rDNA technology 1. Prokaryotic cells or eukaryotic cells: cloning

rDNA is a DNA molecule constructed with DNA from
different sources. rDNA technology refers to techniques that
are used to manipulate, move, recombine, and propagate DNA. This technology produces
a desired protein in large quantities

The foreign DNA sequences can be inserted into plasmid
vectors by opening the circular plasmid with the restriction endonuclease. The
ends of the linearized plasmid DNA are then joined with the ends of foreign
sequence, reclosing the circle to create a chimerical plasmid.

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Tools of rDNA technology

1. Prokaryotic
cells or eukaryotic cells: cloning vectors: plasmids, bacteriophages, and yeast
artificial chromosomes.

2. Restriction enzymes

3. DNA ligases

4. DNA polymerases

Advantages:

§  Express greater concentrations
of protein product (compared to eukaryotic cells).

§  Require relatively simple media
components.

Limitations:

§  Do not perform many important
post-translational modifications (ex: glycosylation).

§  It is not possible to express
large proteins in E. Coli .

Restriction enzymes

Restriction endonucleases are enzymes isolated from
bacteria. In vivo, these enzymes are involved in recognizing and cutting up
foreign DNA, thus protecting bacteria against phage and virus. They: recognize
specific base sequences in the dsDNA: they
break the phosphodiester bonds
between 2 nucleotides within the sequence. These recognition sequences may be
4, 5 or 6 nucleotides long. Over 350 different enzymes have been isolated so
far and each recognizes a different base sequence.  Some enzymes cut the DNA at a different position
in the 2 strands, producing a single stranded overhang or a “sticky end”: Eco RI

 Other enzymes produce blunt-ended
fragments with no sticky ends.:
PvuII

 

Restriction
sites are methylated in bacteria, and thus protected. They are denoted by three
letter names derived from the bacterial strain they originate from
(genus-species-strain).Restriction sites are usually palindromes of 4-, 6- or
8-base pairs. 

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