Download E. Coli - mrkeay

DNA Technology
Biotechnology Tools & Techniques
Recombinant DNA
– fragment of DNA composed of DNA
sequences originating from at least two
different sources
Restriction Endonucleases
– aka restriction enzymes
– molecular scissors that cut double-stranded
DNA at a specific base-pair sequence
Biotechnology Tools & Techniques
• Recognition Site
- A specific sequence within double-stranded
DNA where restriction enzymes recognize
and cut
- Usually palindromic (same front & back…eg.
level, stats, racecar)
Biotechnology Tools & Techniques
• Blunt ends
- DNA fragment from a restriction enzyme
where the ends are base paired
- Eg. SmaI, AluI
• Sticky ends
- DNA fragment from a restriction enzyme
where the ends contain short, singlestranded overhangs
- Eg. EcoRI, SalI, HindIII
Restriction Endonucleases
•
•
•
•
Isolated and purified solely from bacteria
Essentially a bacteria’s immune system
Also how a virus can break down host DNA
Named according to the bacteria from which
they originate. Example, in Escheria coli (E.
Coli), the restriction enzyme is called EcoRI
• BAMHI comes from Bacillus
amyloliquefaciens; H is the strain, 1 is b/c it’s
the first endonuclease isolated from it
Restriction Endonuclease Cut Sites
Restriction Endonucleases
• Recognize and bind to sequences which are
4 to 8 nucleotides long
• Eg. EcoRI looks for 5’ GAATTC 3’
3’ CTTAAG 5’
and cleaves (cuts) between G and A
• A 6 base-pair sequence like this would occur
every 4x4x4x4x4x4 = 46=4096 base pairs
Methylases
• Specific enzymes found in prokaryotes &
eukaryotes
• In prokaryotes, they modify the recognition site
(add a methyl group to one of the bases) which
prevents the DNA from being cleaved
• When foreign DNA is inserted, it is not methylated,
so the desired gene fragment is protected from
being cleaved
• In eukaryotes, methylases control transcription
DNA Ligase
• Enzyme used to join the cut strands of DNA
together (best for sticky ends)
• T4 DNA ligase (from T4 bacteriophage virus)
works better for blunt ends
Gel Electrophoresis
• Process of
separating DNA
on the basis of
size by sorting
through a gel
meshwork
• Like a molecular
sieve
Gel electrophoresis
• DNA is neg. charged due to phosphate group
• DNA already subjected to restriction
enzymes (pieces of various sizes) are loaded
in wells with a buffer solution containing
glycerol (dye to view the DNA)
• Larger fragments move through the agarose
gel more slowly, smaller fragments move
faster and farther
Gel electrophoresis
• Uses DC current to place a neg charge at the
near end (closest to DNA); pos charge at the
far end
• Once complete, fragments are made visible
by staining (often ethidium bromide which
fluoresces under UV light
• Not just nucleic acids – can run proteins
using polyacrylamide gels
Plasmids
• Recall that bacteria contain a singular
circular chromosome, along with many
small, circular pieces of DNA called plasmids
• Plasmids carry genes which confer antibiotic
resistance, as well as resistance to toxic
heavy metals and industrial chemicals
• We can use plasmids for biotechnology,
since bacteria are able to express foreign
genes inserted into plasmids
Plasmids
• Relationship between bacteria and plasmids
is endosymbiotic – both benefit
• Engineered plasmids contain a region that
can be cut by many restriction
endonucleases and found nowhere else on
the plasmid – called the multiple cloning site
• In which gel lane was this plasmid run?
Steps in Recombinant DNA
1. Plasmid DNA is cut open using R.E.
2. Gene fragment to be inserted is opened
using same R.E., therefore ends will match
3. DNA ligase joins fragment to the plasmid
4. Plasmid is introduced into bacterial cell via
transformation
Recombinant DNA
Transformation – introduction of foregin DNA,
usually by a plasmid or virus, into a bacterial
cell
Vectors – vehicles by which DNA is introduced
into host cells (bacteria or viruses)
Host cell – the cell that contains the foreign
DNA and whose machinery is being used to
express that DNA
Homework
• Good summary chart on Pg. 289 - 290
• Do Q 1-6, 8, 11, 14, 15, 16, 17
Polymerase Chain Reaction (PCR)
PCR
• Amplification of DNA sequence by repeated cycles of
strand separation and replication
• Proposed by Kary Mullis in 1987; awarded Nobel
Prize in Chemistry in 1992 for the discovery
• Direct replication of a DNA sequence without
insertion into a plasmid
Steps in PCR
1. Heat DNA to 94-96°C to separate strands
2. Two primers (chunks of single-stranded DNA) are
made which correspond to the beginning and end
of DNA to be copied
3. Heat to 72°C to extend primers using Taq
polymerase
4. Separate strands and anneal (join) primers
5. Extend primers
6. Repeat steps 4-6 for 30 – 40 cycles
PCR
• Useful in criminal investigations (forensics), medical
diagnoses, and genetic & evolutionary biology
(common ancestry)
• Only need small amounts of DNA
Restriction Fragment Length Polymorphism
(RFLP)
RFLP
• Polymorphism – any difference in DNA sequence that
can be detected between individuals
• Only identical twins would not be polymorphic
• RFLP is a process whereby differences in DNA
fragment lengths between individuals are compared
Steps in RFLP
1. DNA extracted from sample and cut by R.E.’s
2. Digested fragments are run on a gel, which is placed
in a denaturing solution against a nylon membrane
3. Single stranded DNA migrates from gel to nylon
membrane using electric current = “Southern
blotting”
4. Membrane soaked in solution with radioactive
probes
Steps in RFLP
5.
6.
7.
8.
Radioactive probes anneal at specific locations
Membrane placed against X-ray film for 2 – 3 weeks
Radioactive probes burn image in X-ray film
Developed X-ray film called an autoradiogram
Homework
• Summary charts Pg. 300 and 303
Medical Applications
• Development of insulin by GE in the 1980’s
• HIV can be detected by PCR soon after infection
• Genetic mutations can be detected before the
manifestation of symptoms
• Being able to pinpoint the location of genes will lead
to more developments in gene therapy (eg. Gene
inserted into adrenal gland cells which secrete a
transmitter which inhibits the sensation of pain)
Agricultural Applications
• Transgenic plants – 1981 (Nestor and Chilton)
Ex. Flavr Savr Tomato (gene for ripening in fruit)
• Crops have been genetically engineered to increase
yield, hardiness, uniformity, herbicide tolerance, and
insect and virus tolerance
• Insert polyester gene into a cotton plant to make
poly-cotton fibers
• Transgenic corn – produces Bt toxin which kills
butterflies!
Forensic Applications
• 1987 – first court case using RFLP where accused was
convicted
• Valuable tool for investigators, but cannot convict on
its own
• Both PCR and RFLP are used in criminal investigations
depending on state of the DNA sample
• Both techniques provide a ‘fingerprint’