Dna cloning


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Dna cloning

  1. 1. DNA CLONINGINTRODUCTION TO DNADNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all otherorganisms. Nearly every cell in a person’s body has the same DNA. Most DNA is located in thecell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found inthe mitochondria (where it is called mitochondrial DNA or mtDNA).The information in DNA is stored as a code made up of four chemical bases: adenine (A),guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, andmore than 99 percent of those bases are the same in all people. The order, or sequence, of thesebases determines the information available for building and maintaining an organism, similar tothe way in which letters of the alphabet appear in a certain order to form words and sentences.DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Eachbase is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, andphosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiralcalled a double helix. The structure of the double helix is somewhat like a ladder, with the basepairs forming the ladder’s rungs and the sugar and phosphate molecules forming the verticalsidepieces of the ladder An important property of DNA is that it can replicate, or make copies ofitself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequenceof bases. This is critical when cells divide because each new cell needs to have an exact copy ofthe DNA present in the old cell.DNA is a double helix formed by base pairs attached to a sugar-phosphate backbone. 1 CMR COLLEGE OF PHARMACY
  2. 2. DNA CLONINGA modern working definition of a gene is "a locatable region of genomic sequence,corresponding to a unit of inheritance, which is associated with regulatory regions, transcribedregions, and or other functional sequence regions ". Colloquial usage of the term gene (e.g."good genes", "hair color gene") may actually refer to an allele: a gene is the basic instruction, asequence of nucleic acids (DNA or, in the case of certain viruses RNA), while an allele is onevariant of that gene. Referring to having a gene for a trait, is no longer the scientifically acceptedusage. In most cases, all people would have a gene for the trait in question, but certain peoplewill have a specific allele of that gene, which results in the trait variant.GENEA gene is a molecular unit of heredity in a living organism. It is a name given to some stretchesof DNA and RNA that code for a type of protein or for an RNA chain that has a function in theorganism. Living beings depend on genes, as they specify all proteins and functional RNAchains. Genes hold the information to build and maintain an organisms cells and pass genetictraits to offspring, although some organelles (e.g. mitochondria) are self-replicating and are notcoded for by the organisms DNA. All organisms have many genes corresponding to variousdifferent biological traits, some of which are immediately visible, such as eye color or number oflimbs, and some of which are not, such as blood type or increased risk for specific diseases, orthe thousands of basic biochemical processes that comprise life.RNA genes and genomesWhen proteins are manufactured, the gene is first copied into RNA as an intermediate product. Inother cases, the RNA molecules are the actual functional products. For example, RNAs known asribozymes are capable of enzymatic function, and microRNA has a regulatory role. The DNAsequences from which such RNAs are transcribed are known as RNA genes.Some viruses store their entire genomes in the form of RNA, and contain no DNA at all. Becausethey use RNA to store genes, their cellular hosts may synthesize their proteins as soon as they areinfected and without the delay in waiting for transcription. On the other hand, RNA retroviruses,such as HIV, require the reverse transcription of their genome from RNA into DNA before theirproteins can be synthesized. In 2006, French researchers came across a puzzling example ofRNA-mediated inheritance in mice. Mice with a loss-of-function mutation in the gene Kit havewhite tails. Offspring of these mutants can have white tails despite having only normal Kit genes.The research team traced this effect back to mutated Kit RNA.[3] While RNA is common asgenetic storage material in viruses, in mammals in particular RNA inheritance has been observedvery rarely. 2 CMR COLLEGE OF PHARMACY
  3. 3. DNA CLONINGFunctional structure of a geneThe vast majority of living organisms encode their genes in long strands of DNA. DNA(deoxyribonucleic acid) consists of a chain made from four types of nucleotide subunits, eachcomposed of: a five-carbon sugar (2-deoxyribose), a phosphate group, and one of the four basesadenine, cytosine, guanine, and thymine. The most common form of DNA in a cell is in a doublehelix structure, in which two individual DNA strands twist around each other in a right-handedspiral. In this structure, the base pairing rules specify that guanine pairs with cytosine andadenine pairs with thymine. The base pairing between guanine and cytosine forms threehydrogen bonds, whereas the base pairing between adenine and thymine forms two hydrogenbonds. The two strands in a double helix must therefore be complementary, that is, their basesmust align such that the adenines of one strand are paired with the thymines of the other strand,and so on.Due to the chemical composition of the pentose residues of the bases, DNA strands havedirectionality. One end of a DNA polymer contains an exposed hydroxyl group on thedeoxyribose; this is known as the 3 end of the molecule. The other end contains an exposedphosphate group; this is the 5 end. The directionality of DNA is vitally important to manycellular processes, since double helices are necessarily directional (a strand running 5-3 pairswith a complementary strand running 3-5), and processes such as DNA replication occur in onlyone direction. All nucleic acid synthesis in a cell occurs in the 5-3 direction, because newmonomers are added via a dehydration reaction that uses the exposed 3 hydroxyl as anucleophile.The expression of genes encoded in DNA begins by transcribing the gene into RNA, a secondtype of nucleic acid that is very similar to DNA, but whose monomers contain the sugar riboserather than deoxyribose. RNA also contains the base uracil in place of thymine. RNA moleculesare less stable than DNA and are typically single-stranded. Genes that encode proteins arecomposed of a series of three-nucleotide sequences called codons, which serve as the words inthe genetic language. The genetic code specifies the correspondence during protein translationbetween codons and amino acids. The genetic code is nearly the same for all known organisms. 3 CMR COLLEGE OF PHARMACY
  4. 4. DNA CLONINGThis above diagram shows a gene in relation to the double helix structure of DNA and to a chromosome(right). The chromosome is X-shaped because it is dividing. Introns are regions often found in eukaryotegenes that are removed in the splicing process (after the DNA is transcribed into RNA): Only the exonsencode the protein. This diagram labels a region of only 50 or so bases as a gene. In reality, most genesare hundreds of times larger.All genes have regulatory regions in addition to regions that explicitly code for a protein or RNAproduct. A regulatory region shared by almost all genes is known as the promoter, whichprovides a position that is recognized by the transcription machinery when a gene is about to betranscribed and expressed. A gene can have more than one promoter, resulting in RNAs thatdiffer in how far they extend in the 5 end.[4] Although promoter regions have a consensussequence that is the most common sequence at this position, some genes have "strong" promotersthat bind the transcription machinery well, and others have "weak" promoters that bind poorly.These weak promoters usually permit a lower rate of transcription than the strong promoters,because the transcription machinery binds to them and initiates transcription less frequently.Other possible regulatory regions include enhancers, which can compensate for a weak promoter.Most regulatory regions are "upstream"—that is, before or toward the 5 end of the transcriptioninitiation site. Eukaryotic promoter regions are much more complex and difficult to identify thanprokaryotic promoters.Many prokaryotic genes are organized into operons, or groups of genes whose products haverelated functions and which are transcribed as a unit. By contrast, eukaryotic genes aretranscribed only one at a time, but may include long stretches of DNA called introns which are 4 CMR COLLEGE OF PHARMACY
