Download Replication of the DNA

Chapter 9. Messing about
with DNA
Prepared by Woojoo Choi
DNA extraction
1) get a bacterial culture or hack off a small sample
2) Disrupt the cell – using blender or chemicals
– Lysozyme: an enzyme that digests the cell wall
– Detergent: molecule that dissolves the greasy cell membrane
DNA extraction
3) Purify and conentrate DNA – Centrifuge, Phenol(to remove the protein),
ribonuclease(an enzyme to get rid of RNA), alcohol(to puch DNA out of
solution
Cutting up the DNA
1) Restriction enzyme
– An enzyme which binds to DNA at recognition site (a specific base
sequence), and then cuts the DNA
– Recognition site is usually four, six or eight bases long and an
inverted repeat
– Each restriction enzyme has its own specific recognition site.
Where it the DNA cut?
1) Type I restriction enzyme
– Cut the DNA a thousand or more bases pair away from its
recognition sequence
– The base sequence at the cut site is not fixed.
– These enzymes are suicidal.
– So, not of much use to molecular biologists
Where it the DNA cut?
2) Type II restriction enzyme
– Cut the DNA within its recognition sequence
– Normally used in genetic engineering
– There are two different ways of cutting the recognition site.
– Blunt ends and sticky ends
Where it the DNA cut?
3) Enzymes that generate sticky ends are more useful.
– The same sticky ends would be generated on both fragments.
– This allows two pieces of DNA to be bound together by hydrogen
bonding and matching the sticky ends.
– This give time for the permanent covalent bonds.
How are fragments of DNA joined together?
1) DNA ligase
– An enzyme that joins DNA
fragments end to end
– DNA with sticky ends tend to stay
attached and DNA ligase join
them permanently without much
trouble
– Ligating blunt ends takes a very
long time and very inefficient
– Bacterial ligase cannot join blunt
ends
– T4 ligase is used because it is
easier to use and can join blunt
ends
Where do restriction enzymes come from?
1) The bacteria usually fight back by chopping up the DNA of the virus.
2) This restricts the entry of the virus and so the enzymes that chopped up
the DNA were called restriction enzyme
Protection of the cell’s own DNA
1) Modification enzyme
– Whenever a bacterial cell makes
a restriction enzyme, this
enzyme is also made
– An enzyme that chemically alters
a base in the recognition site
– This protects all the DNA in a
bacterial cell from being cut
How can individual fragments of DNA be separated out??
1) Electrophoresis
– movement of charged molecules toward an electrode of the opposite
charge
– Used to separate nucleic acids or proteins or any molecule that has a
charge
– Agarose gel electrophoresis can be used to purify DNA or it can be
used to measure the sized of fragments.
How can individual fragments of DNA be separated out??
– Fragments of DNA have the same number of charges per unit length.
– They all cruise along at the same speed toward the positive electrode.
How can individual fragments of DNA be separated out??
– To separate the fragments we run them through a gel.
– Gelatin sets due to a microscopic meshwork formed by its own
protein fibers.
– Gel for DNA work are made of agarose.
How can individual fragments of DNA be separated out??
– The larger molecules find it more difficult to squeeze through the
gaps but the smaller ones are slowed down much less.
– The result is that the DNA fragments separate in order of size.
How can individual fragments of DNA be separated out??
– Both agarose and DNA are naturally colorless, so cannot see where
the DNA has ended up.
– To find DNA fragments ethidium bromide(Etbr), a dye that stains
DNA and RNA when viewed under UV light, is used.
How can individual fragments of DNA be separated out??
– After the bands are located, they are cut out of the agarose slab and
the DNA is extracted to yield a pure fragment.
– To find the size of DNA, we run a set of standard DNA fragments of
known sizes alongside, on the same gel.
Making a restriction map
1) Restriction map
– diagram of DNA showing the cut sites for a series of restriction
enzymes
– Once a map of all the restriction sites is obtained, DNA can be cut as
we wish for analysis and for cloning any useful genes.
Making a restriction map
Making a restriction map
Choosing a vector for our own special purposes
1) Vector
– molecule of DNA which can replicate and is used to carry cloned
genes or DNA fragments
– Bacterial plasmids are the most widely used vector.
– ColE1 plasmid: a particular plasmid whose derivatives have been
widely used as vectors
Choosing a vector for our own special purposes
2) Requirements
– The vector should be a reasonably small and manageable DNA
molecule.
– Moving the vector from cell to cell should be easy.
– Growing and purifying large amount of vector DNA should be
straightforward.
Detecting and selecting vectors
1) To improve the original ColE1 plasmid
– First we get rid of the genes for colicin E1, as these are not needed.
– Add a gene for resistance to the antibiotic ampicillin, the ampR gene
which is gene encoding B-lactamase which degrades penicillin class.
– Transform this new vector into bacterial cells.
– Those cells containing a plasmid survive, while those which did not
get a plasmid are killed.
Inserting genes into vectors
1) DNA ligase: enzyme which
joins ends of DNA strands
together
Inserting genes into vectors
2) We have some problems to use this
– Our vector would be chopped into pieces, not merely opened
coveniently if there were more than one cut site in the vector
– We must avoid inserting the cloned gene into any of the genes
needed by the plasmid for its own replication and survival within the
cell
– We need flexibility using many different restriction enzyme in order
to have a wide choice
Inserting genes into vectors
3) Let’s fix all these problems
– We put into ourplasmid a stretch of artificially synthesized DNA
about 50 base pairs long which contains cut site for seven or eight of
our favorite restriction enzymes, Polylinker or multiple cloning
site(MCS)
Inserting genes into vectors
– One other thing to do is to make
sure that there are no extra cut sites
on the plasmid for any of the
enzymes represented in MCS
• The couch potato’s way to do
this is to choose for the
polylinker only enzymes with
zero cut sites in the plasmid
• Altenatively, we can get rid of
unwanted cut sites by the
approach shown in Figure 9.25
Detecting insertions into vectors
1) There are basically three approaches
– Brute force and ignorance
• Extract plasmid DNA from each of colonies
• Cut the plasmid DNA with the restriction enzyme
• Electrophoresis and check out the fragments
• If the vector contains inserted DNA, we will get two pieces of
DNA, one being the original plasmid and the other the inserted
DNA fragment
Detecting insertions into vectors
– Antibiotics
• Cells that receive a
plasmid without an insert
will be resistance to both
antibiotics
• Those receiving a plasmid
with an insert will be
resistance to only the first
antibiotics
Detecting insertions into vectors
– Color screening
• Plasmids without a DNA insert
will produce B-galactosidase
and the cells carrying them
will turn blue
• Plasmids with an insert cannot
make B-galactosidase and the
cells will stay white
Moving genes between organisms: Shuttle vectors
1) Shuttle vectors
– a vector able to survive in more than one type of host cell
– the detailed requirements for vectors will vary depending on the host
organism, but the general ideas will be the same.
Yeast artificial chromosomes
1) Genes of higher organisms are often relatively huge.
2) Typical bacterial genes are around a thousand base pairs, the genes for
hemophilia may take up a million or more base pairs of DNA.
3) The largest bacterial plasmids that can be manipulated are around
50 kbp.
Yeast artificial chromosomes
4) To carry huge pieces of DNA, construct yeast artificial
chromosomes(YACs)
– linear DNA which mimics a yeast chromosome by having yeast
specific origin, centromere and telomere sequences
– YACs will survive not only in yeast but also in mice.