8.

1 – Manipulating and Cloning

DNA
Pages 366-375
 Diabetes Mellitus
• Overall: inability to produce insulin
• Type 1 – body cannot produce insulin (will
take by mouth or injection)
• Type 2 – body does not produce enough
insulin or cannot use the insulin produced
• Can lead to many health complications:
blindness, organ problems, limb amputations,
and death
 Banting and Best
• Dr. Frederick Banting and Dr. Charles Best (U of
T)
• Helen Free: invented a method to test blood
sugar by dip-test urinalysis
• Prior to this – doctors tasted urine to
determine diabetes
• Humans needed insulin – this was obtained
from pancreases of pigs and cows
• Some humans were allergic to insulin from
animals
• Mass producing human insulin gene by
inserting into bacteria – GENETIC
ENGINEERING
• Bacteria are good for reproducing – quickly,
often, inexpensive to maintain
• E. coli is common to make biosynthetic human
insulin
• Safflowers are also used to produce human
insulin
 Restriction Enzymes
• We need to cut out or isolate the DNA
fragment that contains desired gene
• Restriction enzymes (restriction
endonucleases) are used
• Acts like a scissor and cuts at specific locations
• The enzyme will look for a specific site:
recognition site
• Cuts it into RESTRICTION FRAGMENTS
• Restriction enzyme will cut at the one
recognition site and in one direction
• Will cut the phosphodiester bonds in the DNA
backbone between G and A
• The sites on the DNA are palindromic (reads
the same the other way)
• Another enzyme will cut the complementary
DNA strand leaving few H bonds
• This creates “sticky” ends
• 2 results could occur by cutting: blunt ends
(straight across strand), and sticky ends (zigzag
cuts)
• Different enzymes create different ends (Table
1 Page 367)
• Sticky ends are preferred
• Easier to join strands and easier to join with
pieces cut by the same enzyme
• There are about 2500 restriction enzymes
• Restriction enzymes protect the cells in which
they are found
• These enzymes are highly specific
• It’s named after its cell of origin, plus the
Roman numeral if more than one restriction
enzyme has been isolated from the species
• EcoRI = eco-R-one
https://www.youtube.com/watch?v=iuZEpGYOY
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 DNA Ligase
• Joins cut strands of DNA
• Works best with sticky ends
• T4 DNA ligase works well with blunt ends
• Hydrogen bonds are required to form between
the complementary bases
• Phosphodiester bonds are the stronger bonds
holding the strands together (forming between
the backbones of the double strands)
• Water is produced when DNA ligase joins the DNA
strands
 Plasmids
• Small circular pieces of DNA
• Found in bacteria
• Code for proteins that provide resistance to
antibiotics
• Competent cells – can take up foreign DNA
[like E. coli]
• Vector – plasmid that will transfer for foreign
genetic material
 Restriction Maps
• Diagram that shows the relative locations of
all the known restriction enzyme recognition
sites on a particular plasmid, and the distances
(measured in base pairs) between them
 Transformation
• Successful introduction of DNA from another
source
• The cell that received the DNA is said to be
transformed