  5. 5. DNA CLONINGtranscribed but never translated into protein (they are spliced out before translation). Splicing canalso occur in prokaryotic genes, but is less common than in eukaryotes.ChromosomesThe total complement of genes in an organism or cell is known as its genome, which may bestored on one or more chromosomes; the region of the chromosome at which a particular gene islocated is called its locus. A chromosome consists of a single, very long DNA helix on whichthousands of genes are encoded. Prokaryotes—bacteria and archaea—typically store theirgenomes on a single large, circular chromosome, sometimes supplemented by additional smallcircles of DNA called plasmids, which usually encode only a few genes and are easilytransferable between individuals. For example, the genes for antibiotic resistance are usuallyencoded on bacterial plasmids and can be passed between individual cells, even those ofdifferent species, via horizontal gene transfer. Although some simple eukaryotes also possessplasmids with small numbers of genes, the majority of eukaryotic genes are stored on multiplelinear chromosomes, which are packed within the nucleus in complex with storage proteinscalled histones. The manner in which DNA is stored on the histone, as well as chemicalmodifications of the histone itself, are regulatory mechanisms governing whether a particularregion of DNA is accessible for gene expression. The ends of eukaryotic chromosomes arecapped by long stretches of repetitive sequences called telomeres, which do not code for anygene product but are present to prevent degradation of coding and regulatory regions duringDNA replication. The length of the telomeres tends to decrease each time the genome isreplicated in preparation for cell division; the loss of telomeres has been proposed as anexplanation for cellular senescence, or the loss of the ability to divide, and by extension for theaging process in organisms.Whereas the chromosomes of prokaryotes are relatively gene-dense, those of eukaryotes oftencontain so-called "junk DNA", or regions of DNA that serve no obvious function. Simple single-celled eukaryotes have relatively small amounts of such DNA, whereas the genomes of complexmulticellular organisms, including humans, contain an absolute majority of DNA without anidentified function.[7] However it now appears that, although protein-coding DNA makes upbarely 2% of the human genome, about 80% of the bases in the genome may be expressed, so theterm "junk DNA" may be a misnomer.VECTORA vector is a DNA molecule used as a vehicle to transfer foreign genetic material into anothercell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes. 5 CMR COLLEGE OF PHARMACY
  6. 6. DNA CLONINGCommon to all engineered vectors are an origin of replication, a multicloning site, anda selectable marker.The vector itself is generally a DNA sequence that consists of an insert (transgene) and a largersequence that serves as the "backbone" of the vector. The purpose of a vector which transfersgenetic information to another cell is typically to isolate, multiply, or express the insert in thetarget cell. Vectors called expression vectors (expression constructs) specifically are for theexpression of the transgene in the target cell, and generally have a promoter sequence that drivesexpression of the transgene. Simpler vectors called transcription vectors are only capable ofbeing transcribed but not translated: they can be replicated in a target cell but not expressed,unlike expression vectors. Transcription vectors are used to amplify their insert.Insertion of a vector into the target cell is usually called transformation for bacterialcells, transfection for eukaryotic cells, although insertion of a viral vector is oftencalled transductionCharacteristicsTwo common vectors are plasmids and viral vectors.PlasmidsPlasmids are double-stranded generally circular DNA sequences that are capable ofautomatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin ofreplication that allows for semi-independent replication of the plasmid in the host and also thetransgene insert. Modern plasmids generally have many more features, notably including a"multiple cloning site" which includes nucleotide overhangs for insertion of an insert, andmultiple restriction enzyme consensus sites to either side of the insert. In the case of plasmidsutilized as transcription vectors, incubating bacteria with plasmids generates hundreds orthousands of copies of the vector within the bacteria in hours, and the vectors can be extractedfrom the bacteria, and the multiple cloning site can be cut by restriction enzymes to excise thehundredfold or thousandfold amplified insert. These plasmid transcription vectorscharacteristically lack crucial sequences that code for polyadenylation sequences and translationtermination sequences in translated mRNAs, making protein expression from transcriptionvectors impossible. plasmids may be conjugative/transmissible and non-conjugative: conjugative: mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids. nonconjugative- do not mediate DNA through conjugation, e.g., many R and col plasmids. 6 CMR COLLEGE OF PHARMACY
  7. 7. DNA CLONINGViral vectorsViral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNAthat has been rendered noninfectious, but still contain viral promoters and also the transgene,thus allowing for translation of the transgene through a viral promoter. However, because viralvectors frequently are lacking infectious sequences, they require helper viruses or packaginglines for large-scale transfection. Viral vectors are often designed for permanent incorporation ofthe insert into the host genome, and thus leave distinct genetic markers in the host genome afterincorporating the transgene. For example, retroviruses leave a characteristic retroviralintegration pattern after insertion that is detectable and indicates that the viral vector hasincorporated into the host genomeTYPES OF VECTORS 1. Plasmids: Have a capacity of 15 kb. 2. Phage (lambda)s: Have a capacity of 25 kb. 3. Cosmids or Fosmids: Have a capacity of 35-45 kb. 4. Bacterial artificial chromosomes (BAC)(P-1 derived): Have a capacity of 50-300 kb. 5. Yeast artificial chromosomes (YAC): Have a capacity of 300- >1500 kb. 6. Human artificial chromosomes (HAC): Have a capacity of >2000 kb.INTRODUCTION TO CLONING-:Cloning is the process of moving a gene from the chromosome it occurs in naturally to anautonomously replicating vector. In the cloning process, the DNA is removed from cells,manipulations of the DNA are carried out in a test-tube, and the DNA is subsequently put backinto cells. Because E. coli is so well characterized, it is usually the cell of choice formanipulating DNA molecules. Once the appropriate combination of vector and cloned DNA orconstruct has been made in E. coli, the construct can be put into other cell types.The following steps in the cloning process:1 How is the DNA removed from the cells?2 How is the DNA cut into pieces?3 How are the pieces of DNA put back together?4 How do we monitor each of these steps?Isolating DNA from cellsPlasmid DNA isolation 7 CMR COLLEGE OF PHARMACY
  8. 8. DNA CLONINGThe first step in cloning is to isolate a large amount of the vector and chromosomal DNAs.Isolation of plasmid DNA will be examined first. In the general scheme, cells containing theplasmid are grown to a high cell density, gently lysed, and the plasmid DNA is isolated andconcentrated. When the cells are growing, the antibiotic corresponding to the antibioticresistance determinant on the plasmid is included in the growth media. This ensures that themajority of cells contain plasmid DNA. Without the antibiotic selection, an unstable plasmid (i.e.one without a par function) can be lost from the cell population in a few generations.Cells can be lysed by several different methods depending on the size of the plasmid molecule,the specific strain of E. coli the plasmid will be isolated from, and how the plasmid DNA will bepurified. Most procedures use EDTA to chelate the Mg++ associated the outer membrane anddestabilize the outer membrane. Lysozyme is added to digest the peptidoglycan and detergentsare frequently used to solubilize the membranes. RNases are added to degrade the large amountof RNA found in actively growing E. coli cells. The RNase gains access to the RNA after theEDTA and lysozyme treatments. This mixture is centrifuged to pellet intact cells and large piecesof cell debris. The supernatant contains a mixture of soluble cell components, including theplasmid, and is known as a lysate. The methods used to purify the plasmid DNA from the celllysate rely on the small size and abundance of the plasmid DNA relative to the chromosome, andthe covalently closed circular nature of plasmid DNA. Most plasmids exist in the cytoplasm ofthe cell as circular DNA molecules that are highly supercoiled. The lysate is treated with sodiumhydroxide to denature all of the DNA, and with detergent, SDS. The pH is then abruptly lowered,causing the SDS to precipitate and bring with it denatured chromosomal DNA, membranefragments, and other cell debris. Most of the plasmid DNA reanneals to form dsDNA becauseeach strand is a covalently closed molecule and the two strands are not physically separated fromeach other. The small size of the plasmid allows the plasmid molecules to remain in suspension.The supernatant, which contains plasmid DNA, proteins, and other small molecules can betreated in a number of different ways to purify the plasmid. The most common protocol relies ona column resin that binds DNA. A small amount of the resin is mixed with the plasmid-containing supernatant and the plasmid-bound resin is collected in a small column.The remaining cell components are washed away and the plasmid is eluted from the resin. Thisprocedure is quick, simple, and reliable and can be easily carried out on a large number ofsamples. Many modifications of this procedure have been devised.Chromosomal DNA isolationTo isolate chromosomal DNA, cells are lysed in much the same way as for plasmid DNAisolation. The cell lysate is extracted with phenol or otherwise treated to remove all of theproteins. The chromosomal DNA is precipitated as long threads. The chromosomal DNA is very 8 CMR COLLEGE OF PHARMACY
