Recombinant DNA Technology
Review
• What are enzymes? What function do they
serve?
• What makes up the DNA sequence?
• How is the genetic code determined in an
organism?
Recombinant DNA Technology
http://www.youtube.com/watch?v=x2jUMG2E-ic
Discovery of restriction enzymes
• Bacteriophages infect bacteria by inserting their
genetic material (DNA) into the DNA of bacteria
• In the 1960s, microbiologists discovered that some
bacteria are protected from bacteriophage infection
because they can restrict phage replication
• Scientists proposed that restricted growth of phages
occurred cause the bacteria contained enzymes that
could cut viral DNA into small pieces and prevent
replication
• These enzymes were called restriction enzymes
Restriction Enzymes
• Primarily found in bacteria
• Given names based on genus and species names of bacteria
from which they are isolated
• Cut DNA by cleaving the phosphodiester bond (in sugarphosphate backbone) that joins nucleotides in DNA strand
• Show specificity for certain DNA substrates
• Enzymes bind to, cut (digest) DNA within specific sequences
of bases called recognition sequence or restriction site
• Typically recognize 4, 6, or 8 nucleotide sequences
Restriction Enzymes
• Restriction enzyme EcoRI,
named because it was
discovered in Escherichia
coli
Restriction Enzymes
• Some restriction enzymes cut DNA to create DNA
fragments with overhanging single-stranded ends
called “sticky” or cohesive ends
Table 3.1 – Cohesive Ends
Restriction Enzymes
• Some enzymes generate fragments with nonoverhanging or blunt ends
Restriction Enzymes
• Enzymes that produce cohesive ends are often
favored over blunt-end cutters because DNA
fragments with cohesive ends can easily be
joined together.
• DNA from any source, bacteria, humans, dogs,
frogs, dinosaurs, and ancient human remains
can be digested by a particular restriction
enzyme as long as it has the restriction site for
that enzyme!
Restriction Enzymes
• DNA cutting enzymes
– Cuts double stranded DNA
– Two incisions to cut DNA at sugar-phosphate bond on
each strand
• Very specific
– Enzymes recognize short nucleotide sequences
– Enzymes cut the DNA at specific points within DNA
• For example – if the restriction enzyme cuts
between a C and G then
5'GGCC3'
3'CCGG5'
5'GG CC3'
3'CC GG5'
5'GG CC3'
3'CC GG5'
Restriction Enzymes
• Each restriction enzyme recognizes a
specific restriction site, a short nucleotide
sequence
• Some restriction enzymes cut straight
across the DNA
• Some restriction enzymes cut at offset
nucleotides
• For example GAATTC
G AATTC
G
CTTAAG
C TTAAG
CTTAA
AATC
G
Restriction Enzymes
• Sequences that are cut at offset points
produce overhanging pieces of DNA called
“sticky ends”
Sticky end
G
CTTAA
AATC
G
Restriction Enzymes
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html
Restriction Fragments
• Each cut of DNA creates restriction
fragments
• A long strand of DNA may be cut several
times by a restriction enzyme to create
several restriction fragments
Why are restriction fragments useful?
