DNA Extraction and Gels
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Transcript of DNA Extraction and Gels
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