Download Manipulating DNA

Manipulating DNA
• Recombinant DNA: a combination of DNA in
ways that “nature never intended”
– Usually, DNA from different organisms combined
• Terminology
– Clones: organisms, cells, or molecules that are
identical copies (in this case, usually DNA)
• Tools: restriction enzymes, vectors
– Procedures: gel electrophoresis, Southern blot,
PCR, bacterial and cell culture methods
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Restriction enzymes
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• Produced by bacteria to destroy virus DNA
– “restriction endonucleases”
– Named after bacterium, e.g. EcoR1 from E. coli
• Recognize specific sequences in DNA
– Recognition regardless of source of DNA
– Sequences usually 6 bases, feature palindromes
• Enzymes usually make staggered cuts
– Produce “sticky ends” which are free to base pair
with other DNA cut the same way.
• Different DNA molecules cut with same enzyme
can be spliced together.
Restriction enzymes
http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif
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Cloning
• Having only a few DNA molecules is not too useful
– Can’t find them on a gel
– Absorb to things and get lost
– Destroyed by stray enzymes
• Carrying out genetic manipulation requires multiple
copies of the same molecule
– Identical copies: clones
– Naked pieces of DNA are easily lost or destroyed
by enzymes and cannot be replicated.
– Insertion into a “vector” solves those problems.
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Cloning and vectors
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• Traditional cloning: to get multiple copies of
DNA of interest, must get it into a cell
– Vector: means for moving DNA into a cell where
multiple copies can be made
– Vectors may be plasmids
• Easily shuttled into bacteria or plants (Ti plasmid)
• Plasmids are replicated within cells
– Vector may be a bacteriophage
• Protein coat in which DNA is packaged, then
delivered
Plasmid vectors
• Plasmids are circular DNA molecules
• Plasmids can be cut with restriction enzymes
and piece of DNA inserted
• Plasmids can be taken up by bacterial cells
– When cell multiplies, so does plasmid with DNA
• Plasmids can multiply to high numbers in cell
– Thus making multiple copies of your DNA
• Bacterial clone (colony) that has your DNA
must be selected for and screened for.
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Getting a plasmid into bacteria is one thing
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How do you know which of
the few bacteria actually
took up the plasmid?
How can you know your
gene of interest is actually
in the plasmid?
(cut plasmid can recircularize w/o insert)
http://wilfred.berkeley.edu/~gordon/BLOG-images/bacteria1.jpg
Cloning with pUC18
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Selection: bacteria
that don’t have the
plasmid are
unwanted (and
numerous). Ampicillin
in culture medium
kills those.
Screening: Not all plasmids
contain inserted DNA. Betagalactosidase (lac Z) turns
“X-gal” blue; insert in lac Z
gene = white colonies.
http://www.isu.edu/~shiemalc/courses/Molecular%20Biotechnology/Images/pUC18.gif
Screening with X-gal
www.mun.ca/.../scarr/ Blue_&_White_Colonies.html
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Using phage
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• DNA can be cloned into Bacteriophage
– When virus replicates, makes multiple copies of its
DNA including the DNA to be cloned
– Plaque contains new viruses and cloned DNA
http://www.tk2.nmt.ne.jp/~czar/phage_
gif/plaq_p7.jpg
YAC
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• Yeast artificial chromosome
– Has a centromere, telomeres, restriction sites, and
lots of room to put cloned DNA in.
– Carries very large pieces, good for eukaryotic DNA
libraries
• A set of clones containing most to all of an
organism’s genome
• DNA cut with restriction enzymes, cloned;
individual clones have to be identified later.
– Yeast = eukaryote and safe; good for making
proteins, especially glycoproteins.
YAC structure
YAC starts circular.
Contains a centromere, an
origin to allow its replication,
and two BamH1 sites that open
up the circle leaving telomeres
at the end.
DNA can be cloned into a
restriction site (e.g. EcoR1).
http://www.inrp.fr/Acces/biotic/biomol/outilsbm/images/yac4.gif
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PCR: identification, amplification, or cloning
of DNA through DNA synthesis
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• DNA synthesis, whether PCR or DNA replication in a
cell, is carried out by DNA polymerase.
• DNA polymerases have three requirements:
– Only work in a 5’ to 3’ direction
– Require a template: DNA to copy.
– Require a primer: a free 3’ OH end to add to.
• PCR uses Taq polymerase, a heat stable polymerase
from the thermophilic bacterium Thermus aquaticus.
Facts about PCR
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• PCR requires sequence information
– Primers on either side of area of interest must be
complementary, thus you must know sequence.
• Works best on fairly small fragments of DNA
• Theoretically, can work on a single DNA molecule.
– This is where PCR really shines.
– Is a method for amplification: make enough DNA to
see a band on a gel; substitute for traditional
cloning.
