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Chapter 9


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Ryerson University
CHY 204
Mario Estable

CHAPTER 9: DNA-BASED INFO TECHNOLOGIES -Techniques for DNA cloning paved the way to the modern field of genomics & proteomics, the study of genes & pro on the scast of whole cells & organisms. Paul Berg, Herbert Boyer & Stanley Cohen developed the 1 techniques for recombinant DNA. 9.1 DNA cloning: The Basics -A clone is an identical copy. DNA cloning involves separating a specific gene/DNA segment form a larger chromosome, attaching it to a small molec of carrier DNA & then replicating modified DNA 1000s or millions of times through both an inc in host cell #s & creation of multiple copies of cloned DNA in each cell resulting selective amplification of a particular gene/DNA segment. Cloning of DNA from any organism involves 5 general procedures: 1. Cutting DNA at precise locations: restriction endonucleases provide necessary molecular scissors 2. Selecting a small mole of DNA capable of self-replications: cloning vectors which is a deliver agent, typically plasmids or viral DNAs. 3. Joining the 2 DNA fragments covalently: DNA ligase linking cloning vector & DNA to be cloned. Recombinant DNAs are composite DNA molec comprising covalently linked segments from 2/more sources. 4. Moving recombinant DNA from test tube to host cell 5. Selecting/identifying host cells that contain recombinant DNA. *E.coli is the most common host cell b/c its DNA metabolism is well understood, many naturally occurring vectors are assoc w/ E.coli (plasmids & bacteriophages), techniques are available for moving DNA from 1 bacterial cell to another. -Restriction endonucleases & DNA ligase yield recombinant DNA: restriction endonucleases/restriction enzymes recognize & cleave DNA at specific seq (recognition seq/restriction sites) to generate a set of smaller fragments. DNA ligases joins the DNA fragment to be cloned to a suitable cloning vector; the recombinant vector is introduced into host cell which amplifies fragment via cell division. In the host cell’s DNA, the seq that would be recognized by its own RE is protected from digestion by methylation of DNA catalyzed by a specific DNA methylase. The RE+methylase=restriction-modification system. 3 types of RE, Type I,II,III: Type I & III are generally large multisubunit complexes containing both endonuclease & methylase activities; both req ATP. Type I RE cleave DNA at random sites that can be more than 1000bp from the recog seq; Type III RE cleave DNA ~25bp from the recog seq; & Type III RE are simpler, req no ATP & cleave DNA w/in the recog seq itself, ie. Cuts at palindromes. Some RE makes stanggered cuts on 2 DNA strands leaving 2-4 nucleotides of 1 strand unpaired at each resulting end, these unpaired strands are called sticky ends; they can base pair w/ each other or w/ other complementary sticky ends of other DNA fragments. RE cleave both strands of DNA at the opposing phosphodiester bonds leaving no unpaired bases on the end called blunt ends. A common intermediate step in cloning of a specific gene/DNA segment is the construction of a DNA library (collection of DNA clones) -Cleavage of DNA molecules by RE: target DNA fragment is isolated & DNA ligase can be used to join it to a similarly digested cloning vector, ie. Vector digested by the same RE, eg. A fragment generated by EcoRI will not link to a fragment generated by BamHI. DNA ligase catalyzes formation of new phosphodiester bonds in rxn that uses ATP or another similar cofactor. The base pairing of complementary sticky ends greatly facilitates litigation rxn but blunt ends less eff. Researchers can create new DNA seq by inserting synthetic DNA fragments (called linkers) b.w ends that are being ligated. Polylinkers are inserted DNA fragments w/ multiple recog seq for RE. -Fig9-2: RE recognize & cleave only specific seq leaving sticky ends or blunt ends. Fragments can be ligated to other DNAs, eg. Cleaved plasmid cloning vector. The rxn is facilitated by annealing of complementary sticky ends; ligation is less eff for DNA fragments w/ blunt ends than for those w/ complementary sticky ends & DNA fragments w/ diff sticky ends generally are not ligated. A synthetic polylinker w/ recog seq for diff RE can be inserted into the plasmid that has been cleaved by a RE; the insert is a linker & insert w/ multiple restriction sites is called a polylinker. -Cloning vectors allow amplification of inserted DNA segments: 3 popular cloning vectors commonly used w/ ecoli are plasmids, bacteriophages & bacterial artificial chromosomes & a vector used to clone large DNA segments in yeast. -Plasmids are circular DNA molec that replicate separately from the host chromosome, eg. Constructed E.coli plasmid pBR322 (can clone 10-20 copies per cell); can be introduced into bacterial cells via transformation—cells & plasmid DNA are incubated together at 0° in CaCl2 soln then subjected to shock vs. electroporation—cells incubated w/ plasmid DNA are subjected to high-voltage pulse making bacterial membrane permeable to large molec. The usual strategy is to use a plasmid that incl a gene that the host cell req for growth under specific conditions, eg. Gene w/ resistance to an antibiotic; only cells transformed by the recombinant plasmid can grow in the presence of antibiotic