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 Cloning 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

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. 

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

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.

Gene Cloning

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.

Selection of Recombinant Plasmid

LacZ, White Blue Selection Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue. Resistant to ampicillin, has (ampr gene) 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.

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.

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 method utilizing 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.

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Transformation

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.

Transformant Selection