PCR
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Template DNA
Primers
dNTPs
Taq polymerase
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2.
3.
4.
http://www.pitt.edu/~heh1/PCR.jpg
Denature (high heat)
Anneal primers
Synthesize
Repeat
Some PCR details
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• Primers: 18-22 nucleotides long, typically
• Geometric increase in DNA: from 1 molecule, 34
million in 25 cycles.
• 5000 bp limit in size
• Most reliable when starting with at least 10-100 DNA
molecules.
• http://faculty.plattsburgh.edu/donald.slish/PCR
mov.html
• MANY animations online.
Making a genomic library
• DNA from organism is cut with restriction
enzymes; vector DNA (e.g. YAC) also cut.
– Restriction fragments inserted into vectors
– Vectors inserted into cells which grow
• Multiple copies of DNA obtained
• Library of expressed DNA using cDNA
– If expressed, then can be collected as mRNA
– Reverse transcriptase can make DNA from RNA
template
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Action of reverse transcriptase
Poly-T synthetic
oligonucleotide acts
as primer, mRNA is
the template.
DNA-RNA hybrid is denatured, RNA is digested away,
and DNA polymerase makes a ds DNA of the ss DNA.
This piece of DNA, representing an active gene, can
now be cloned.
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Finding a clone
• Finding a clone anywhere involves using a
probe.
– A probe is a nucleic acid that is sufficiently
complementary to the DNA of interest that it will
base-pair with it.
– The probe must be labeled or else the binding will
be invisible (and thus worthless)
– Labeling is usually with radioactive isotopes or
fluorescent compounds
• Detection is with x-ray film
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DNA hybridization
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• Use of a probe results in a ds molecule where the
strands are from different sources: hybrid.
• Heat (or alkali) required to denature DNA (ss)
• Done at high salt
– Na+ stops PO4- from repelling each other
• Done at elevated temperature
– Incorrect (weak) matches between strands come
apart, allow correct ones to form.
• Complementary strands “zip up” once they find
each other.
• If you have primers, you can use PCR instead to
find your DNA: the one that gets amplified.
Colony (in situ) hybridization
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• For finding e.g. bacterial colonies that contain
your clone.
• Cells in colonies on a plate are lysed, the DNA
is denatured and transferred to a membrane.
• Membrane is mixed with probe (complementary
and labeled)
– Loose probe is washed away
– Membrane covered with x-ray film
– Colony with clone of interest makes spot on film
Characterizing cloned DNA
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• Restriction maps
• In this example, a
1650 bp clone is cut
by SalI into 2 pieces,
large and small.
• Cutting with KpnI
makes 3 pieces, so 2
sites must be present.
• Combination of
enzymes allow pieces
to be put in order.
http://www.umanitoba.ca/faculties/afs/plant_science/courses/39_314/l07/map2digest.gif
Southern Blot
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DNA sequencing
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• DNA sequencing is a method that reveals the
sequence of nucleotides in a stretch of DNA.
• Sequencing requires DNA synthesis; you
actually determine the sequence of a
complementary strand.
• A template and primer are mixed with a
polymerase, dNTPs, and a smaller proportion of
ddNTPs
DNA sequencing-2
ddNTPs (Dideoxy NTPs) are missing an
-OH group at the 3’ position.
Remember that this is where the next
nucleotide must attach during DNA
synthesis.
• There are many DNA molecules in a test
tube, all being copied at the same time. If
a ddNTP is inserted into the growing DNA
strand, synthesis of that molecule
STOPS.
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/dideoxy.jpg
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DNA sequencing-3
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• ddNTPs are inserted at random (that is, when G is
called for, sometimes the enzyme will grab ddGTP)
• This process results in a collection of DNA molecules
of different lengths that can be separated on a gel.
• Since all 4 ddNTPs are present, growth of the DNA
can stop after any base, producing a large number of
DNA molecules that differ in size by 1 base.
• Each ddNTP is fluorescently labeled with a different
color. The whole mixture is separated in a run-off gel,
and the different colors marking which base
terminated the reaction can be detected.
http://www.dnalc.org/ddnalc/resources/shockwave/cycseq.html
DNA sequencing-4
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This picture shows a few of the different sized molecules that
can result.
Notice that each ends with a particular ddNTP; in modern
practice, this will have a color associated with it and thus be
identified.
derived from opbs.okstate.edu/.../ img015.jpg
DNA sequencing gel
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Inexpensive, older
method:
Four reaction tubes, each
containing a different
ddNTP:
ddATP, ddCTP, etc.
Contents of each tube run
in a different lane, stained
to see the bands.
Gel read from bottom up.
carnegieinstitution.org/.../ sequencinggel.html
DNA sequencing w/ fluorescent labels
ddCTP = blue
ddATP = green
ddTTP = red
ddGTP = grey
medlib.med.utah.edu/.../biochem/ Formosa/menu.html
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