BIOL 112 Lecture Notes - Lecture 12: Polymerase Chain Reaction, Cystic Fibrosis, Restriction Fragment Length Polymorphism

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20 Apr 2012
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To make an organism express a new phenotype
1)
To understand how the gene itself works (in isolation)
2)
this will be helpful when we don't know what's wrong with the disease….
To discover the biochemical basis of an inherited disease, cystic fibrosis.
--->TYPE II : Positional cloning technology - genetic engineering to study gene in isolation
Why clone?
Respiratory infections which result from an inability to move mucus out of the lungs.
Before modern treatment, only treated the symptoms not the underlying cause.
Most common autosomal disease among Caucasians
Recessive; 1/25 Caucasians carry a mutant allele.
Something to do with salt regulation. It's said that midwives knew that if an infant's sweat was excessively salty, it
would die young.
We don't know its genetic sequence...
About it….
Sturtevant and his genetic mapping might allow us to identify and clone a gene without knowing anyhing else about it
except it's phenotype and its map position.
If we find two genes that flank the CF gene and code for genes we know, then somewhere in the DNA between those genes
is the CF gene.
Example: Cystic Fibrosis
Map the gene for cystic fibrosis in humans.
Since non-coding DNA is not selected for, the restriction sites tend to
vary a lot from one individual to the next. Since the restriction sites are
determined by DNA sequence, variations in these sites will segregate in a
basically Mendelian fashion.
Problem
Not many traits in humans that show simple Mendelian
inheritance against which you can map your disease trait.
You can't do controlled matings in humans.
Solution
We can create phenotypes --> Restriction fragment length
polymorphisms (RFLPs) - there are restriction sites randomly
distributed (~every 4kb) in DNA
Find RFLPs by looking for changes in the size of resulting DNA fragments after
digesting genomic DNA with restriction enzymes.
[Melting--> Annealing of primers--> Elongation (DNA polymerase)]
Make DNA primers that flank the stretch of DNA of interest,
1)
complementary to each of the strands.
Mix the primers with a single strand of template DNA.
2)
Melt the DNA by heating allow primers to anneal/hybridize to the 3' end of the DNA to be amplified on each
complementary strand.
3)
Use DNA polymerase to initiate polymerization of complementary strands in opposite directions. The direction of synthesis
is toward the sequence of the other primer so both primers are making copies of the sequence in between the primers.
4)
Now you have two double strands of the DNA of interest! The new strand has incorporated the primer which is not
5)
Problem
If all the genomic DNA is digested wit Bam HI and there will be
millions of fragments (the smear in the electrophoresis lab 12).
So how do you test for the presence of a articular RFLP?
Solution
Amplify a small stretch of DNA so that you can study it:
Polymerase Chain Reaction (PCR) - a method of producing large
quantities of a short stretch of DNA from a known template DNA
molecule.
DENT LECTURE 12: Biotechnology: positional cloning
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