2. 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).
3. 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…
6. Restriction enzyme Recognize and cut at DNA specific sequence
DNA ligase Join compatible ends of DNA fragment. Use
ATP
Alkaline phosphatase Remove phosphate group from strand of
DNA
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
thermostable microbe.
7. 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
from EcoRI
EcoRI is so named because it was isolated from
Escherichia coli strain called RY13.
Restriction Enzymes
8. Classify restriction enzymes
Type I enzymes are complex, multisubunit, combination restriction-and-
modification enzymes that cut DNA at random far from their recognition
sequences.
Type II enzymes cut DNA at defined positions close to or within their
recognition sequences.
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.
9. Restriction Enzymes
Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restriction
enzymes”.
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
"six-base cutter”.
11. 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.
14. Results of Type II Digestion
Enzymes that cut at same position on both strands
leave “blunt” ends
SmaI
Isochizomer XmarI
5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’
3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’
23. Electrophoresis
Gel electrophoresis is a method for separation and
analysis of macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and charge.
24. Gel: Agarose or Acrylamide
Dye
Nucleic acid stain: EtBr
Marker
Running time
25.
26.
27.
28. Electrophoretic mobility of form of
plasmid DNA
Circular DNA
Linear DNA
Supercoil DNA
Hypersupercoil DNA
29.
30. Uncut plasmid DNA can be in five forms
Nicked
Circular
Linear covalently close
Supercoil
Hyper supercoil