  9. 9. DNA CLONINGfragile and breaks easily. For these reasons, the chromosomal DNA is not usually purified usingcolumns. Rather, the precipitated threads are collected by centrifugation.The discovery of restriction enzymesRestriction enzymes were discovered in E. coli in the 1950s by scientists studying bacteriophage.Bacteriophage l can be grown on an E. coli K12 strain and titered on E. coli K12 to determinethe number of phage per milliliter. A hightiter phage lysate will contain approximately 1010plaque forming units per ml (pfu/ml). If this phage lysate is titered on an E. coli B strain, the titerwill drop to 106 pfu/ml. One of the phage that forms a plaque on E. coli B can be used to make ahigh-titer lysate on E. coli B.The lysate grown on E. coli B will titer on E. coli B atapproximately 1010 pfu/ml but will titer on E. coli K12 at 106pfu/ml. This four-log drop inplating efficiency can be traced to genes encoded by the bacterial chromosome of each strain.The system is known as host restriction and modification. Host restriction is carried out by arestriction endonuclease and modification is carried out by the protein that modifies therestriction site.Cutting DNA moleculesOnce DNA has been purified, it must be cut into pieces before the chromosomal DNA and theplasmid DNA can be joined. The problem is to cut the DNA so that it will be easy to join the cutends of the chromosomal DNA to the cut ends of the plasmid DNA. A group of enzymes, calledrestriction enzymes, are used for this purpose. Restriction enzymes are isolated from differentbacterial species.Bacteria use restriction enzymes and modification enzymes to identify their own DNA from anyforeign DNA that enters their cytoplasm. The restriction part of the system is an enzyme thatrecognizes a specific DNA sequence or restriction site and cleaves the DNA by catalyzingbreaks in specific phosphodiester bonds. The cleavageis on both strands of the DNA so that adouble-stranded break is made. The modification part of the system is a protein that recognizesthe same DNA sequence as the restriction enzyme. The modification enzyme methylates theDNA sequence so that the restriction enzyme no longer recognizes the sequence. Thus, thebacteria can protect its own DNA from the restriction enzyme. Any DNA that enters the bacteriaand contains the unmethlyated restriction site is cut and degraded. There are three types ofrestriction–modification systems (Table 14.1). The types are distinguished based on the numberof proteins in the system, the cofactors for these proteins, and if the proteins form a complex. 9 CMR COLLEGE OF PHARMACY
  10. 10. DNA CLONINGType I restriction–modification systemsType I systems are the most intricate and very few of them have been described. Three differentproteins form a complex that carries out both restriction and modification of the DNA. Thecomplex must interact with a cofactor, S-adenosylmethionine, before it is capable of recognizingDNA. The S-adenosylmethionine is the methyl donor for the modification reaction and allknown Type I systems methylate adenine residues on both strands of the DNA. The restrictionreaction requires ATP and Mg++ for cleavage of the DNA. The complex also has topoisomeraseactivity. The DNA sequence recognized by Type I enzymes is also complex. The sequence isasymmetric and split into two parts (Fig. 14.1a). The first part is a 3 bp sequence, next is a 6–8bpspacer of nonspecific sequence, and finally there is a 4–5 bp sequence. Cleavage of the DNAoccurs randomly, usually no closer than 400 bp from the recognition sequenceand sometimes as far away as 7000 bp.Fig. 14.1 The sequences recognized by restriction enzymes. (a) Type I restrictionenzyme sites. (b) Type II restriction enzyme sites. (c) Type III restriction enzyme sites. 10 CMR COLLEGE OF PHARMACY
  11. 11. DNA CLONINGType II restriction–modification systemsType II systems are composed of two independent proteins. One protein is responsiblefor modifying the DNA and one for restricting the DNA. Modification of the DNA uses S-adenosylmethionine as the methyl donor. The Type II modification enzymes methylate the DNAat one of three places, with each specific modification enzyme methlyating the same residueevery time. The modifications that have been found are 5-methlycytosine, 4-methylcytosine, or6-methlyadenosine. The DNA sequence recognized by Type II restriction enzymes is symmetricand usually palindromic (Fig. 14.1b). The DNA sequence is between 4 and 8 bp in length, withmost restriction enzymes recognizing 4 or 6 bp. Both the cleavage of the DNA and modificationof the DNA occur symmetrically on both strands of the DNA within the recognition sequence.Restriction enzymes function as dimers of a single protein so that each protein monomer caninteract with one strand of the DNA. Thus, both strands of the DNA are cleaved at the sametime, generating a double-stranded break. Several thousand Type II systems have been identified.Type II restriction enzymes are the most useful for cloning because they generate DNAmolecules with a specific sequence on the ends (Fig. 14.2).Fig. 14.2 Type II restriction enzymes generate DNA molecules with specific sequences on bothends. These ends can be rejoined to regenerate the restriction site 11 CMR COLLEGE OF PHARMACY
  12. 12. DNA CLONINGType IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than onesubunit. They cleave DNA on both sides of their recognition to cut out the recognition site. Theyrequire both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleaveDNA following interaction with two copies of their recognition sequence.One recognition siteacts as the target for cleavage, while the other acts as an allosteric effector that speeds up orimproves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restrictionendonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleaveboth sequences at the same time. Type IIG restriction endonucleases (Eco57I) do have a singlesubunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylatedDNA. Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from theirnon-palindromic asymmetric recognition sites. These enzymes may function as dimers.Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two differentsubunits. Some recognize palindromic sequences while others have asymmetric recognition sites.Restriction–modification as a molecular toolType II restriction enzymes have several useful properties that make them suitable for cuttingDNA molecules into pieces for cloning experiments. First, most cloning ex-periments requiremanipulation of DNA molecules in a test-tube. The fact that the Type II restriction enzymes are asingle polypeptide aids in the purification of the enzyme for in vitro work. Second, Type IIrestriction enzymes recognize and cleave DNA at a specific sequence. The cleavage is on bothstrands of the DNA and results in a double- stranded break. Cleavage of the DNA leaves one ofthree types of ends, depending upon the specific restriction enzyme (Fig. 14.3a). Some enzymesleave a 5¢ overhang, some a 3¢ overhang, and some leave blunt ends. The ends with either a 12 CMR COLLEGE OF PHARMACY
  13. 13. DNA CLONING5¢ or 3¢ overhang are known as sticky ends. Any blunt end can be joined to any other blunt endregardless of how the blunt end was generated. Sticky ends can be joined to other sticky ends,provided that either the same Type II restriction enzyme was used to generate both sticky endsthat are to be joined or that the bases in the overhang are identical and have the correct overhang(Fig. 14.3b).Fig. 14.3 Cleavage of DNA by a Type II restriction enzyme leaves one of three types of ends,depending on the enzyme used. (a) The ends generated can be blunt ends, sticky ends with a 5¢overhang, or sticky ends with a 3¢ overhang. (b) The sticky ends from two molecules cut withtwo different restriction enzymes can be joined if the overhangs can hybridize. In this example,the hybrid site formed is no longer a substrate for either enzyme.Type III restriction–modification systemsType III systems are composed of two different proteins in a complex. The complex isresponsible for both restriction and modification. Modification requires S-adenosylmethionine, isstimulated by ATP and Mg++, and occurs as 6-methyladenine. Type III modification enzymesonly modify one strand of the DNA helix. Restriction requires Mg++ and is stimulated by ATPand S-adenosylmethionine. The recognition sites for Type III enzymes are asymmetric and 5–6bp in length. The DNA is cleaved on the 3¢ side of the recognition sequence, 25–27 bp away 13 CMR COLLEGE OF PHARMACY
