Download 03 Biotechnology Note

Biotechnology (chapter 6)
Recombinant DNA
– a fragment of DNA composed of sequences originating from at
least 2 different sources
– scientists use this DNA to mess with (manipulate) genes and
proteins
Restriction Endonucleases
 AKA restriction enzymes
 molecular scissors that can cut double stranded DNA at a
specific base pair sequence
 each type of restriction enzyme (RE) recognizes a
characteristic sequence of nucleotides know as the
recognition site
 this site is usually palindromic and is 4 to 8 nucleotides
long
 once bound, the RE cuts the DNA creating 2 DNA fragments
Blunt Ends
Sticky Ends
 RE’s were found in bacterial cells as part of their immune
system
 they recognize the foreign DNA of a virus and cut it up into
little pieces
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DNA Ligase
 enzyme used to join the cut strands of DNA
 2 strands of DNA cleaved by the same RE will naturally
want to join but help is need to connect the sugarphosphate backbone
 molecular biologists use T4 DNA ligase which originated
from the T4 bacteriophage
Gel Electrophoresis
 the separation of DNA fragments through a gel meshwork
(agarose)
 solution containing different sized DNA fragments is mixed
with a dye and glycerol and is placed in wells in the gel
 an electric current is then run through the gel and the
fragments move towards the positive cathode
 the smaller the fragment, the further it will travel
 the fragment lines are then stained and compared to other
gels
Plasmids
 small circular pieces of DNA that can enter and exit
bacterial cells
 plasmids often carry genes for resistance to toxins and
drugs and it is by passing these from bacteria to bacteria
that resistance can spread
 we put genes we want expressed into the plasmids and them
put them in bacterial cells
 insulin is made this way (human gene being expressed by
bacteria)
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Transformation
 is the introduction of DNA into a bacterial cell and having
that cell accept the new DNA
Genetic Engineering
History
 principals experimentally established in early 1970’s by
Stanly Cohen and Herbert Boyer
 experiments resulted in a method of selecting, recombining
and introducing new genes into bacteria using plasmid
vectors
Products of Genetic Engineering
 diabetes: more than 90% of diabetics rely on insulin
produced by bacteria
 AIDS: used to rebuild muscle lost to deterioration
(somatropin)
 agriculture: crops are sprayed with bacteria that have been
engineered to disable them from forming an ice nucleating
factor
 ice nucleation factor is a protein found on bacterial cell
walls that allows ice to seed and crystallize
Somatropin
 somatotropin is the human growth hormone
 we used to harvest this hormone from the pituitary gland of
cadavers -expensive
 somatropin in an engineered gene that is produced in
bacteria
 this drug is identical to natural human growth hormone and
is used to treat growth disorders
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 abused by athletes as it helps build muscle mass
 some US states allow the injection of bovine somatotropin to
help boost milk production – illegal in Canada
Advanced Molecular Biological Techniques
The Polymerase Chain Reaction (PCR)
 proposed by Kary Mullis in 1987, Nobel Prize in 1992
 the PCR cycle allows for the rapid coping of DNA
 a sample of DNA is place in a solution containing
nucleotides and enzymes
 this broth is heated to 94oC – 96oC which causes the DNA
strands to separate, the temperature is then reduced
 using a polymerase enzyme from the bacterium Thermus
aqauticus, found in hot springs, each strand is then paired
with complementary nucleotides resulting in 2 complete
strands of DNA
 repeat
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Restriction Fragment Length Polymorphism (RFLP)
 polymorphism is any difference in a DNA sequence that
can be detected between individuals
 so in humans, we have the same genes but different alleles
 but for forensics, scientists actually look at the non-coding
regions of DNA which contain variations in the number of
tandem repeats
 by running a DNA analysis in a process similar to gel
electrophoresis, scientists can look at 13 or so different noncoding regions
 this is called Restriction Fragment Length Polymorphism
Analysis
 then we catch the bad guy
DNA Sequencing
 determining the exact sequencing of base pairs for a certain
gene in order to analyze gene structure and its relation to
gene expression and protein formation
 Human Genome Project – used the Sanger dideoxy method
which sort-of counts the A T G’s and C’s
 Venter created the “whole-genome shotgun cloning method”
which was faster (computers and such to aid in the results)
which is why he is credited with deciphering the genome on
April 6th, 2000
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Applications
Medical Applications
 Genetic Screening – process by which an individual’s DNA is
scanned for genetic mutations
 Gene Therapy – the alteration of a genetic sequence in an
organism to prevent or treat a genetic disorder (not really
used yet)
Agricultural Applications
 make transgenic plants which are organisms that have
foreign DNA
 why? increased yield, frost resistance, insect resistance etc
 fallout is on insects and animals that eat these plants…
Forensics
 RFLP and PCR are used to create DNA fingerprints
 1987 – 1st court case (Britain) to use DNA fingerprinting
 it is now illegal in Canada to refuse to give a DNA sample if
the fuzz asks for one
The Future
…
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