. Recombinant DNA technology is commonly used to make gene cloning possible, whereas genetic engineering often relies on recombinant DNA technology and gene cloning to modify an organism's genome. Often, the terms recombinant DNA technology and genetic engineering are used interchangeably. The word clone is derived from a Greek word that describes a cutting (of a twig) that is used to propagate or copy a plant. A modern biological definition of a clone is a molecule, cell, or organism produced from another single entity. The laboratory methods required for gene cloning as described in this chapter are differ- ent from the techniques used to clone whole organ- isms, which we discuss in Chapter 7. Restriction Enzymes and Plasmid DNA Vectors Many nearly simultaneous discoveries and collaborative cfforts among several researchers led to the discovery of ‘nwo essential components that made recombinant DNA techniques and gene cloning possible—restriction enzymes and plasmids (plasmid DNA). Restriction enzymes are DNA-cutting enzymes, and plasmid DNA is a small, circular form of DNA naturally present in many bacteria, which scientists have learned to manip ulate in order to carry and clone other pieces of DNA. ‘Much of what we know about DNA replication and DNA-synthesizing enzymes that made cloning possible was learned from studying DNA structure and replication in bacteria and in bacteriophages. Bacteriophages, often simply called phages, are vieuses that infect bacterial cells, Microbiologists in the 1960s discovered that some bacteria are protected from destruction by bacterio- phages because they can restrict phage replication. Swiss scientist Werner Arber proposed that restricted growth of phages occurred because these bacteria contained enzymes that could cut viral DNA into small pieces, thus preventing vital replication, Because of this ability, these enzymes were called restriction enzymes. In 1970, working with the bacterium Havinophilus influenzae, Johns Hopkins University researcher Hamilton Smith isolated Hindill, the first restriction enzyme to be well characterized and used for DNA cloning. Restriction enzymes are also called restriction endonucleases (endo, “within”; nuclease, “nucleic acid-cutting enzyme") because they cut within DNA molecules at specitic sequences as opposed to enzymes (exonucteases) that ‘cut beginning at the free ends of DNA molecules, and82 Chapter 3 Recombinant DNA Technology and Genomics not at specific sequences. Smith demonstrated that HindIll could be used to cut or digest DNA into small fragments, In 1978, Smith shared a Nobel Prize with Werner Arber and Daniel Nathans for their discoveries of restriction enzymes and their applications. Restriction enzymes are primarily found in bacte- ria, and they are given abbreviated names based on the genus and species names of the bacteria from which they are isolated. For example, one of the first restric- tion enzymes to be isolated, EcoRI, is so named because it was discovered in the Escherichia coli (E. coli) strain called RY13. Restriction enzymes cut double-stranded DNA by cleaving the phosphodiester bond (in the sugar-phosphate backbone) that joins adjacent nucleo. Uides in a DNA strand, However, restriction enzymes do Rot just randomly cut DNA, nor do all restriction ‘enzymes cut DNA at the same locations, Like other enzymes, restriction enzymes show specificity for certain substrates. For these enzymes, the substrate is a specific sequence within double. stranded DNA. As shown in Figure 3.ta, restriction enzymes bind to, recognize, and cut (digest) DNA Within specific sequences of bases called restriction sites. Why don’t restriction enzymes digest DNA in bacterial cells? Bacteria protect their DNA from restric. tion enzyme digestion because some of the nucleotides in thelr DNA contain methyl groups that block restric tlon enzymes from digestion (Figure 3,1b). @ 7 Restioton, enzyme) Econ / —z 3" EcoRi Unmethyiated si retin . | ee Cohesive ends Er eS 8 8 FIGURE 3.1. Rest n Sites and Restriction Enzyme by Eco! produces DNA fr site by the enzyme EcoRI methylase blocks DNA cleavey EcoRI restriction site occurs on an adenine nucleotide, Pecurs on cytosine nucleotides, ion lati 19° by Ech I ras But Methylation methylation Restriction enzymes are commonly referred to 3 {our- or six-base-pair cutters because they typically reco nie restriction sites with a sequence of four or six nude tides, Eight-base-pair cuters have also been identifed Each restriction site is a Palindrome—the arrangement 1 nucleotides reads the same forward and backward ol Shposite strands of the DNA molecule, (Remember the Word madam or the phrase a Toyous as ‘examples of palit HME) Some restriction enzymes, such ee ERE DNA to create DNA. fragments with overhanging sing Stranded ends called “sicky= iD tH nicd “sticky” or cohesive ends (s Figure 3.1a); other cate Fea MeS generate fragments wil feat yeanded ends called blunt ends, resttiction sine SOU" mictoorganisms, and the the table tes: Notice that the tiret tees enaymesit molecules wine ease-Pait cutters that produce DNA (Tag) isa ait €hesive ends, The fearih, entym ends, and te b38¢-Pair cutter that produces cohest® ended DNA he lower three enzymes produce blunt sive ends arg SEEMS. Enzymes that produce coh Many cloning oP |vored over bluntecmd eutets with cohesive oPetiments: because DNA tragimen DNA from any nS SN easily be joined togethel me UY so at dogs, eats, “Tiygs°MEE—such as bacteria, Il ns emaingva nS dinosaurs hun ena —82N be digest enzyme a ed by "8 as the DNA’ restrictio?