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Biology Articles » Methods & Techniques » Fast and non-invasive PCR sexing of primates: apes, Old World monkeys, New World monkeys and Strepsirrhines » Methods

Methods
- Fast and non-invasive PCR sexing of primates: apes, Old World monkeys, New World monkeys and Strepsirrhines

Primer region identification and design

Primate Y chromosomes were downloaded from Ensembl [16] and compared to the mouse Y chromosome using BLAST with strict parameters (W = 18 (primer length), e = 1E-100). The region with the highest identity was found to be in the UTY gene in humans (248 bp with 95.56% identity to mouse, Human Y: 13909061-13909308), and this gene and the X homolog (UTX) was selected for further analysis.

8 UTY and 11 UTX sequences were downloaded from GenBank and aligned (UTY: Eulemur fulvus, Macaca fascicularis, Ateles geoffroyi, Hylobates lar, Pongo pygmaeus, Gorilla gorilla, Pan troglodytes, Homo sapiens UTX: Lemur catta, Cheirogaleus medius, Colobus sp., Hylobates lar, Presbytis entellus, Leontopithecus rosalia, Ateles geoffroyi, Macaca fascicularis, Gorilla gorilla, Pongo pygmaeus, Homo sapiens. The alignment files are available as 1, 2).

Additional File 1. Full UTX/UTY sequence set. The unaligned set (fasta format) of 8 UTY and 11 UTX sequences (UTY: Eulemur fulvus, Macaca fascicularis, Ateles geoffroyi, Hylobates lar, Pongo pygmaeus, Gorilla gorilla, Pan troglodytes, Homo sapiens UTX: Lemur catta, Cheirogaleus medius, Colobus sp., Hylobates lar, Presbytis entellus, Leontopithecus rosalia, Ateles geoffroyi, Macaca fascicularis, Gorilla gorilla, Pongo pygmaeus, Homo sapiens).

Format: TXT Size: 25KB Download file

Additional File 2. Cropped UTX/UTY alignment. The cropped alignment (fasta format) of 8 UTY and 11 UTX sequences (see also figure 1A, UTY: Eulemur fulvus, Macaca fascicularis, Ateles geoffroyi, Hylobates lar, Pongo pygmaeus, Gorilla gorilla, Pan troglodytes, Homo sapiens UTX: Lemur catta, Cheirogaleus medius, Colobus sp., Hylobates lar, Presbytis entellus, Leontopithecus rosalia, Ateles geoffroyi, Macaca fascicularis, Gorilla gorilla, Pongo pygmaeus, Homo sapiens).

Format: TXT Size: 5KB Download file

Adequate regions were selected manually (figure 1A) and degenerate primers were designed using the Primo online software [17]. The resulting primers were designed with relatively high annealing temperatures to allow possible mismatches to the template in distantly related species (table 1).

DNA extraction and PCR

DNA was extracted from shed or plucked hairs, ear tissue or blood. From hair samples DNA was extracted using a slight modification of the Chelex protocol [18]: Approximately 5 mm of the root portion of 5–8 hairs from each individual was cut directly into 1.5 mL Eppendorf tubes containing 200 μl of 20% Chelex resin solution (BIO-Rad, Hercules, CA, USA). Tubes were vortexed briefly, boiled for 12 min, centrifuged 3 min at 13000 rpm and stored at minus 20°C. From tissue samples DNA was extracted using DNeasy Tissue Kit (cat. no. 69506, Qiagen), and from blood samples DNA was extracted using proteinase K digestion, followed by a protein salting-out step as described in [19]. PCR was performed in a total volume of 10 μl and contained 1 μl buffer (1.5 mM MgCl2), 1.6 μl dNTP (1.25 mM of A, C, G, T, respectively), 4, 1 and 0.25 μl of UTY, UTXY and UTX primers (10 pmol/μl), 0.1 μl Taq polymerase (Amersham Pharmacia Biotech), topped up with distilled water to 8 μl and 2 μl template DNA was added. The different volumes of the three primers were found after optimization for equal intensity of the X and Y fragments. Cycling conditions were 94°C for 3 min, and 35 cycles of 94°C for 30 s, 57°C for 40 s and 72°C for 1 min and a final extension step of 72°C for 7 min. PCR fragments were separated on 2 % agarose gels (120 V, 1 hour 45 minutes) and visualized using SybrGreen staining.


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