The ability to identify the sex of a DNA sample is an important tool
in molecular ecology and conservation genetics. The optimal marker
would work on small amounts of non-invasive samples that are likely to
include highly degraded DNA and be applicable in many species.
Molecular sex identification normally works by PCR amplification of
sex specific regions that differ in length. The PCR products can then
be visualized using standard electrophoresis, revealing females as
homozygotes (XX) and males as heterozygotes (XY). Generally, in order
to detect the male sex, a Y chromosomal fragment must be amplified.
This can be done by (a) amplification of a homolog region on X and Y
with a length difference, (b) a triple primer PCR with a common primer
X/Y and a Y specific primer (short fragment) and X specific primer
(longer fragment) or (c) a multiplex PCR with a Y region and an
positive control (autosomal or X region).
Several loci have been used for sex identification in humans and closely related species, e.g. the amelogenin system [1-3] the zinc-finger protein [4,5], the SRY locus  or a combination . Detection of sex-specific restriction patterns 
requires some pre-analysis development (i.e. sequencing to identify
restriction sites) and enzyme restriction of PCR products, which is
time consuming. The SRY locus requires co-amplification of external
control regions [6,7], which may be unreliable for non-invasive DNA samples with DNA of low quality.
The widely used amelogenin system 
has recently been found to provide ambiguous results in humans such as
null-alleles, primer mutations or amplification failure, resulting in
erroneous gender determination [9-11]. It works in closely related apes , but not orang-utans , baboons or more distantly related species .
Also, the Sullivan amelogenin system amplifies a very small X-Y size
difference (6 bp), which does not consistently resolve well on agarose
gels. Therefore this system needs more time-consuming and expensive
acrylamide gel- or capillary electrophoresis. Recently, a new general
multiplexing method was published, using the amelogenin locus, the SRY
locus and group specific primers and suitable for non-invasive samples . Non-identification of males may result from non-amplification of the Y fragment and it has been argued 
that all "females" (XX or XY with no Y amplification) should be
verified with a second independent sex test. This calls for the
development of multiple independent tests that can be carried out in
We have previously developed a new primer pair for a different
region of the amelogenin gene suitable for sexing lemurs and humans and
therefore possibly most primate species . However, like the zing-finger protein system ,
the resulting fragments are too long (> 250 bp) for non-invasive
samples, which often contain highly degraded DNA. Alternatively, we
then designed primers for a small region of the DEAD-BOX gene, which
are able to sex apes and monkeys, but these primers do not work in
prosimians – probably due to primer region mutations .
At present, genomic sequence information is only available for a few
primates (human, chimpanzee, macaque) and rodents (mouse, rat) or even
more distantly related species. So far, we find that it is impossible
to identify suitable homolog XY regions with the desired degree of
conservation for a standard two-primer PCR design that will work in all
The objective of this study was to identify a suitable region for a
triple primer PCR design with a shared XY primer in combination with an
X- and Y-specific primer. The primers should amplify a short region and
be widely conserved through primate evolution, such that the method
would work on non-invasive samples from all primate species.