  14. 14. DNA CLONINGfrom the recognition sequence. Type III restriction enzymes require two recognition sites ininverted orientation in order to cleave the DNA (Fig. 14.1c).Naming restriction enzymesRestriction enzymes are named for the species and strain inwhich they are first identified.Forexample, BamHI was the first enzyme found in Bacillusamyloliquefaciens H. ClaI wasthe firstrestriction enzymefound in Caryophanon latum.Because the enzymes are named after the speciesthey come from, the first three letters in the restriction enzyme are always italicized. Somespecies encode more than one restriction enzyme in their genome. Hence, the names, DraI andDraIII or DpnI and DpnII.Generate double-stranded breaks in DNA by shearing the DNAUsually restriction enzymes are used to cut the chromosomal DNA for cloning. The drawback ofthis approach is uncovered when the gene contains a restriction site for the enzyme being used toconstruct the library. One way to solve this problem is to only partially digest the chromosomalDNA with the restriction enzyme. Another way is to shear the chromosomal DNA and clone therandomly sheared DNA into a blunt end restriction enzyme site in the vector. One way to shearchromosomal DNA is by passing it quickly through a small needle attached to a syringe.Joining DNA moleculesAs described above, both plasmid and chromosomal DNA can be independently isolated fromcells and digested with restriction enzymes. If, however, DNA with doublestranded ends issimply transformed back into E. coli, E. coli will degrade it. The double-stranded ends must becovalently attached. A version of this reaction is normally carried out in the cell by an enzymeknown as DNA ligase. During DNA replication, the RNA primers are replaced by DNA (seeChapter 2). At the end of this process, there is a nick in the DNA that is sealed by DNA ligase.The double-stranded break formed by the restriction enzyme can be thought of as two nicks, eachof which is a substrate for ligase.If a plasmid molecule that has been digested with a restriction enzyme is subsequently treatedwith ligase, the plasmid moleculeends can be covalently closed by ligase (Fig. 14.4). Ligation isan energy-requiring reaction that occurs in three distinct steps. In the first step, the adenylylgroup from ATP is covalently attached to ligase and inorganic phosphate is released. Next, theadenylyl group is transferred from ligase to the 5¢ phosphate of the DNA in the nick. Lastly, thephosphodiester bond is formed when the 3¢ OH in the nick attacks the activated 5¢ phosphate.AMP is released in the process. Because of this mechanism, ligase requires both a 3¢ OH and a5¢ phosphate. If the 5¢ phosphate is missing (see below), the nick cannot be sealed by ligase.Fig. 14.4 The ends of DNA molecules can be joined and the phosphate backbone of the DNAreformed by an enzyme called DNA ligase. Ligase uses three steps to reform the backbone. (Step 14 CMR COLLEGE OF PHARMACY
  15. 15. DNA CLONING1) A ligase molecule and an ATP molecule interact and the adenylyl group of ATP is covalentlyattached to the amine group of a specific lysine residue in the ligase protein. (Step 2) Theadenylyl group is transferred to the 5¢ phosphate in the nicked DNA. (Step 3) The 3¢ OH attacksthe activated 5¢ phosphate, reforming the backbone and releasing AMP.Manipulating the ends of moleculesWhile the goal of cloning is to construct a vector carrying the piece of DNA of interest, thereactions of the cloning process are concerned with the DNA ends. How the DNA ends areformed is very important because it dictates if the ends can be ligated to form the desiredconstruct. The physical state of the DNA ends can be manipulated in vitro to influence theligation reaction (Fig. 14.5a). For example, blunt ends cannot be ligated to sticky ends. Thesticky ends can be made into blunt ends by the reaction of sev-eral different enzymes. If thesticky end contains a 5¢ overhang, then any one of several different DNA polymerases can beused to add the missing bases to the 3¢ OH using the 5¢ overhang as a template. A 3¢ overhangcannot be filled in, rather the overhang must be removed. Many DNA polymerases have a 3¢ to5¢ exonuclease activity and this activity can be used to remove 3¢ overhangs. The 5¢ phosphatecan also be manipulated (Fig. 14.5b). If DNA molecules are missing the 5¢ phosphate, thephosphate can be added by an enzyme called T4 polynucleotide kinase. T4 polynucleotide kinaseis an ATP-requiring enzyme that was originally identified in the bacteriophage T4. Moleculesthat have been phosphorylated by T4 polynucleotide kinase can be ligated to other molecules byligase. When pieces of chromosomal DNA are mixed with cut vector DNA, ligation of severaldifferent molecules can take place. The vector DNA ends can be ligated to reform the vector, theends of a piece of chromosomal DNA can be ligated to each other, the ends of several vector orseveral chromosomal molecules can be ligated, or the ends of a piece of chromosomal DNA canbe ligated to the ends of a piece of vector DNA. The ligation mix is usually put back into cells bytransformation. And the antibiotic marker on the vector is selected for. Only cells transformed by 15 CMR COLLEGE OF PHARMACY
  16. 16. DNA CLONINGmolecules that contain vector DNA will form colonies. Of the molecules in the ligation mix, onlyreligated vector DNA or vector DNA with a chromosomal insert are a possibility in thetransformants. To reduce the number of vector molecules that are relegated without achromosomal insert, the 5¢ phosphates on the vector can be removed by anenzyme known as a phosphatase. The ends of vector molecules that have beendephosphorylated (the 5¢ phosphate has been removed) can only be ligated to chromosomalDNA molecules that have 5¢ phosphates (Fig. 14.5b). These molecules still havea nick on each strand but this nick can be sealed inside the cell.Fig. 14.5 The ends of DNA molecules can be manipulated in vitro to meet the requirements ofthe experiment. (a) 5¢ overhangs can be filled in by DNA polymerase and 3¢ overhangs can beremoved by the 3¢ to 5¢ exonuclease activity of DNA polymerase. (b) 5¢ phosphates arerequired for ligase to function. If the 5¢ phosphate is missing, ligase cannot seal the nick.Phosphates can be removed by phosphatase and added by T4 polynucleotide kinase. Somecloning strategies take advantage of this by removing the 5¢ phosphates from the vector so that itcannot re-ligate without an insert. The insert allows two of the four nicks to be sealed. Thismolecule is stable enough to be transformed into cells where the other two nicks will be sealed.Visualizing the cloning processAt each step of the cloning process, what is happening to the DNA molecules in the test-tube canbe monitored using a technique called gel electrophoresis. In this technique, a gel (Fig. 14.6) 16 CMR COLLEGE OF PHARMACY
  17. 17. DNA CLONINGcontaining small indentations or wells is cast. The DNA is loaded into the wells and the gel isplaced in an electric current. Because DNA is negatively charged, it will move in the gel towardsthe positive pole. The DNA migrates or moves in the electric current based on size and shape.The larger a DNA molecule, the slower it moves. The more compact, or supercoiled a piece ofDNA, the faster it moves.Fig. 14.6 A diagram of an agarose gel after electrophoresis and staining of the DNA with afluorescent dye such as ethidium bromide. Lane 1 contains supercoiled, open circular and linearDNA forms of the same plasmid. Supercoiled DNA runs faster because of its topology. Lane 2contains a 5kb supercoiled plasmid and lane 3 contains a 10 kb supercoiled plasmid. Lanes 4and 5 contain DNA fragments of different sizes. The band in lane 4 is larger than the bands inlane 5, meaning that the larger band contains more base pairs. Lane 6 contains DNA fragmentsof known molecular weights (molecular weight standards). Note that the molecular weightstandards cannot be used to predict the sizes of supercoiled DNA.The gel can be made from several different polymers, depending on the specifics of theexperiment. Agarose forms a matrix that will separate DNA molecules from ~500bp up to entirechromosomes (several million base pairs). If an electric current isconstantly applied to anagarose gel from only one direction, agarose gels will separate DNA from ~500bp to ~25,000 bp.If the direction and the timing of the current are varied over the electrophoresis time, then entirechromosomes can be separated in agarose. An alternative polymer, polyacrylamide, can be usedto separate molecules a few base pairs in length to approximately 1000 bp. 17 CMR COLLEGE OF PHARMACY
  18. 18. DNA CLONINGOnce the DNA has been separated in the gel, the gel is immersed in a solution containingethidium bromide. If ultraviolet light is used to illuminate the gel, the ethidium bromide that isbound to the DNA will fluoresce, indicating the presence of bands of DNA (Fig. 14.6). Eachband is composed of DNA molecules that are similar in size and shape. For example, whenplasmid DNA is extracted from the cell, the majority of it is supercoiled. Supercoiled DNAmigrates very fast in an agarose gel (Fig. 14.6, lane 1). Some of the DNA will get nicked in theprocess of being extracted. The nick allows all of the supercoils to be removed, resulting in anopen circle. Open circles migrate slower than supercoiled DNA. Some of the DNA will have adouble-stranded break after isolation and the resulting molecules are linear. Linear DNAmigrates the slowest of the three forms. If two different plasmids, one 5 kb and one 10 kb areisolated and run in an agarose gel, the supercoiled 5 kb plasmid will migrate faster than the 10kbsupercoiled plasmid (Fig. 14.6, lanes 2, 3). Likewise, the open circle 5kb plasmid speciesmigrates faster than open circle 10 kb plasmid species and the 5 kb linear species will migratefaster than the 10 kb linear species.If a circular plasmid DNA is digested with a restriction enzyme that has two recognitionsites in the plasmid, two linear pieces of DNA will result (Fig. 14.7). The shorter piece willmigrate faster than the longer piece. If the DNA starts as a linear molecule and is digested with arestriction enzyme that recognizes the DNA in two places, then the DNA will be cut into threepieces (Fig. 14.7). Once all of the molecules are linear, they will migrate in the agarose gel basedmainly on size.Fig. 14.7 The number of bands a molecule is cut into depends on if the starting molecule islinear or circular. Lane 1, molecular weight standards. Lane 2, bands from a cut circularmolecule. Lane 3, bands form the same molecule as in lane 2 except the starting molecule waslinear and not circular. 18 CMR COLLEGE OF PHARMACY
