Transcript Finding Your Alleles

PV92
PCR
Finding
Your
Alleles
Listed here are functional
genes (genes that code
for proteins).
PV92 is not functional
(as far as we know) so it
is not shown. It is an
intron.
PV92 is 601 base-pairs long.
Some PV92 segments carry ONE Alu
SINE (Single INterspersed Element; aka:
Alu repeats)
Each Alu is about 300 base-pairs long.
So PV92 with an Alu would be ______
base-pairs long.
10% of your genome is made of
Alu SINE!
Locus =
location of a
gene or element
Possible genotypes:
Alu repeat
Alu repeat
Alu repeat
++
+--
Finding your genotype
1. Get DNA out of hair cells
2. PCR the PV92 segment.
3. Gel Electrophoresis of
amplified DNA regions with
(or without) the Alu repeat.
Need a “hair tag”
Get DNA out of Hair Cells
• Instagene Matrix has beads that cling to Mg2+ .
Without the Mg2+ available, the DNAases that
will come out of lysosomes won’t work (which
is good, because then the DNAases can’t
degrade your DNA!)
lysosome
nucleus
DNA
Mg2+
DNAases
Mg2+
Normal Conditions:
Virus DNA enters
cell, DNAases are
released from
lysosome, Mg2+
activates the
DNAases, &
goodbye virus DNA!
Get DNA our of Hair Cells
• Heating unclumps the cells and helps
deactivate the DNAases.
• High heat breaks down all membranes (cell,
nucleus, lysosomes, etc.) so DNA is
released.
PCR = Polymerase Chain Reaction
• MASTER MIX HAS
• Primers – Forward and reverse, that will latch on
to the top strand and the bottom strand of DNA at
60O (after it is denatured at 92O).
Forward
Primer
Reverse
Primer
Master Mix has
• Taq polymerase – at 72O this enzyme will
add As, Ts, Cs, and Gs to the DNA where
the primers leave off.
• Nucleotides (As, Ts, Cs, and Gs) for Taq
polymerase to add.
• MgCl2 – to stabilize DNA.
Taq
polymerase
Taq stands for Thermus aquaticus, a bacterium
that lives in hotspring. To do PCR, DNA must
be split apart (denatured). This can be done
using high heat. So the enzyme that puts
nucleotides back on to the DNA has to be able to
withstand high heat. Enzymes in bacteria that
live in hotsprings can do this.
PCR Annimation
• http://www.sumanasinc.com/webco
ntent/anisamples/molecularbiology/
pcr.html
Denature – 92O
Anneal – 60O
Extend (or Polymerize) – 72O
• Agarose acts as a
“molecular sieve.”
Small things pass
through it easily,
larger things don’t.
• The smaller the
DNA fragment, the
faster it runs.
• DNA has a charge, so you put
the wells with DNA
at the – end and the
DNA will be
attracted to the +
end.
Gel Electrophoresis
Gel Electrophoresis
Results
Loading dyes are added to every
sample, including the controls, so that
(1) the sample sinks to the bottom of
the well (due to the glycerol in the dye)
and (2) so you know when your run is
complete. Because the loading dye is
so small, it will run faster than any of
the DNA samples. When the loading
dye gets near the end, it’s time to quit
the run before you DNA runs off the
end!
Ladder
(molecular
mass ruler, or
MMR)?
Gel Electrophoresis
Results
Molecular Mass Ruler (MMR) is
placed in the first well. It is made of
several pieces of DNA of known
length. You can compare your DNA
lengths to the MMR to estimate the
lengths of your DNA.
You can also just compare your DNA
lengths to the known + +, + -, and - - .
But it is standard procedure to always
use a MMR.
Molecular
Mass Ruler
Genotype Frequency of Class
Number of
Students
++
+-
-Total # of
students =
Frequency of genotype
(# of students with
genotype/total # of students)
Genotype Frequency US population
+/+
Number of
individuals
2,422
Frequency
+/-
5,528
0.55
-/-
2,050
0.21
Total
10,000
1.00
0.24
How does our class compare?
Total number of Alu alleles in class
Number of
students
++
Total number Frequency of
of Alu alleles Alu alleles
+
-
+-Total # of
alleles =
+
-
Total number of Alu alleles
in US population
Number of
individuals
++
2,422
+-
5,528
--
2,050
Total =
Total
number of
Alu alleles
+
-
Frequency of
Alu alleles
+
-
How does our class compare?
Your Gel Sketch