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Recombinant DNA Technology
Recombinant DNA technology procedures by which
DNA from different species can be isolated, cut and
spliced together -- new "recombinant " molecules are
then multiplied in quantity in populations of rapidly
dividing cells (e.g. bacteria, yeast).
Application of Recombinant DNA
Human gene therapy: recombinant human insulin,
recombinant human growth factor, recombinant
hepatitis B vaccine…
Engineered crop plants: golden rice (β-carotene),
herbicide resistant crops, insect resistant crops…
Recombinant DNA Tool
Enzyme used in molecular biology
Restriction enzyme Recognize and cut at DNA specific sequence
DNA ligase Join compatible ends of DNA fragment. Use
Alkaline phosphatase Remove phosphate group from strand of
Polynucleotide kinase Add phosphate group to a DNA strand in
the 5’ to 3’ direction
DNA polymeraseI DNA synthesize
Exonuclease III Digest nucleotide from a DNA strand in the
3’ to 5’ direction
RNAse Nuclease digest RNA, not DNA
Taq DNA polymerase Heat-stable DNA polymerase isolated from
Restriction enzymes are primarily found in bacteria
and are given abbreviations based on genus and
species of the bacteria.
One of the first restriction enzymes to be isolated was
EcoRI is so named because it was isolated from
Escherichia coli strain called RY13.
Classify restriction enzymes
Type I enzymes are complex, multisubunit, combination restriction-and-
modification enzymes that cut DNA at random far from their recognition
Type II enzymes cut DNA at defined positions close to or within their
Type III enzymes are also large combination restriction-and-modification
enzymes. They cleave outside of their recognition sequences and require two
such sequences in opposite orientations within the same DNA molecule to
accomplish cleavage; they rarely give complete digests.
Type IV enzymes recognize modified, typically methylated DNA and are
exemplified by the McrBC and Mrr systems of E. coli.
Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restriction
Cut up “foreign” DNA that invades the cell.
Type II and III restriction enzymes cleave DNA chains
at selected sites.
Enzymes may recognize 4, 6 or more bases in selecting
sites for cleavage.
An enzyme that recognizes a 6-base sequence is called a
Basics of type II Restriction Enzymes
No ATP requirement.
Recognition sites in double stranded DNA have a 2-fold
axis of symmetry – a “palindrome”.
Cleavage can leave staggered or "sticky" ends or can
produce "blunt” ends.