making any cell that contains plasmid “selectable” under those growth conditions, such gene is called selectable marker. Transformation of typical bacterial cells w/ purified DNA becomes even more less successful as plasmid size inc & difficult to clone DNA segments longer than 15,000bp when plasmids are used as a vector. *Basic DNA cloning: via transformation & electroporation in prokaryotes vs. transfection in eukaryotes. -Bacteriophages lamda has very eff mechanism for delivering its 48,502bp of DNA into bacterium & can be used as a vector to clone some larger DNA segments upo to 23kbp. Researches have developed bacteriophages lamda vrdtors that can be readily cleaved into 3 pcs, 2 contain essential genes (~30kbp) & 3 pc is a filler DNA which is discarded when the vector is to be used for cloning & additional DNA is inserted b/w 2 essential segments to generate ligated DNA molecules long enough to form phage particles, ie. The packaging mechanism selects for recombinant viral DNAs. Once the bacteriophage lamda fragments are ligated to foreign DNA fragments of suitable size, the resulting recombinant DNAs can be packaged into phage particles by adding them to crude bacterial extracts that contain all the pro needed to assemble a complete phage, in what is known as in vitro packaging. -Bacterial artificial chromosomes (BAC) are simply plasmid designed for cloning very long segments (typically 100 to 300kbp of DNA); generally incl selectable markers such as resistance to antibiotic chloramphenicol (Cmr) & very stable ori that maintains plasmid at 1 to 2 copies per cell. DNA fragments are cloned into the BAC vector, large circular DNAs are introduced into host bacteria by electroporation which use host bacteria w/ mutations that compromise the structure of their cell wall permitting uptake of the large DNA molec. Fig 9-6: The par genes derived from the F plasmid assist in the even distribution of plasmids to daughter cells at cell division which inc likelihood of each daughter cell carrying 1 copy of the plasmid even when few copies are present. This low # of copies are useful in cloning large segments of DNA b/c it limits opportunities for unwanted recombination rxns that can unpredictably alter large cloned DNAs over time. The BAC incl selectable markers, eg. LacZ gene is situated in the cloning region such that it is inactivated by cloned DNA inserts. Intro of recombinant BACs into cells by electroporation is promoted by the use of cells w/ altered/porous cell wall. Recom DNAs are screened for Cmr; plates also contain a substrate for beta-galactosidase that yields a coloured product. Colonies w/ active beta-galactosidase & hence no DNA insert in the BAC vector turn blue & those w/o it, hence w/ desired DNA inserts are white. -Yeast artificial chromosomes (YACs): shuttle vectors are plasmids that can be propagated in cells of 2/more diff species. YAC vectors are high capacity vectors that contain all elements needed to maintain a eukaryotic chromosome in yeast nucleus (yeast ori, 2 selectable markers & specialized seq. Vector is propagated as a circular bacterial plasmid, cleavage w/ BamHI removes a length of DNA b/w 2 telomere seq (TEL) leaving telomeres at the ends of linearized DNA. Cleavage at another internal site, EcoRI divides the vector into 2 DNA segments referred to as vector arms, each w/ a diff selectable marker. Genomic DNA is prepared by partial digestion w/ RE to obtain suitable fragment size which are then separated by pulsed field gel electrophoresis; the DNA fragments of appropriate size are mixed w/ prepared vector arms & ligated; the mixture is then used to transform treated yeast cells w/ very large molec (prepared by removal of cell wall to form spheroplast) which are then selected for X/Y. The culture on the med that req presence of both selectable marker genes ensure growth of only those yeast cells that contain YAC clone. Stability of YAC clone inc w/ size; YACs that lack a telomere at either end are rapidly degraded. -Specific DNA seq are detectable by hybridization: a probe is a radioactive labelled DNA/RNA & complementary to DNA of interest. An agar plate w/ bacterial coloniesNitrocellulose paper is pressed onto an agar plate containing many individual colonies from the library, each w/ its own recombinant DNA; some cells from each colony adhere to the paperpaper is treated w/ alkali to disrupt the cells & expose denatured DNARadiolabelled DNA probe is incubated w/ paper & then washedadded radioactive DNA probe anneals only to its complementary DNA & after unannealed probe is washed away, the hybridized DNA can be detected by autoradiography. -B/c more than 1 DNA seq can code for any given aa seq, the genetic code is said to be degenerate thus the correct DNA seq for a known aa seq can’t be known in advance. The probe is designed to be complementary to a region of the gene w/ minimal degeneracy, ie. The region w/ the fewest possible codons for the aa (2 at most); oligos are synthesized w/ selectively randomized seq so that they contain either of the 2 possible nucleotides at each posn of pot degeneracy, eg. Fig9-9; 1 of 8 will complement gene perfectly & all eight will match at least 17 of 20 posns. -Expression of cloned genes produces large quantities of pro: Most eukaryotic genes lack the DNA seq elements, eg. Promoters, seq that instruct RNA polymerase