DNA extraction & gels.ppt

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 6 months ago)

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After discovering DNA was the carrier of
genetic info, it became apparent that control
over its mechanisms would be essential


We have ways of isolating DNA, determining
the order of its nucleotides, and can even
insert of piece of DNA from one organism to
another


To get at DNA, we have to remove the two
membranes that protect it


Cell membrane and nuclear envelope


These are made of phospholipid bilayers


Detergents are used to dissolve the lipids to
expose the genetic material


Next, a protease is used to dissolve unwanted
proteins


Sodium acetate further precipitates the
remaining protein


Next, the sample is centrifuged, and the
protein can be removed


DNA can be precipitated by the addition of
cold ethanol


This both precipitates the DNA and washes the salt


Finally, the solution is centrifuged, and the
pellet of DNA is removed


The DNA extracted is often very long


Too long to be useful in many cases


Restriction enzymes are used to cut the DNA


However, it is not a random cut site, rather a
very specific sequence


There are many different enzymes (~1000),
many with their own unique sequence they
attach to


They do not cleave the DNA straight down,
instead the cut the bond between adjacent
nucleotides


The uneven cutting creates what is known as
“sticky ends”


They are “sticky” because they are single
stranded


DNA is normally doubled stranded, and naturally
anneals (reforms hydrogen bonds) into double


There is a random number of cut sites on
every strand of DNA


But, in the end, the sample is cut into smaller
pieces


These smaller segments can be run through a
gel


The gel is a semi
-
solid, porous medium that
DNA can move through


Electrodes are set at either end to make one
positive, the other negative


DNA is slightly negative, and will be attracted
to the positive end


The smaller samples can move through the
gel faster, and will make it farther down


Each band represents a strand of DNA, of a
specific length


The bands furthest down are the smallest


Gels are normally run to compare a sample of
DNA to a known sample, cut with the same
enzymes


If the bands line up, it is likely that the
samples are the same


This is very useful in DNA fingerprinting