® or ancient io @ particular restrict Nas a restriction sil ) (eon EeoRt will not clea? methylated DNA, (al Dj est, 0 oF ation of Noe! th® Econ Of DNA Nr N of frequen ofthe83 Introduction to Recombinant DNA Technology and DNA Cloning 3a oh PRE ‘Common Restriction Enzymes Ey ES Restriction Site Haemopnis eqyptvs serena84 Chapter 3 Recombinant DNA Technology and Genomics Restriction enzymes are sophisticated “scissors” that molecular biologists se to manipulate DNA. Working with restriction enzymes has become much easier since Hamilton Smith and others pioneered their use. Now over 600 restriction enzymes are com- mercially available rather inexpensively. These enzymes are readily available because they have been cloned using recombinant DNA technology, so they are made and isolated in large quantities. Commer- cially prepared enzymes come in conveniently sized Prepackages with buffer solutions that provide all the components necessary for optimal enzyme activity. f researchers must work with an enzyme with which they are unfamiliar, they can use restriction enzyme databases such as REBASE (http://rebase.neb.com), en outstanding tool for locating technical specifica, tions and suppliers of enzymes. |n addition, a variety of software packages and Websites are available to assist scientists who work with restriction enzymes and DNA sequences. For example, imagine thet you are a molecular biologist who just cloned and sequenced a 7,200-base-pair (bp) Piece of DNA and you want to see if there is an Snayme that will cut your gene to create a 250-bp that enzyme, restriction enz the “scissors 1 In the simplest sense, the discovery of ‘ymes provided molecular biologists with needed to catty out gene cloning, In the early 1970s, Paul Berg, Herbert Boyer Stanley GoheR. and colleagues at Stantord University wet gene Gloning to change molecular biology. forevex Berg {ounded recombinant DNA technology when he created Ie Of recombinant DNA by joining together, or spc. ma DNA from the E.coli chromosome andl DNA Ione a paeate virus called simian virus 40 (SV40). Berg first Solated chromosomal DNA from 2. eo and DN from $140. Then he cut both DNA samples with FeoRl added fc and viral DNA fragments to a reaction wie with the enzyme DNA ligase, and succeeded in cre Pybrid molecule of SV40 and F. ca DNA. Thee TRICE Of this discovery was fully recognized wih Berg won the 1980 Nobel Prize amtiment, which demonstrated that DNA could he cut from different sources with the same enzyme and that the 'estrction fragments. could be joined ne create a Wambinant DNA molecule. Berg shared thie Prize with Walter Gilbert and Fredetick Sanger, who independently developed methods for sequencing DNA—a dliscuss later in this chapter ‘ating a impor- n Paul in Chemistry for this process we STE SuiLY NS) Restriction Enzymes piece of DNA to use as a Probe for detecting similar Genes in other species. Not too long ago, it you had @ {ot of enzymes in your freezer, you could digest this PNA and run gels to see if you could get a 250-bp piece, restriction site of interest — @ very time-consuming and eye-straining effort! Ty he Internet makes this task While Berg worked phen was int Fete nh a temnosomal DNA ested in the molecu a olecular biology of s™ Ce of DNA known as plasmids, Plasmid sonny i Bacteria, Plasinids are c0l” in the bacterial eyes PNA because then a reset chromosome. pra ct™ IN addition to vine bocterl sm Plasmid Small; most average approximately In size and they remlganetely 000 bp in size and they af® catiged {He chromosome. cohe? ns the transfer af plasmids la, and the © contrib, all 0 tHe spread of arntity resists ee. sistan Cohen postulate ostttlated $ Vectors—piece: that plasmids could be used : and replir cate (done) worked with Cohen and Boye? Successfully, "7 na Plasmids 10 clone DNA experiments in ed results mal bitth Of req, ¥ conside 2 restrict Boye are hey i describing thes? "this work the infor” Previous SMMology. Using FR! nie IY isolated by Herbet gether ad then joined fragment’ 7 SB DNA Tigase 10 erealé Plasmids. “Recall fro" Alyzes the formation &! ION. en, they from each pl new ligase cay3.1. Introduction to Recombinant DNA Technology and DNA Cloning 35 n nucleotides. phosphodiester bonds betwe igase can tetracycline resistance and restriction sites for several join together DNA with cohesive ends as well as blunt- enzymes including EoRI and HindIll. In subsequent ended fragments. As a result of these and other experi- work they used similar experiments to done DNA from menis, Cohen and Boyer produced the first plasmid the South African claw-toed frog Xenopus laevis (another vector for cloning purposes, called pSC101 and named important model organism in genetics and developmen. “SC” for Stanley Cohen. pSC101 contained a gene for tal biology) into the ERI site of pSC101. Figure 32 EcoRl [sition sites —— 41) Restriction enzyme cuts (digests) ch at double-stranded DNA at its particular restriction site Human DNA’ cut cut 2) Digestion