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Recombinant DNA
Gene isolation
Genome mapping
Gel electrophoresis
Genetic engineering
Gene therapy
Recombinant DNA Technology
• Technique that allows DNA to be combined
from different sources
Recombinant DNA/Restriction Enzyme
Example
• In the early 1970s, a group of scientists joined
together DNA from E. coli chromosomes and
DNA from a primate virus
– They isolated the DNA from each organism and
cut them into fragments with EcoRI
– They added the E. coli and viral DNA fragments to
a reaction tube and added DNA ligase
– They created a hybrid of the two DNA types,
recombinant DNA
Recombinant DNA Technology
• Plasmids are considered extrachromosomal
DNA because they are present in the bacterial
cytoplasm in addition to the bacterial
chromosome
• Plasmids can be used as vectors, pieces of
DNA that can accept, carry, and replicate
(clone) other pieces of DNA
Recombinant DNA Technology
• Plasmids are considered extrachromosomal
DNA because they are present in the bacterial
cytoplasm in addition to the bacterial
chromosome
• Plasmids can be used as vectors, pieces of
DNA that can accept, carry, and replicate
(clone) other pieces of DNA
Plasmid Vectors
– used to
clones specific
DNA
http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html
NIH and RAC
• Scientists were concerned about:
– with what might happen if recombinant bacteria
were to leave the lab
– if such bacteria could transfer their genes to other
cells
– survival in other organisms including humans
• National Institutes of Health (NIH) formed the
Recombinant DNA Advisory Committee (RAC)
to evaluate the risks of recombinant DNA
technology and establishing guidelines
• http://oba.od.nih.gov/rdna_rac/rac_about.html
Transformation
• Transformation – process for inserting foreign DNA
into bacteria
– Treat bacterial cells with calcium chloride solutions
– Add plasmid DNA to cells chilled on ice
– Briefly heat the cell and DNA mixture, plasmid DNA will
enter the bacterial cell
– Once inside bacteria, plasmids replicate and express their
genes
Selection
• Joining of DNA fragments and plasmids by
transformation can be inefficient
– Some plasmids will rejoin and recircularize and not bond
with any foreign DNA
– During transformation, many cells will not take up DNA
• Recombinant bacteria, those transformed with a
recombinant plasmid, must be separated from
nontransformed bacteria as well as cells with
plasmids without foreign DNA
• This process is called selection because it is designed
to identify (select for) recombinant bacteria and
prevent the growth of nontransformed bacteria and
bacteria without foreign DNA
Polymerase Chain Reaction
• Technique for making
copies or amplifying
a specific sequence of
DNA in a short period
of time.
PCR (Polymerase Chain Reaction)
1. Target DNA to be
amplified is added to a thinwalled tube and mixed with
deoxyribonucleotides (dATP,
dCTP, dGTP, dTTP), buffer
and DNA polymerase.
Primers are added to the
mixture; they are
complimentary to
nucleotides on opposite ends
of the target DNA.
2. Tube is placed in a
thermal cycler (gene
cycler). This will take the
sample through a series
of reactions called the
PCR cycle.
Thermal Cycler
A.
B.
C.
Denaturation – tube is
heated to ~94-96ºC
separating DNA into single
strands
Hybridization (annealing) –
tube is cooled to ~60-65ºC
allowing primers to bond to
ends of DNA
Extension (elongation) –
temperature raised slightly
and DNA polymerase copies
the DNA by binding to
primer and making a new
strand
DNA polymerase used in this reaction is Taq
DNA polymerase from Thermus aquaticcus.
Bacteria is adapted to live in hot springs it has
enzymes that can withstand high temperatures
necessary for PCR.
PCR
• At the end of one
cycle, amount of DNA
has doubled.
• Researchers can
repeat cycle (usually
20-30 times) to
amplify millions of
copies of DNA
Uses of PCR
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Studying gene expression
Detection of viral or bacterial infections
Diagnosing genetic conditions
Amplifying trace amounts found at crime
scenes
• Amplifying trace amounts found in ancient
DNA
http://www.dnalc.org/resources/animations/pcr.html
http://www.youtube.com/watch?v=_YgXcJ4n-kQ
http://www.maxanim.com/genetics/PCR/pcr.swf
http://practicality.wordpress.com/2008/01/13/the-pcr-song-with-lyrics/
Gel Electrophoresis
• Used to separate and visualize DNA fragments
based on size
• Agarose gel is a semisolid material with small
pores through which DNA can travel;
fragments are separated by size – smaller
fragments can travel further through pores
than larger fragments
1. To run a gel, it is submerged in buffer solution
that will conduct electricity
2. DNA samples are loaded into small wells and an
electric current is passed through the gel
3. The sugar-phosphate backbone renders DNA
with a negative charge; therefore DNA will
move to the positively charged end
4. Because migration distance is inversely
proportional to the size of the DNA fragment,
large DNA fragments migrate short distances
and small fragments migrate faster
5. Dyes are added to monitor DNA migration
Gel Electrophoresis Animations
• http://www.dnai.org/b/index.html
• http://www.teachersdomain.org/resource/tdc
02.sci.life.gen.creatednafingerprint/
• http://learn.genetics.utah.edu/units/biotech/
gel/
• http://www.youtube.com/watch?v=2UQIoYh
OowM