  19. 19. DNA CLONINGFig. 14.8 The steps in a Southern blot. (a) DNA (usually chromosomal DNA) is digested withdifferent restriction enzymes and run on a gel. The DNA is cut in many different places and leadsto many fragments of different sizes. (b) The DNA is transferred from the gel to a nylon ornitrocellulose filter. (c) A fragment of DNA that contains the gene of interest is tagged andused as the probe. The probe is added to the membrane containing DNA and allowed to hybridizeto any DNA fragment on the membrane to which it has homology. Excess probe is washed awayand the probe is detected. If any fragments with homology are present, the size of the fragmentscan be determined based on where probe is detected.Constructing libraries of clonesSometimes it is desirable to make clones of all of the genes from an organism and subsequentlyto fish out the clone of interest. A large group of clones that contains all of the pieces of achromosome on individual vector molecules is known as a library. To construct a library, vectorDNA is isolated and digested with a restriction enzyme that only cuts the vector once.Chromosomal DNA is isolated and digested with the same restriction enzyme. The cutchromosomal DNA and the cut vector DNA are mixed together and treated with ligase. Thismixture is transformed into E. coli and plated on agar containing the antibiotic that correspondsto the antibiotic resistance determinant on the vector. E. coli cells that are capable of formingcolonies must either contain the vector without a chromosomal DNA insert or a vector with achromosomal DNA insert. If the correct ratio of chromosomal DNA to vector DNA is used(usually two chromosomal molecules for every one vector molecule), rarely does a vector havetwo distinctpieces of chromosomal DNA inserted into it. Every clone should carry a unique pieceof chromosomal DNA. If enough independent colonies are isolated, the entire sequence of thechromosome should be represented by the population of colonies. Libraries can be made fromany kind of DNA, regardless of species. How many independent clones are needed so that the 19 CMR COLLEGE OF PHARMACY
  20. 20. DNA CLONINGlibrary contains the entire genome? This depends on several factors and can be calculated usingthe formula:where:P = probability of any unique sequence being present in the library.N = number of independent clones in the library.F = fraction of the total genome in each clone (size of average insert/total genomesize). For example, a library with ~10 kb inserts is made from E. coli, which has a genomesize of 4639 kb. To ensure that any given E. coli gene has a 99% probability of being ona clone in the library would require 2302 independent clones.DNA detection—Southern blottingIn 1975, E.M. Southern described a technique to detect sequence homology betweentwo molecules, without determining the exact base sequence of the molecules (Fig. 14.8). Thetechnique relies on fractionating the DNA on an agarose gel and denaturingthe fractionated DNA in the agarose. The denatured DNA is transferred to a solid support, suchas a nylon or nitrocellulose filter. A second DNA, called the probe, is labeled with a tag,denatured, and applied to the filter. Probes can be tagged with radioactivity and detected with X-ray film. They can also be labeled with fluorescent nucleotides or enzymes such as alkalinephosphatase or horseradish peroxidase. The enzymes are then detected with special substratemolecules that change color or emit light when cleaved by the enzyme. The probe will hybridizewith any DNA on the filter that has complementary base sequences. Once the excess, non-hybridized probe is washed away, the tag attached to the probe can be detected.Southern blotting can be used in many types of experiments. For example, if you have a cloneof your favorite gene and you want to know if your gene exists in other species. You can isolatethe chromosomal DNA from all of the species you want to test, digest the DNA with one orseveral restriction enzymes, and prepare a Southern blot. The clone of your favorite gene is usedas the probe. If another species has sequence homology to your gene, then the bandcorresponding to the fragment containing the homology will be detected. The different species 20 CMR COLLEGE OF PHARMACY
  21. 21. DNA CLONINGcan be as diverse as bacteria, yeast, mice, rats, plants, humans, or as simple as several differenttypes of bacteria. Blots with many different species of DNA included have become known aszoo blots!A version of Southern blots can be used to identify clones from other species that are related toany gene of interest (Fig. 14.9). This technique is known as colony blotting. A population ofcells containing a library from another species is plated on several agar plates and the cells areallowed to grow into colonies. The colonies are transferred to either a nylon or nitrocellulosefilter and lysed on the membrane. The DNA from the lysed colonies is attached to the membraneby either UV crosslinking for nylon or heat for nitrocellulose. A plasmid carrying the gene ofinterest is labeled with a tag and used as the probe. The labeled probe will hybridize with DNAfrom a lysed colony only if the colony carries a clone that contains complementary sequences.Once the appropriate clone is identified, that colony from the agar plate is purified and used as asource of the cloned gene of interest. For example, a cloned yeast gene can be used to fish out ofa human library a related human clone.Fig. 14.9 A diagram of a colony blot. (a) The cells to be tested are plated on agar plates andincubated until they form colonies. (b) The colonies are transferred to a filter and lysed, releasingtheir DNA. The DNA is attached to the filter. (c) A tagged probe is added to the filters and thecolonies that carry DNA that is homologous to the probe can be identified. 21 CMR COLLEGE OF PHARMACY
  22. 22. DNA CLONINGDNA amplification— polymerase chain reactionIn 1993, the Nobel Prize in Chemistry was awarded for a novel and extremely importantdevelopment called polymerase chain reaction (PCR, Fig. 14.10a). PCR allows almost any pieceof DNA to be amplified in vitro. Normally DNA replication requires an RNA primer, a DNAtemplate, and DNA polymerase. PCR uses the DNA template, two DNA primers, and a uniqueDNA polymerase isolated from a bacterium that grows at 70°C. The unique properties of thispolymerase are that, unlike most DNA polymerases, it is capable of synthesizing DNA at 70°Cand it is stable at even higher temperatures. In PCR, many cycles of DNA synthesis are carriedout and these cycles are staged by controlling the temperature that the reaction takes place. Forexample, the DNA template is double-stranded DNA and the two strands must be separatedbefore any DNA synthesis can take place. The PCR reaction mix is first placed at 90–95°C tomelt the DNA. The temperature is lowered to ~40–55°C to allow the primer to anneal to thesingle-stranded template. Finally, the temperature is raised to 70°C to allow the DNA to besynthesized from the primer. This cycle of melting the template, annealing the primers, andsynthesizing the DNA is repeated between 25 and 35 times for each PCR reaction.What happens to a template molecule in each cycle of DNA synthesis? In the first cycle, the twostrands of the template separate, one primer anneals to each strand and two dsDNA moleculesare produced (Fig. 14.10b). One strand of each dsDNA molecule is synthesized only from theprimer to the end of the template. In the second cycle, all four strands are used as templates. FourdsDNA molecules are produced, two are similar to the dsDNA molecules produced in the firstcycle and the other two have three out of the four DNA ends delineated by the primers. In thethird cycle, the eight strands are used as templates and eight dsDNA molecules are produced.Two of the dsDNA molecules are similar to the products produced in cycle one, four of thedsDNA molecules have three out of four ends delineated by the primers, and the other twomolecules now have all four ends delineated by the primer. In the remaining cycles, the numberof dsDNA molecules continues to increase linearly. In cycle 4, 8/16 molecules have all four endsdelineated by the two primers, in cycle 5, 24/32, cycle 6, 56/64, cycle 7, 120/128, and cycle 8,248/256. After 35 cycles, the dsDNA molecule with all four ends delineated by the primers is thepredominant molecule in the PCR reaction mix. The DNA primers used in PCR are chosen sothat the piece of DNA of interest is amplified. The DNA primers are synthesized in vitro. Bycarefully choosing primers, the exact base pairs at either end of the amplified fragment can bepredetermined. The template DNA can be from any source. Only a small amount of template isneeded. Once a fragment of DNA has been amplified, it can be cloned into an appropriate vector,used as a probe, restriction mapped, or used in a number of other techniques. The DNAreplication that takes place in PCR, like in vivo DNA replication, is not 100% accurate.Occasionally, a mistake is made. If the amplified fragment is to be cloned, the resulting clonesmust be sequenced to ensure that they carry a wild-type copy of the gene. If the amplifiedfragment is to be used as a probe, a few mutant copies in a mixture that contains a large number 22 CMR COLLEGE OF PHARMACY