produces DNA fragments with cohesive ends. DNA from another source, perhaps a Hydrogen bondin ‘bacterial plasmid lydroae 9 ‘of cohesive ends 3) When DNA fragments cut by the same restriction enzyme come together, they can join by base pairing, 4) The joined fragments will usually form either linear molecule of a cicular one, as shown here fora plasmid. Other combinations of ‘fragments can also occur, however. Covalent attachment of DNA, backbones by DNA ligase 5) The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing @ molecule of recombinant DNA Containing human and plasmic DNA. Bacteral Haina plasmid DNA DNA ‘Recombinant DNA ating Recortbinert DNA’ EcoR nds tow apie rection sk RUGURE 32, Crestng Ree the DNA bctboneprodusng ONA agents hs single Fae a sya fagmontscan orm hyorogon bond wh ear other bees they faded ons of he Oru pas DNA ligase ca the ate he formation of ovale a are backbones) ofthe ragmentst crest a plete of recombinant DNAa6 Chapter 3 Recombinant DNA Technology and Genomics illustrates how recombinant DNA can be formed in a process similar to that used in the Cohen and Boyer experiments. Cohen and Boyer had created the first DNA clon- ing vector—a vehicle for the insertion and replication of DNA—and in 1980 were awarded patents for PSCIOI and for the gene splicing and cloning tech- niques they had developed. These experiments ush~ ered in the birth of modern biotechnology, because Many of the current techniques used for gene cloning and gene manipulations are based on these funda. mental methods of recombinant DNA technology. In 1974, as a direct result of the Berg, Cohen, and Boyer experiments, gene cloning pioneers and ctitics voiced concerms about the safety of genetically modified organisms. Scientists were concerned about what might happen if recombinant bacteria were to leave the lab or Hf such bacteria could transfer their genes to other cells or survive in other organisms, including humans, in 1975, an invited group of well-known molecular biolo- Sists, virologists, microbiologists, lawyers, and journal, ists gathered at the Asilomar Conference Center in Pacific Grove, California, to discuss the benefits and Potential hazards of recombinant DNA technology, As a result of the historic Asilomar mecting, the National Institutes of Health (NTH) formed the Recombi- nant DNA Advisory Committee (RAC), which was charged with evaluating the risks of recombinant DNA technology and establishing guidelines for recombinans DNA research. In 1976, the RAC published a set of auldelines for working with recombinant organisme the RAC continues to oversee gene cloning research, and compliance with RAC guidelines is mandatory fo; Scientists working with recombinant organisms, Transformation of Bacterial Cells and Antibiotic Selection of Recombinant Bacteria Cohen also made another important contribution to gene Cloning, which made the psciot cloning paberiments possible. His laboratory demonstrates how transformation, a process for inserting foreign BNA into bacteria, could be used to reliably inten {ice DNA into bacteria. Cohen discovered that Il he treated bacterial cells with calcium chloride. soln. jlans, added plasmids to cells chilled on ice, and then briefly heated the cell and DNA mixture, plasmids entered bacterial cells. Once inside bacteria, plastic, are replicated by the cell and their genes expressed. Transformation techniques {h greater detail later (Chapter 5). A more modern Hanslormation method, called electroporation, ‘volves applying a brief (mittisecond) pulse. af are are explained high-vollage electricity 10 create tiny: holes in tf bacterial cell wall that allow DNA to ente Peration can also be used to introduce DNA itll animatian cells and to transtorm plant cells Ligation of DNA fragments and transformation any method are somewhat inellicient During ligation create alte digested plasmid ligates back to its During ccCulatized plasmid that lacks foreign DN During transformation, » majority of cells do not 12 UP DNA. Now that we have seen how DNA call nserted into a vector and introduced into bactel eels ve ed oder how recombinant bacteria—oe vite with 2 Fecombinant plasmid—ean be & bacteria and to 2@'8° number af nontransfonm™ ria and bacterial cully that contain plasmid DN wou 1orcigh DNA. This screen > process is cll ication PCAUE iis designee oS ere fication nt (clectng ested to foititate th a 8 recombinant bacteria Preventing the s 7 eee OF (Selecting ayaien nonttal formed bacteria and i Teeting ayainst) ae ‘without foreign Dyn Cohen and poy. technique in whieh Plated on aga way to ig formed ¢ eteria that contain ph sca Sed antibiotic selection © lates w2hstormed: bacterial cells @ Plates with different antibiotics, on nant bacteria and nome Sats antibiotic velection 8 p Modern cloning teelnia™ Bes, sual ate Other Mo i White” demtify arate” Popular selection election (the rease" Y Soon be x scl iS cloney of vious). In blue-white 5 : strated jg tO 4 resitiction site in the 4 nodes gi 'N Figure 3.3. Recall that the 1 rarades the disacene’ ase (B-galy- an enzytm | 7 charides glucage Patience into the mone tional pS, UDted by ptt nt” "al Begal enzyme et at inserted gene, n9 TaNstormed. pq.) PFOduced : hat “4 bacier pat ble. 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