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
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).