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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 » Results

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

A region of UTY is highly conserved between human and mouse

The comparison of the repeat masked human, chimpanzee and mouse Y chromosome yielded surprisingly few results. The best hit was 248 bp with 95.56% identity between mouse and human. This region is a part of the Y-chromosomal isoform of the ubiquitously transcribed tetratricopeptide repeat protein gene (UTY). Hence, UTY and the X-chromosomal homolog (UTX) were selected for further analysis.

The UTX/UTY region is suitable for triple primer design

A small region in the alignment of 19 primate UTX/UTY sequences (see methods) showed three distinct patterns, useful for primer design (figure 1): The UTX/UTY primer is located in a region with high conservation between primate X and Y homologs (marked in green in figure 1A). The UTY primer is located in a region conserved between Y homologs but with conserved differences between the X and Y homologs (marked in blue in figure 1A) and the UTX primer is located in a region conserved between X homologs but with conserved differences between the X and Y homologs (marked in red in figure 1A). The resulting primers (Table 1, figure 1B) were chosen to be able to bind to all sequences in the alignment and to yield both short fragment sizes as well as fragments with differing lengths. The two fragments (consensus fragment length: Y = 86 bp, X = 127 bp) are easily separated on agarose gels (figure 1C).

Figure 1. UTY/UTX alignment.A) Alignment of primate UTX/UTY regions. B) Top: Primers. Middle: UTY consensus sequence. Bottom: UTX consensus sequence. C) The resulting consensus fragments amplified by the three primers.

Table 1. PCR primers, degeneracy and annealing temperatures (Ta).

Successful sexing of all primate species tested

The primers work in all species tested yielding amplifications of both X and Y fragments (figure 2). Amplifications were successful regardless of template DNA concentration, hence the method is suitable for analysing non-invasive samples.

Figure 2. Successful sexing in XX primate species. The primers were successfully tested in apes, New World monkeys, Old World monkeys and prosimians using identical PCR conditions. Males: two bands (XY), females: one band (XX).

Since the primers were designed in well conserved regions, no species-specific optimization was necessary and a shared PCR protocol and identical annealing temperature was used (table 1).


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