where to bind, req for expression their expression in E.coli cells so bacterial regulatory seq for transcription & translation must be inserted at appropriate posn relative to the eukaryotic gene in the vector DNA. Expression vectors are cloning vectors w/ transcription & translation signals needed for the regulated expression of a cloned gene. The rate of expression of cloned gene is controlled by replacing the gene’s promoter & regulatory seq w/ more eff & convenient versions supplied by the vector. Fig 9-10: The gene to be expressed is inserted into 1 of the restriction sites in the polylinker, near the promoter w/ the end encoding the amino end proximal to the promoter. The promoter allows eff transcription of the inserted gene, the transcription termination seq sometimes improves the amt & stability of mRNA produced. The operator permits regulation by means of repressor that binds to it. The ribosome binding site provides seq signals needed for eff translation of mRNA derived from the gene. The selectable maker allows the selection of cells containing the recombinant DNA, eg. Antibiotic resistance. -Alterations in cloned genes produce modified pro: Specific aa may be replaced individually by site-directed mutagenesis (SDM—by Michael Smith, won NP 1992) which changes aa seq of pro by altering the DNA seq of cloned gene. 2 approaches: a) if appropriate restriction sites flank the seq to be altered, researchers can simply cleave a DNA segment in a recombinant DNA plasmid, replace it w/ a synthetic DNA fragment w/ specific-base pair change & ligate it to the plasmid so now the plasmid contains the gene w/ the desire base pair change. B) when suitably located restriction sites are not present, researchers can perform oligonucleotide-directed mutagenesis where you can create a specific DNA seq change. You have a single strand of recombinant plasmid DNA w/ specific DNA seq, CAG, an oligonucleotide w/ the specific base change (GCC) is annealed to the specific DNA seq. The annealed strand serves as a primer for synthesis of a strand complementary to the plasmid vector; its fused via DNA polymerase, dNTPs (deoxynucleotide triphosphates) & DNA ligase. Plasmid vector w/ annealed strand undergoes transformation of E.coli cells repaired by cellular DNA enzymes. In E.coli cells, about ½ of plasmids will have gene w/ desired base-pair change. *Fusion pro is parts of 2 diff genes ligated to create new combos. -Terminal tags provide binding sites for affinity purification: affinity chromatography is one of the most eff methods of pro purification; the usstof fusion pro has made it possible to purify almost any pro by affinity chromatography. 1 , the gene encoding the target pro is fused to a gene encoding a peptide/pro that binds to a known ligand w/ high affinity & specificity; peptide/pro used may be attached at either amino/carboxyl end called a terminal tag/tag. The general procedure is attachment of tag w/ GST (glutathione-S-transferase) , a small enzyme that binds to glutathione/GSH. The GST tag is fused to carboxyl end of the target pro via genetic engineering; the tagged pro is expressed in host cells & is present in crude extract when the cells are lysed. The extract is subjected to affinity chromatography on a column containing medium of immobilized glutathione; the GST-tagged pro binds to the glutathione retarding its migration through the column while other pro wash through rapidly. The tagged pro is subsequently eluted from the column w/ a soln containing elevated salt [ ] or free glutathione to compete w/ the immobilized ligand for GST binding (only held by noncovalent interactions allowing for gentle elution). The tag can be partially/completely removed from the purified fusion pro using a protease that cleaves the seq near the junction b/w target pro & fused tag. -His tags bind tightly & specifically to Ni2+; are short tags w/ simple seq of 6/ more His residues. Chromatography media w/ immobilized Ni2+ can be used to eff seperate His tagged pro from other pro in extract. 9.2 From genes to genomes -A DNA library is a collection of DNA clones, gathered together as a source of DNA for seq, gene discovery or gene func studies; a genomic library is produced when the complete genome of an organism is cleaved into 1000s of fragments & all are cloned by insertion into a cloning vector. 1 step in preparing a genomic library is partial digestion of DNA by RE. Using hybridization methods, researchers can order individual clones in a library by identifying clones w/ overlapping seq; a set of overlapping clones represents a catalo for a long continuous segment of a genome, aka contig. STS (sequence-tagged sites) are DNA segments of known seq incl known genes. Fig 9-13: A segment of chromosome from organism X has markers AQ representing STS; below it is an array of BAC clones, numbered 1-9. ordering the clones on the genetic map is a many-stage process; presence/absence of an STS on an individual clone can be determined by hybridization, eg. Probing each clone w/ DNA complementary to the STS. Once the STSs on each BAC clone are identified, the clones can be ordered on the map. Eg. Clones 3,4 &5 have marker E, 4&5 also have marker F & 5 also has marker G which indicates order of the sites E,F,G. The clones partially overlap & their order must be 3,4
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