  23. 23. DNA CLONINGof wild-types copies will not present a problem. Thus, depending on the use of the PCRfragment, these contaminating mutant copies of the fragment must be accounted for.Fig. 14.10 A diagram of the polymerase chain reaction. (a) The general reaction taking place ineach cycle. The template must be denatured, the primers annealed, and the DNA synthesized.(b) The fate of the template molecules in the first three cycles. The circle and triangle attachedto the ends of the molecules represent the DNA ends that are delineated by the primers. (c)How many molecules of each type are formed in successive cycles. There will always be twomolecules that match the ones formed in cycle 1. The number of molecules with three of thefour ends matching the primer ends increases by two in each successive cycle. The number ofmolecules with all four ends determined by the primers is amplified dramatically. At the end of35 cycles, >99% of the DNA molecules will have all four ends specified by the primers. 23 CMR COLLEGE OF PHARMACY
  24. 24. DNA CLONINGFig. 14.11 PCR can be used to add specific sequences to the end of a DNA molecule. Thesespecific sequences can be restriction enzymes sites or any other sequence of interest. In cycle 1,the added bases simply do not hybridize to anything. In cycle 2, the added bases are replicated,effectively making them a part of the PCR products. In subsequent cycles, the added sequenceswill be replicated as part of the DNA molecules. 24 CMR COLLEGE OF PHARMACY
  25. 25. DNA CLONINGSite-directed mutagenesis using PCRPCR can be used to introduce a specific mutation into a specific base pair in a cloned gene (Fig.14.12). A primer is designed that contains the mutant base pair in place of the wild-type basepair. This mutant primer is used in a PCR reaction with the plasmid containing the cloned gene.PCR is used to synthesize the entire plasmid and after several rounds of replication, the largemajority of plasmid molecules contain the mutation. This mixture of mutant and wild-typemolecules is transformed into E. coli and the plasmids in individual colonies are tested for thepresence of the mutation by determining the DNA sequence of the cloned gene.Fig. 14.12 One strategy for site-directed mutagenesis using PCR. Primers are designed tosynthesize both strands of the plasmid containing the sequence to be mutagenized. Included in theprimers are the base changes to be incorporated into the final mutant product. These primers areused in a PCR reaction to synthesize both strands of the plasmid with the incorporated change.Amplifying DNA using PCR: functional uses of PCRAt its most basic level, PCR is used to amplify a small amount of DNA into a large amount ofDNA. The large amount of DNA is then analyzed by restriction mapping, cloning, sequencing,or someother technique. PCR is not limited to laboratory uses; it has had profound impacts on 25 CMR COLLEGE OF PHARMACY
  26. 26. DNA CLONINGmany other disciplines. PCR is used to amplify DNA from crime scenes. The small amounts ofDNA found in unlikely places can now be analyzed to help prove guilt or innocence. DNAextracted from Egyptian mummies can be amplified by PCR. The relationships betweenindividuals buried in the pyramids have been studied using PCR and restriction mapping. Manydisease-causing microbes cannot be grown in the laboratory or take a long time to grow. Notknowing the microbes present in a sick person can prevent them from getting the proper courseof treatment in time. PCR can identify the presence of specific bacterial species simply by usingspecies specific primers in a PCR reaction and a sample (sputum, blood, etc.) from the sickperson. A PCR test can be conducted in a few hours or in time to drastically alter the course oftreatment. In any discipline where the amount of DNA has traditionally been limiting, PCR canbe used to circumvent that problem and help provide information.GENOMIC LIBRARYA genomic library is a population of host bacteria, each of which carries a DNA molecule thatwas inserted into a cloning vector, such that the collection of cloned DNA molecules representsthe entire genome of the source organism. This term also represents the collection of all ofthe vector molecules, each carrying a piece of the chromosomal DNA of the organism, prior tothe insertion of these molecules into the host cells.CREATING A LIBRARYThe DNA molecules of an organism of interest are isolated. The DNA molecules are thenpartially digested by an endonuclease restriction enzyme. Sometimes, the DNA molecules aredigested for different lengths of time in order to ensure that all the DNA has been digested tomanageable sizes. The digested DNA molecules are separated by sizeusing agarose electrophoresis, and a suitable range of lengths of DNA pieces are isolated andligated into vectors. The vectors can then be taken up by suitable hosts.The hosts are kept inliquid media and can be frozen at -80°C for a long period of time. Usually the hostsare bacteria that do not contain any plasmids, so as to be sensitive to antibiotics.The process of subdividing genomic DNA into clonable elements and inserting them into hosts iscalled creating a library, a clone bank or a gene bank. A complete library of host cells willcontain all of the genomic DNA of the source organism. 26 CMR COLLEGE OF PHARMACY
  27. 27. DNA CLONINGGENE EXPRESSIONExpression cloning is a form of cloning in which a scientist reproduces DNA of particularinterest and implants it into a cell so that the DNA can be studied in action to learn more about it.While the term “cloning” often conjures up an image of reproductive cloning, in which a geneticcopy of a living organism is created, expression cloning only clones a segment of DNA, not anentire organism. It is used in scientific research, and it has contributed significantly to manystudies in the field of DNA and the genome of living organisms.In expression cloning, a scientist first selects a DNA segment of interest, and then attaches it to aplasmid which can penetrate a cell to carry the DNA inside. These plasmids are known ascloning vectors or expression vectors. Once a cell has been transfected, as this process is called,the scientist can culture the cell to create numerous cloned cells which can be further studied.Each cloned cell will follow the instructions in the introduced DNA, producing a response whichcan be analyzed in the lab.This type of cloning can also be combined with recombinant DNA technology, in which asection of DNA is altered for scientific study. Using this technique, a researcher could dosomething like change the proteins expressed by a particular segment of DNA in order to figureout how an organism becomes resistant to antibiotics. Expression cloning can also be used tocreate a library of cloned DNA which can be distributed to other laboratories for additionalstudy.Scientists can also create an assortment of clones, with each one expressing a specific protein, inthe quest for a particular gene. In these instances, the cells can be cultured and then studied, withthe cells which produce the relevant gene being isolated for further study. One could think of thistype of study as a series of cake recipes which are experimented with to find a cake with thedesired properties.Unlike reproductive cloning, expression cloning does not produce a viable organism, but only asmall section of DNA. For people who struggle with the ethical issues involved in reproductivecloning, expression cloning is sometimes viewed as acceptable, because it does not createsomething which could be considered “alive.” Expression cloning can also be used in therapeuticcloning, and it will theoretically play a role in gene therapy, in the event that gene therapybecomes viable. cloning is a technique in DNA cloning that uses expression vectors to generate alibrary of clones, with each clone expressing one protein. This expression library is thenscreened for the property of interest and clones of interest recovered for further analysis. Anexample would be using an expression library to isolate genes that could confer antibioticresistance. 27 CMR COLLEGE OF PHARMACY
