We DNA sexed each sample using the 1237L/1272H primer set (Kahn et al. 1998).
The following 15µl reaction (PCR 1) was used to amplify DNA from blood, liver, hatched eggshell membranes, mouth swabs, and autolytic tissue samples: 7.96µL millipore water, 1.0µl 25mM MgCl2, 1.5µl 10X Buffer, 1.2µl 2mM dNTP mix (consisting of all 4 dNTPs), 0.12µl 20mM forward primer, 0.12µl 20mM reverse primer, 0.1µl 5 unit taq polymerase, and 3µl extracted DNA.
The following 15µl reaction (PCR 2) was used to amplify DNA from plucked contour feathers, molted contour feathers, plucked chick down, and predated eggshell membranes: 0.96µl millipore water, 1.0µl 25mM MgCl2, 1.5µl 10X Buffer, 1.2µl 2mM dNTP mix (consisting of all 4 dNTPs), 0.12µl 20mM forward primer, 0.12µl 20mM reverse primer, 0.1µl 5 unit taq polymerase, and 10µl extracted DNA.
All PCRs were performed in a Perkin Elmer 9600 thermocycler. The PCR conditions were as follows: 94˚C for 2 minutes (initial denaturing step) followed by 30 cycles of 94˚C for 30 seconds, 56˚C for 1 minute, and 72˚C for 2 minutes (Kahn et al. 1998). This was followed by a 10 minute extension step at 72˚C and a final ramp to 4˚C (Kahn et al. 1998).
Test PCRs for DNA Sexing
(When you begin working on a species, start with several PCR reactions with varying amounts of MgCl2 and DNA to find the conditions that work best for your species of interest). Click here for test PCR reactions.
• For DNA sexing on 3% agarose gels, a minimum of 3µl of extracted DNA is required from any sample type (see above)
• For DNA sexing on acrylamide gels, DNA sexing can normally be done with 3µl of extracted DNA from all sample types
References
Kahn NW, St. John J, and Quinn T. (1998) Chromosome-specific intron size differences in the avian CHD gene provide an efficient method for sex identification in birds. Auk, 115, 1074-1078.
Information on this page should be cited as: Bush, K.L., Vinsky, M.D., Aldridge, C.L., and Paszkowski, C.A. 2005. A comparison of sample types varying in invasiveness for use in DNA sex determination in an endangered population of Greater Sage-Grouse (Centrocercus urophasianus). Conservation Genetics, 6: 867-870.
