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PHARMACOBIOTECHNOLOGY
 Recombinant
DNA (rDNA) is constructed
outside the living cell using enzymes called
“restriction enzymes” to cut DNA at specific
sites and enzymes called “ ligases” to insert
the cleaved piece of DNA (the “insert” ) into
“ vector DNA” i.e., plasmid or viral DNA that
will be able to enter a “host” cell or
microorganism. Once the foreign DNA enters
the host organism it is called “recombinant
organism”.
 Combining
from two
different
organisms
DNA
-type of enzyme in DNA strand.
-produced nucleic acid strand breaks interior
of nucleic acid strand.
-restriction endonucleases-enzyme produced
by bacteria that is used in recombinant DNA.
-cuts open bacterial plasmid.(molecular
Scissors)
-gene construct engineered to plasmid with
ligases. Plasmids back to bacterium.
 Restriction
endonucleases
can cut DNA at
specific sites,
leaving sticky
ends for insertion
of new DNA
Legend:
The action of restriction enzymes
Restriction enzymes, also called restriction
nucleases (EcoRI in this example) , surrounds the
DNA molecule at the point it seeks(sequence
GAATTC). It cuts one strand of the DNA double
helix at one point and the second strand at a
different, complementary point (between the G and
the A base). The separated pieces have single
stranded "sticky-ends," which allow the
complementary pieces to combine.
The newly joined pieces are stabilized by DNA
ligase.
EcoRI, one of many restriction enzymes, is
obtained from the bacteria Escherichia coli.
-carrier for DNA during the recombinant
DNA process.
-plasmid-piece of free-floating DNA in
the cytoplasm of bacteria.
-double-stranded, circular molecules
that replicate independently of the
chromosome.
A cloning vector is a small piece of
DNA into which a foreign DNA
fragment can be inserted. The
insertion of the fragment into the
cloning vector is carried out by
treating the vehicle and the foreign
DNA with the same restriction
enzyme, then ligating the fragments
together. There are many types of
cloning vectors. Genetically
engineered plasmids and
bacteriophages (such as phage λ) are
perhaps most commonly used for this
purpose
 Antibiotic
resistance gene used to
identify recombinant cells
A palindromic number or numeral palindrome
is a 'symmetrical' number like 16461, that remains
the same when its digits are reversed.
The term palindromic is derived from palindrome,
which refers to a word like rotor that remains
unchanged under reversal of its letters.
The first palindromic numbers (in decimal) are:
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 22, 33, 44,
55, 66, 77, 88, 99, 101, 111, 121, 131,
141, 151, 161, 171, 181, 191 etc,
Most restriction enzymes leave sticky
ends ,single strand extensions that will
base pair with the opposite strand of
another piece of DNA cleaved by the
same enzyme , but some leave blunt
ends.
These annealed pieces will have single
strand breaks (called “NICKS”) at the
sites at which they were cleaved.
Treatment of these mixed pieces with
an enzyme called DNA LIGASE
( usually T4 ligase from bacteriophage
named T4) .
In gene cloning procedures, a gene
corresponding to a specific protein will be
joined with a cloning vector so that it can be
transferred into a host cell. A cloning vector
can be a plasmid or a bacteriophage.
Plasmids are self-replicating, double stranded
, circular DNA molecules that are usually
maintained in the host cell independent
extrachromosomal moiety.
Plasmids will be characterized by size, host
specificity (which organism it can exist in),
and copy number, ex. The number of copies of a
given plasmid usually found in a cell. Ones
used for cloning purposes often have genetic
elements such as antibiotic resistance genes
that can be used to determine which cells have
the plasmids.
Bacteriophages (viruses that infect
bacteria ) are also used as cloning
vectors, and foreign DNA inserted
into the viral genome in an
analogous manner. Viral DNA (
carrying as insert) get cells by viral
infection, and the genes are
expressed as the viral genome in
expressed.
Hybridization probe
In molecular biology, a hybridization probe is a
fragment of DNA or RNA of variable length (usually
100-1000 bases long), which is used in DNA or RNA
samples to detect the presence of nucleotide
sequences (the DNA target) that are complementary
to the sequence in the probe. The probe thereby
hybridizes to single-stranded nucleic acid (DNA or
RNA) whose base sequence allows probe-target base
pairing due to complementarity between the probe
and target. The labeled probe is first denatured (by
heating or under alkaline conditions such as exposure
to sodium hydroxide) into single DNA strands and then
hybridized to the target DNA (Southern blotting) or
RNA (northern blotting) immobilized on a membrane.
Specific proteins can be detected by complimentary
antibodies, leading to a color reaction, in a process
called Western blotting.
Molecular DNA- or RNA-based
probes are now routinely used
in screening gene libraries.
A LIBRARY is a collection of
recombinant organisms, each
of which contains a different
fragment of DNA from the
organism which naturally has
the gene of interest in its
chromosomes.
Polymerase Chain
Reaction
Polymerase chain reaction (PCR)
enables researchers to produce
millions of copies of a specific DNA
sequence in approximately two
hours. This automated process
bypasses the need to use bacteria
for amplifying DNA.