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Lecture 6.Recombinant DNA.

Gel electrophoresis. Cloning vectors.

Essential Cell Biology. Chapter 10.

Several illustrations on this page have been taken from the Essential Cell Biology textbook (Garland Publishing, 1998)


Hydrolysis of DNA by restriction enzymes produces a mixture of DNA fragments

Individual DNA fragments can be isolated by electrophoresis in polyacrylamide or agarose gels which allows separation of DNA fragments by size. During gel electrophoresis, small molecules migrate faster through the pores of the gel matrix than larger molecules.

"Mice" run faster through the forest than "elephants"




Polyacrylamide or agarose gels contain a microscopic network of pores through which DNA molecules are pulled by the electric force.

Polyacrylamide gel is usually prepared as a slab cast between two glass plates


Two thin spacers are aligned along the sides of one glass plate


The second glass plate is put over. Thickness of the spacers determines how thick the gel slab will be


A "comb" is inserted between glass plates to form wells into which the sample will be later loaded


Catalyst is added to the liquid gel solution; the solution is poured into the gel cassette where it solidifies


After the comb is removed, pockets or "wells" remain in the gel

 


A sample containing a mixture of DNA fragments is put into the well and electric field is applied: negatively charged DNA moves from katode (-) to anode (+)


Smaller DNA fragments migrate faster; larger DNA fragments migrate slower. DNA fragments of the same size migrate with the same rate and form a band in the gel


The amount of DNA is usually very small and cannot be seen directly. Special dyes are used to stain the gel and visualize DNA bands


Cutting DNA with different restriction enzymes produces a different pattern of DNA bands in a gel. This is used for fast characterization of DNA

 

If you want to learn more about electrophoresis and cloning you can use the following link:

Recombinant DNA at MIT


CLONING VECTORS

In gene cloning, once recombinant DNA is constructed, it is introduced into a bacterial or eukaryotic host. In the host, recombinant DNA has to be maintained, replicated and passed from one generation to another. This is achieved by introducing recombinant DNA into a cell on a DNA vehicle called cloning vector. The most commonly used vectors are plasmid cloning vectors.


Bacterial cells may contain extra-chromosomal DNA called plasmids. Plasmids are usually represented by small, circular DNA.


Some plasmids are present in multiple copies in the cell


ARTIFICIALLY CONSTRUCTED PLASMIDS USED AS CLONING VEHICLES ARE CALLED PLASMID CLONING VECTORS


TWO MAIN FEATURES OF THE CLONING VECTOR:

ORIGIN OF REPLICATION

SELECTABLE MARKER


Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes. Without replication origin, DNA cannot be replicated in the cell.


Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker the plasmid becomes useful for the cell. Under the selective conditions, only cells that contain plasmids with selectable marker can survive. Commonly, genes that confer resistance to various antibiotics are used as selective markers in cloning vectors. For example, genes that render cells resistant to ampicillin, neomycin, or chloramphenicol are among commonly used selective markers.


Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction nucleases located next to each other

 

 

For example, this is a polylinker of one of the commonly used cloning vector plasmids pUC19:

 

HindIII SphI PstI SalI XbaI BamHI SmaI KpnI SacI EcoRI

5'..AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC..3'

3'..TTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCTCGAGCTTAAG..5'

 

Restriction sites of the polylinker are not present anywhere else in the plasmid. Therefore, cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector.



Gene to be cloned can be introduced into the cloning vector at one of the restriction sites present in the polylinker

 


Different types of cloning vectors are used for different types of cloning experiments. The vector is chosen according to the size and type of DNA to be cloned.

Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs.

Bacteriophage lambda vectors are used to clone DNA fragments in the range of 10,000 - 20,000 base pairs.

Yeast Artificial Chromosomes (YACs) can be used for cloning of hundreds of thousands of base pairs.

Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells.


Bacteriophage lambda cloning vectors


Yeast Artificial Chromosomes (YACs)


Retroviral cloning vectors

Retroviruses are RNA viruses. The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome. Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely. Retroviral vectors are widely used to study oncogenes and other human genes.


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