Many species of exotic birds are sexually monomorphic: male and female
birds are indistinguishable. DNA sexing utilizes the fact that male and female birds have
different sex chromosomes. Bird sex determination is the reverse of that in humans; the
female chromosome determines the sex of the bird. Male birds have two Z chromosomes, and
are designated ZZ. Female birds have one Z chromosome and one W chromosome (ZW). The
purpose of this lab was to extract DNA from blood samples of Dr. Allen’s geese, that
he got by clipping a nail on the bird and letting it bleed. The next thing that we had to
do was amplify the CHD genes with the P2 and P8 PCR primers, the procedure for this is
listed below. This last part of this lab, running the PCR products out on a polyacrylamide
electrophoresis gel, Dr Allen performed for us. In theory, female geese should show two
DNA bands and males should only show one.
Procedure
Pipet 20 :l of protease into the bottom of a 1.5 ml microcentrifuge
tube.
Add 200 :l ample to the microcentrifuge tube, 10 :l of blood and 190 :l
of PBS.
Add 200 :l of buffer AL to the sample and mix by pulse-vortexing
for about 15 sec.
Incubate at 56E C for 10 min.
Briefly centrifuge the 1.5 ml
microcentrifuge tube to remove drops from the inside of the lid.
Add 200 :l of ethanol to the sample, and mix again by
pulse-vortexing for 15 sec. After mixing, briefly centrifuge the 1.5 ml microcentrifuge
tube to remove drops from the inside of the lid.
Carefully apply the mixture
from step 6 to the QIAamp spin column in a 2ml collection tube without wetting the rim.
Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. After centrifuging, place
the QIAamp spin column in a clean 2ml collection tube and discard the tube containing the
filtrate.
Carefully open the QIAamp
spin column and add 500 :l of buffer AW1 without
wetting the rim. Close the cap and centrifuge at 6000 x g (8000rpm) for 1 min. After
centrifuging, place the QIAamp spin column in a clean 2 ml collection tube and discard the
collection tube containing the filtrate.
Carefully open the QIAamp
spin column and add 500 :l of buffer AW2 without
wetting the rim. Close the cap and centrifuge at full speed, 20,000 x g (14,000 rpm) for 3
min.
Place the QIAamp spin column
in a clean 1.5 ml microcentrifuge tube and discard the collection tube containing the
filtrate. Carefully open the QIAamp spin column and add 200 :l
of buffer AE or distilled water. Incubate at room temperature for 1 min and then
centrifuge at 600 x g (8000 rpm) for 1 min.
Results
According to the results of the gel above geese three, for, and five should be males
because they only show one band. Geese one and two should be female because they show two
bands.
ZOOLOGY 310 GUIDE TO DNA
SEXING OF GEESE
