Transcript DNA Analysis using Gel Electrophoresis

DNA Analysis using Gel
Electrophoresis
DNA Analysis
• DNA can be analyzed in several different
ways depending on the information that
you want to find out
• DNA sequencing will tell you the letters in
the sequence (Ex: AGTGGATATG . . .)
• Gel electrophoresis will look for specific
sequences that can indicate the presence
of specific genes
DNA sequencing
Gel electrophoresis
Gel electrophoresis
• Works by separating DNA segments (aka
fragments) by placing them in a gel
substance and applying an electrical
current.
• DNA fragments are sorted according to
size
How do we get these fragments?
• To cut DNA in specific places, we use restriction
enzymes (called restriction endonucleases)
• Restriction enzymes cut DNA at specific
sequences (like CCCTTT, they might cut
between the last C and first T)
• If you have that sequence in your DNA it will be
cut, if not then it won’t. This results in different
people having different DNA fragments.
• http://www.dnalc.org/ddnalc/resources/restriction
.html
Why are we cutting the DNA?
• We cut the DNA to see if the person has a
specific sequence of DNA (a gene!)
• We are testing to see which genes people
have and if they have 0, 1, or 2 copies.
What can it tell us?
• If the gene contains the sequence
CCCTTT, we can use a restriction enzyme
that cuts at that sequence.
• Person 1: ATCTATCTCCCTTTGGA
Result = 2 fragments
• Person 2: AGTTGTAGTCCTTGATA
Result = 1 fragment
Gel Electrophoresis Procedures
1) Get DNA sample and amplify it using
PCR.
2) Mix DNA with restriction enzymes and
allow DNA to be cut into fragments.
3) Pour buffer solution into electrophoresis
machine (this will let the electricity flow)
4) Use a micropipettor to load DNA samples
into small holes (called wells) in your gel
(made of agarose)
Micropipettor = used to pick up small, exact amounts of a sample
You can set it to a certain amount and then it will pick up exactly that
amount each time!
Procedures continued . . .
5) Place lid on machine and plug into power
source.
6) Set to the correct voltage and turn on.
7) Let run until you see the sample has moved
through the gel.
8) Turn machine off and unplug it. Remove lid and
take out gel.
9) Stain gel with blue dye so we can see the
fragments.
10) Analyze gel.
How does it work?
• DNA molecules are slightly negative, so they are
attracted to a positive charge.
• The samples are placed at the negative end of
the machine.
• When the current is turned on, the DNA will
move towards the other side of the machine
because that side is positive.
• Smaller fragments move faster than larger ones,
so they are seen farther away from the wells.
http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
Analysis
• People with lines (bands) in the same
spots have the same genes.
• Different positions tell us if the person has
2 copies of the dominant allele, two of the
recessive allele, or one of each.
Summary
Go here to learn more!
• http://learn.genetics.utah.edu/content/labs/gel/