  28. 28. DNA CLONINGGene expression is the process by which information from a gene is used in the synthesis of afunctional gene product. These products are often proteins, but in non-protein coding genes suchas ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, theproduct is a functional RNA. The process of gene expression is used by all known life -eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), possiblyinduced by viruses - to generate the macromolecular machinery for life. Several steps in the geneexpression process may be modulated, including the transcription, RNA splicing, translation, andpost-translational modification of a protein. Gene regulation gives the cell control over structureand function, and is the basis for cellular differentiation, morphogenesis and the versatility andadaptability of any organism. Gene regulation may also serve as a substrate for evolutionarychange, since control of the timing, location, and amount of gene expression can have a profoundeffect on the functions (actions) of the gene in a cell or in a multicellular organism. In genetics,gene expression is the most fundamental level at which the genotype gives rise to the phenotype.The genetic code stored in DNA is "interpreted" by gene expression, and the properties of theexpression give rise to the organisms phenotype.Regulation of gene expression refers to the control of the amount and timing of appearance of thefunctional product of a gene. Control of expression is vital to allow a cell to produce the geneproducts it needs when it needs them; in turn this gives cells the flexibility to adapt to a variableenvironment, external signals, damage to the cell, etc. Some simple examples of where geneexpression is important are: • Control of insulin expression so it gives a signal for blood glucose regulation • X chromosome inactivation in female mammals to prevent an "overdose" of the genes it contains. • Cyclin expression levels control progression through the eukaryotic cell cycle 28 CMR COLLEGE OF PHARMACY
  29. 29. DNA CLONINGMore generally gene regulation gives the cell control over all structure and function, and is thebasis for cellular differentiation, morphogenesis and the versatility and adaptability of anyorganism.Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. The stability of the final gene product, whether it is RNAor protein, also contributes to the expression level of the gene - an unstable product results in alow expression level. In general gene expression is regulated through changes in the number andtype of interactions between molecules that collectively influence transcription of DNA andtranslation of RNA.Numerous terms are used to describe types of genes depending on how they are regulated, theseinclude: • A constitutive gene is a gene that is transcribed continually compared to a facultative gene which is only transcribed when needed. • A housekeeping gene is typically a constitutive gene that is transcribed at a relatively constant level. The housekeeping genes products are typically needed for maintenance of the cell. It is generally assumed that their expression is unaffected by experimental conditions. Examples include actin, GAPDH and ubiquitin. • A facultative gene is a gene which is only transcribed when needed compared to a constitutive gene. • An inducible gene is a gene whose expression is either responsive to environmental change or dependent on the position in the cell cycle.Direct interaction with DNA is the simplest and the most direct method by which a protein canchange transcription levels. Genes often have several protein binding sites around the codingregion with the specific function of regulating transcription. There are many classes of regulatoryDNA binding sites known as enhancers, insulators, repressors and silencers. The mechanisms forregulating transcription are very varied, from blocking key binding sites on the DNA for RNApolymerase to acting as an activator and promoting transcription by assisting RNA polymerasebinding.The activity of transcription factors is further modulated by intracellular signals causing proteinpost-translational modification including phosphorylated, acetylated, or glycosylated. Thesechanges influence a transcription factors ability to bind, directly or indirectly, to promoter DNA,to recruit RNA polymerase, or to favor elongation of a newly synthetized RNA molecule.The nuclear membrane in eukaryotes allows further regulation of transcription factors by theduration of their presence in the nucleus which is regulated by reversible changes in theirstructure and by binding of other proteins. Environmental stimuli or endocrine signals may cause 29 CMR COLLEGE OF PHARMACY
  30. 30. DNA CLONINGmodification of regulatory proteins eliciting cascades of intracellular signals, which result inregulation of gene expression.More recently it has become apparent that there is a huge influence of non-DNA-sequencespecific effects on translation. These effects are referred to as epigenetic and involve the higherorder structure of DNA, non-sequence specific DNA binding proteins and chemical modificationof DNA. In general epigenetic effects alter the accessibility of DNA to proteins and so modulatetranscription.DNA methylation is a widespread mechanism for epigenetic influence on gene expression and isseen in bacteria and eukaryotes and has roles in heritable transcription silencing and transcriptionregulation. In eukaryotes the structure of chromatin, controlled by the histone code, regulatesaccess to DNA with significant impacts on the expression of genes in euchromatin andheterochromatin areas.Expression vectors -:Expression vectors are a specialized type of cloning vector in which thetranscriptional and translational signals needed for the regulation of the gene of interest areincluded in the cloning vector. The transcriptional and translational signals may be syntheticallycreated to make the expression of the gene of interest easier to regulate.Post-transcriptional regulation-: In eukaryotes, where export of RNA is required beforetranslation is possible, nuclear export is thought to provide additional control over geneexpression. All transport in and out of the nucleus is via the nuclear pore and transport iscontrolled by a wide range of importin and exportin proteins.Expression of a gene coding for a protein is only possible if the messenger RNA carrying thecode survives long enough to be translated. In a typical cell an RNA molecule is only stable ifspecifically protected from degradation. RNA degradation has particular importance inregulation of expression in eukaryotic cells where mRNA has to travel significant distancesbefore being translated. In eukaryotes RNA is stabilised by certain post-transcriptionalmodifications, particularly the 5 cap and poly-adenylated tail.Intentional degradation of mRNA is used not just as a defence mechanism from foreign RNA(normally from viruses) but also as a route of mRNA destabilisation. If an mRNA molecule hasa complementary sequence to a small interfering RNA then it is targeted for destruction via theRNA interference pathway. 30 CMR COLLEGE OF PHARMACY
  31. 31. DNA CLONINGTranslational regulation-:Direct regulation of translation is less prevalent than control oftranscription or mRNA stability but is occasionally used. Inhibition of protein translation is amajor target for toxins and antibiotics in order to kill a cell by overriding its normal geneexpression control. Protein synthesis inhibitors include the antibiotic neomycin and the toxinricin.Protein degradation-:Once protein synthesis is complete the level of expression of that proteincan be reduced by protein degradation. There are major protein degradation pathways in allprokaryotes and eukaryotes of which the proteasome is a common component. An unneeded ordamaged protein is often labelled for degradation by addition of ubiquitin.PURPOSE OF EXPRESSIONUsually the ultimate aim of expression cloning is to produce large quantities of specific proteins.To this end, a bacterial expression clone may include a ribosome binding site (Shine-Dalgarnosequence) to enhance translation of the gene of interests mRNA, a transcription terminationsequence, or, in eukaryotes, specific sequences to promote the post-translational modification ofthe protein product. 31 CMR COLLEGE OF PHARMACY
  32. 32. DNA CLONINGFactors taken into account for expression are : (i) Supply of prokar yotic promot er for express ion of eukary otic genes, (ii) Supply of riboson al binding sites for the cloning vector, (iii) Remov al of introns from mRNA obtaine d from eukary otic genes 32 CMR COLLEGE OF PHARMACY
  33. 33. DNA CLONINGCloning and expressing a geneIn many cases, the goal is to not only clone the gene of interest but it is also to have the clonedgene expressed. When cloning a gene behind a promoter, the spacing of the elements needed fortranscription and translation is critical. The closer the spacing of the elements to the optimumspacing for each element, the better the regulation and the better the expression of the clonedgene. The spacing can be manipulated during the cloning process. If a PCR fragment is used as asource of the gene to be cloned, base pairs can be inserted as needed by the design of theprimers. For example, many vectors contain a promoter, mRNA start site, ribosome binding site(Shine– Dalgarno sequence), and ATG start codon (Fig. 14.13a). Each of these elements are inthe correct order with the correct spacing. Following the ATG start codon are the multiplecloning sites (MCS). A gene is cloned into one of the restriction sites in the MCS. In this case,the reading frame must be maintained from the ATG start codon through the MCS and into thecloned gene (Fig. 14.13b). If the cloned gene is out of frame, this can be corrected by changingthe primers used to amplify the fragment (Fig. 14.14). 33 CMR COLLEGE OF PHARMACY
