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_OBRIEN-16__Recombinant DNA _ cloning 118-128

_OBRIEN-16__Recombinant DNA _ cloning 118-128 - Recombinant...

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Unformatted text preview: Recombinant DNA and Cloning A. Introduction . "Cloning" is a way to isolate a specific DNA fi'agment (a "gene"), and produce it in a pure form in large amounts. Cloning makes detailed analysis possible that would otherwise be difficult or impossible without the gene in pure form, separated fi'om the rest of the DNA of the organism. Cloning comprises techniques that provide a powerful approach for 1. Understanding eukaryotic genes 2. Providing large scale production of scarce proteins, e. g. peptide hormones 3. Controlled alteration of genetic composition of cells and whole organisms B. Cloning Specific DNA fragments 1. Some definitions and concepts a. Cloning vectors . . . usually plasmids, DNA molecules capable of autonomous replication G b. Foreign DNA. . . fragment to be studied, that is not originally part of cloning vector c. Recombinant DNA DNA molecules constructed in vitro, consisting of DNA from 2 or more sources a? ll? Join the foreign DNA to the vector (ligase) WW Introduce the recombinamleflm into cells (transfection, transformation) in which they can replicate Identify and isolate cells that contain the recombinant vector. From these, other cells (or clones) may be obtained. am a. b. C. Bacterial plasmids; pBR 322, p SP6 Bacterial viruses; Lambda gt 11 Animal viruses; SV40 4.21a51m'd5 a b. Small circular ds DNA molecules Contain genes for antibiotic resistance, to allow selection, by growth on media containing (lacking) antibiotics Contain several restriction sites Can be replicated (amplified) in bacteria I I? Example: pBR322 (4362 bp) Pail ‘9 fat V‘ army“ 3' G CTGCA - OH f——_— Can circulaxize I.) /‘ CTGCAG CTGCA G ampr gene inactivated or combine with other (foreign) DNA that has been cut VVlth Pst 1 [my 5607/ ACGTC GACGTC [V/VZ / jf/y ff??? Which can circularize and be used to transfonn bacteria M a, / r ( {e% 1 fl \ W Recombinant Plasmid (use ligase to close gaps) / 2 O Recombinant plasmid has 1) Foreign DNA 2) Two Pst 1 sites 3) Split ampr gene (inactive) Plasmid is tetr but amp5 6. lntrodnctimmfifiecombinamilasmid into E. eoli 4° + Capt-r 42°w37° to raise efficiency of transformation (~ 106—4402) AmpS Host Tets 7. Select bacteria with recombinant plasmid Growth on media containing tetracycline Lacking ampicillin (-+)4/2V (—44 277/ 8. Obtain elones from single cells icell growth in nutrient broth 09 cells (~ 3 plasmids/cell) chloramphenicol (blocks protein synthesis on bacterial ribosomes) 109 cells (~ 1000 plasmids/cell) IZI 9. Isolation of cloned fragment a. Concentrate bacteria with recombinant plasmid b. Lyse 4/ J/ c. Differential precipitation of plasmid DNA Q d. Cut out foreign DNA with Pst 1 a“ y e. Separate plasmid and foreign DNA on agarose gel {7er 47 y a . (fl) 53> (4-) C. Expression of foreign genes in E. coli 1. Expression vectors Vectors with a regulated bacterial promoter (eg. [3 gal) upstream from insertion site insertion site \ / Can construct plasmids that contain sequences causing expressed proteins to be secreted (to minimize '. degradation) 2. Limitations of Eukaryotic gene expression in bacteria a Nosplicjng. Genes with introns cannot be expressed properly. Can be circumvented by using cDNA. b. W or modify eukaryotic proteins (e.g., glycosylation). 3. Examples of eukaryotic proteins produced in E. mli. Somatostatin human growth hormone insulin interferons D. W (complementary DNA) fiommRNA mRNA 3' 5‘ AAAAAAA 3' $ anneal oligo (dT) primer 5' AAAAAAA 3' TITFIT 3' 5' i reverse transcriptase mRNA AAAAAAA TTITFIT ss cDNA /23 5'mBl\_IA_______AAAAAAA3' 3' TTITITT KeflM/fi KM E t 3' TITTIT 5' g Rnase H (degrades RNA 33 W i in hybrid) E Q ~ and P01 1 TTITIT 5' 5' __...._ AAAAA 3' 3'C ireverse transcriptase TITI'IT i Pol I T'ITITT 5' 5' AAAAAA AAAAAA 3' 3' TITITT S1 nuclease ds cDNA TI’ITT (j _____ AAAAA ds cDNA Can add tails (oligo C, G) with Wings: - or add “linkers” with ligase to facilitate coupling with vector DNA 5' xxxxxxx3' moocxx 5' CCCCCC + (ligase) CCCCCC W Can be inserted behind dim, responsive to diflerenLreginatoanignals 12 4 SITE-DIRECTED MUTAGENESIS The ability to change individual codons or groups of codons in expressed coding sequences. CA6 m Cloned gene (cDNA) in ss DNA M vector (Ml3). Hybridize a primer to the region On desired to mutagenize Jflff D/M fp/7fl, ”ff; Primer contains one (or only a few) 10/57 07¢ 51¢, mismatched bases Extend primer with DNA pol, ligate, to generate ds DNA with one (or few) mismatches Replication of primer strand will introduce mutation at the site of the mismatch. Variations of this procedure are used to alter amino acids at active sites of enzymes (one at a time), to introduce new restriction enzyme cleavage sites and other directed, specific alterations [25 Alteration of Genetic Composition: Transgenic mice: Original demonstration of the insertion of foreign gene, under control of a separate promoter, into the pronucleus of a fertilized mouse ovum r 1. Metallothionine (MT) gene in mouse control region MT gene S'Qfl o o \ W111 activate MT (33 % cysteine) MT gene in presence of MT complexes Zinc ion heavy metals (Zn2+) 6 Zn” \ excreted 2. Rat Growth Hormone (RGH) control region RGH gene 5 ' :fl—E 3. 1n 3mm recombination: Place the rat RGH gene under control of the mouse MT promoter MT control RGH gene 5' :t—CZS region 4. Insert into pBR 322 and clone in E. 9211 recombinant plasmid Microinject cloned plasmid into pronuclei of fertilized mouse ova (~600 copies/egg) (140 eggs) Implant in (pseudopregnant) foster mothers Result: 21 offspring (from 140 implanted eggs) Test for presence of RGH gene (i.e., are the animals "transgenic"? Wean and give Zn++ RGH+ ' RGH" 7 14 “Super mouse” Normal ® /Z7 TO ISOLATE GENES and To Produce/Engineer SCARCE PROTEINS Using information derived from protein SCREEN cDNA KT) EXPRESSION LIBRARY PROTEIN +AA SEQUENCE -> PEPTIDE GENE SYNTHESIS (cDNA) i deduced SCREEN cDNA PROBE -‘> cDNA, LIBRARY EXPRESSION SYSTEM 4.2.219 2~2 ’7’ 2 e MET GLU ASN TRP VAL HIS ASP PRO LYS LEU ATG GAG AAC TGG GTA CAC GAC CCA AAA TTA A T C T T C G G T T CTA G G C T . Ego)“ G th11 LIBIUXRY 5);” Q? Iffec/ @Jf'flo . “ INFECT. 7} Fof’fi“: ,, O (7 .3 ...
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