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Recombinant DNA Technology & Cloning

LECTURE 3 :. Recombinant DNA Technology & Cloning. Biotechnology; 3 Credit hours Atta- ur - Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST). What are clones?. Clones

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Recombinant DNA Technology & Cloning

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  1. LECTURE 3: Recombinant DNA Technology & Cloning Biotechnology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

  2. What are clones? • Clones • Genetically identical molecules, cells, or organisms all derived from a single ancestor • Cloning • The production of identical copies of molecules, cells, or organisms from a single ancestor

  3. What is DNA cloning? • DNA cloning is a technique for reproducing DNA fragments.  • It can be achieved by two different approaches:  ▪ cell based ▪ using polymerase chain reaction (PCR) • a vector is required to carry the DNA fragment of interest into the host cell. 

  4. What is Gene Cloning? • A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule. • The vector transport the gene in the host cell, which is usually a bacterium, although other type of cells can also be used • Within the host cell the vector multiplies, producing numerous identical copies not only itself but also the gene it carries

  5. What is Gene Cloning? • When the host cell divides, copies od recombinant DNA molecule are passed to the progeny and further vector replication take place • After a large number of cell divisions, a clony or clone, of identical host cell is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by recombinant DNA molecule is said to be cloned.

  6. Gene Cloning

  7. Selection of Recombinant Plasmid • Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule. • The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules. • The insertion of a DNA fragment into the polylinker disrupts part of the -galactosidase(lacZ+) gene, leading to nonfunctional  -galactosidase in E. coli. • The blue–white color selection test can be used to select for vectors with or without inserts.

  8. Selection of Recombinant Plasmid

  9. LacZ, White Blue Selection • Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue. • Resistant to ampicillin, has (amprgene) • Contains portion of the lac operon which codes for beta-galactosidase. • X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium. • Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white.

  10. X-gal and Beta-galactosidase • The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional β-galactosidase. • Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the production of LacZ.

  11. Transformation • The process of transferring exogenous DNA into cells is call “transformation” • There are basically two general methods for transforming bacteria. • The first is a chemical methodutilizing CaCl2 and heat shock to promote DNA entry into cells. • A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

  12. MoUBetween NUST, MoST and PCST • Establishment of “National Analytical Laboratory for Substance of Abuse” • By Mir Changez Khan Jamali & DrMudassirIsrar • Dope Testing • Drug Analysis • Drug Abuse • Diagnosis

  13. Transformation

  14. Transformation; Electroporation To electroporate DNA into cells, washed E. coli are mixed with the DNA to be transformed and then pipetted into a plastic cuvette containing electrodes. A short electric pulse, about 2400 volts/cm, is applied to the cells causing smalls holes in the membrane through which the DNA enters.

  15. Transformant Selection

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