  34. 34. DNA CLONINGFig. 14.13 Cloning vectors can incorporate many different features including transcription andtranslation signals. (a) In this example, the cloning vector pBAD24 is shown. It contains aregulated promoter, mRNA start site, ribosome binding site, and a start codon. The multiplecloning sites are located after the start codon. (b) To clone into this vector and have your proteinof interest be expressed from the regulated promoter, your DNA sequence must be cloned so thatthe reading frame of the gene is maintained.Fig. 14.14 If there are no restriction sites that leave the reading frame intact, then the fragment tobe cloned can be amplified by PCR and the correct number of bases added to the primer. 34 CMR COLLEGE OF PHARMACY
  35. 35. DNA CLONINGDNA sequencing using dideoxy sequencingDNA sequencing is the determination of the exact sequence of bases in a given DNA fragment.The start of the DNA sequence is determined by the placement of a DNA primer. Normaldeoxyribonucleoside triphosphate precursors (dNTPs or dATP, dTTP, dCTP, and dGTP) andDNA polymerase are added to carry out DNA synthesis (Fig. 14.15a). In addition to the normaldNTPs, four special dideoxyribonucleosides (ddATP, ddTTP, ddCTP, and ddGTP) are includedin small amounts. The special ddNTPs have a fluorescent tag attached to them. ddGTP has afluorescent tag that fluorescese at one wavelength, ddATP a tag that fluoresces at a differentwavelength, ddTTP a tag with a third wavelength, and ddCTP a tag with a fourth wavelength.ddNTPs do not have a 3¢ OH and therefore block further synthesis of DNA. The fluorescentlytagged ddNTPs are randomly incorporated into the growing DNA molecules (Fig. 14.15b). Theresults of DNA synthesis in the presence of tagged ddNTPs are a collection of DNA moleculesdifferent from each other by one base. The fluorescently tagged ddNTP at the end of eachmolecule is dictated by the sequence of the template DNA. The tagged fragments aresubsequently separated on either a polyacrylamide gel or on a very thin column. At the base ofthe column or polyacrylamide gel is located a laser. As the DNA fragments run off the column orgel, they pass through the laser beam, fluoresce, and the wavelength of the fluorescence isrecorded and sent to a computer.The order of the fluorescently tagged molecules coming off the column reflects the sequence ofthe template DNA. The automation of DNA sequencing has greatly simplified the process andmade it much faster. Approximately 350 to 500bp of DNA sequence can be read from oneprimer. By carrying out separate sequencing reactions using primers located every 350 to 450 bpon the template, the sequence of the entire template can be determined.Fig. 14.15 Dideoxy DNA sequencing using fluorescently tagged ddNTPs. (a) Adideoxyribonucleoside triphosphate does not contain a 3¢ OH and as such terminates DNAreplication. (b) The fluorescently tagged ddNTPs are randomly incorporated into growing DNAmolecules. This leads to a collection of molecules and in this population are molecules that areonly a few bases long all the way up to about 500 bases. The fragments will differ in size by only 35 CMR COLLEGE OF PHARMACY
  36. 36. DNA CLONINGone base pair. The population is separated in a gel or column using an electric current. Thesmallest molecules migrate fastest and will reach the bottom of the gel or column first. A laser ispositioned at the bottom of the gel or column and detects the fluorescent bases as they migratethrough. The different bases are tagged with different colored dyes. The signalsdetected by thelaser are relayed to a computer that records what color of dye was attached to each sizedfragment 36 CMR COLLEGE OF PHARMACY
  37. 37. DNA CLONINGFig. 14.16 Sequences that are very similar over the entire length of protein indicate that the twoproteins may have a similar function. The greater the similarity, the greater the chance thefunctions are the same. 37 CMR COLLEGE OF PHARMACY
  38. 38. DNA CLONINGDNA sequence searchesTo date, many millions of base pairs of DNA from many species have been sequenced. Forexample, the chromosomes of at least 50 bacterial species, several yeasts, and the large majorityof all human chromosomes have been determined. These sequences contain an incredible amountof information. So much in fact that special computer programs had to be designed to helpinterpret just a fraction of the data. When a DNA sequence is published in a scientific journal, itis also deposited in a computer database known as GenBank. When a sequence is placed inGenBank, the known and predicted features of the sequence are also indicated. These includepromoters, open reading frames, and transcription factor binding sites. Just a listing of As, Cs,Gs, and Ts are known as a raw sequence and the sequence with all of the features indicated isknown as an annotated sequence.It is possible to search the sequences in GenBank using several different programs. You cansearch by the name of an interesting gene very easily. If you have the sequence of a gene ofinterest, it is possible to search for related sequences. GenBank can be searched using a DNAsequence or using that DNA sequence translated into the protein sequence. The programs usedfor these searches are capable of identifying not only exact matches but also sequences that havediffering degrees of similarity. What can be learned from sequence searches? First, DNAsequence searches are more stringent than protein sequences. Two DNA sequences either havean adenine in the same position or they do not. Protein sequences can have the same amino acidin the same place and are, thus, identical at that position. Proteins can also have similar aminoacids in one position, such as valine in one protein and alanine in the other. Because both aminoacids are hydrophobic, they can frequently carry out the same functions. In this case, the proteinsare said to be similar in a given position. If two proteins have similarity over a large segment oftheir sequences, they may have similar functions (Fig. 14.16). This kind of analysis is especiallyuseful if the function of one of the proteins has been identified. Knowing the function of one ofthe proteins suggests that the other protein should also be checked for this function. More limitedregions of sequence similarity or identity can indicate the presence of a cofactor binding site.Sequence similarities can provide very valuable information about an unknown sequence anddramatically influence the direction of experiments on the novel gene or protein. 38 CMR COLLEGE OF PHARMACY
  39. 39. DNA CLONINGConclusion-:In 1962, the Nobel Prize in Medicine and Physiology was awarded toWatson and Crick for the discovery of the structure of DNA. Thetechnology developed in the 49 years since has revolutionized howbiological research is conducted. The ability to manipulate genes invitro has greatly increased not only the experiments that are nowpossible but also how scientists think about biological problems.Each of the techniques described above allows scientists tomanipulate a novel gene in many different ways with the goal ofuncovering its unique role in the cell.Because of recent technological advancements cloning in the animalsand humans has been an issue and is strictly banned, Scientists havemade some major achievements with cloning, including the asexualreproduction of sheep and cows. There is a lot of ethical debate overwhether or not cloning should be used. However, cloning, or asexualpropagation has been common practice in the horticultural worldfor hundreds of years 39 CMR COLLEGE OF PHARMACY
  40. 40. DNA CLONINGREFERENCE 1. Biochemistry by U.Satyanarayana and U.Chakrapani,edition 2008,Molecular Biology and Biotechnology Pg no’s-523-695 2. Lobban, P. and Kaiser, A.D. 1973. Enzymatic end-to-end joining of DNA molecules. Journal of Molecular Biology, 78: 453. 3. Fundamentals of Biochemistry by J.L.Jain ,edition 2005,S.Chand publications,Nucleic acids pg no’s 280-332 4. Cell Biology by Stephen.R.Bolsover,second edition,Recombinant and genetic engeneering,DNA Cloning pg no’s-129-160 5. http://en.wikipedia.org/wiki/Gene 6. http://en.wikipedia.org/wiki/Dna 7. Molecular Biology of the cell @NCBI,Digitally signed by Jesil Mathew.A, Basic genetic mechanisms ,Recombinant DNA technology and control of gene expression PDF 8. Biochemistry by Jeremy Berg, John and Lubert, Fifth edition, Unit’s 27,28,29 DNA Replication,Recombination,RNA synthesis and splicing and Protein synthesis,Portable Document File 9. Harper’s Illustrated Biochemistry by Robert,Daryl,peter and victor,26th edition,Mc graw hill, Section 4,structure,functional,replication of informational macromolecules pg no’s 33-41 10. Lehninger,Principles of Biochemistry,edition 4th,Chapter 9,DNA based informational technologies pg no 306-330 40 CMR COLLEGE OF PHARMACY