A Y-STR Triplex for Use After Autosomal Multiplexes
School of Biological Sciences
Flinders University of South Australia
Bedford Park, Australia
Senior Forensic Scientist
Forensic Science Centre
School of Biological Sciences
Flinders University of South Australia
Bedford Park, Australia
in the evidential utility of Y-STR multiplexes does not rise with
loci number the same way as it does with unlinked autosomal loci.
This might encourage consideration for using small Y-STR multiplexes.
In this study, one practical Y-STR triplex that consists of DYS438,
DYS390, and DYS439 was used to analyze 13 evidentiary stains from
two sexual assault cases after using the AmpFlSTRâ
Profiler PlusTM (PE Biosystems,
Foster City, California) multiplex. In Case 1, the Y-STR profiles
established the minimum number of male contributors in four out
of five multiple-source stains and confirmed the identity of the
suspects who contributed to the mixed autosomal profiles. In Case
2, the Profiler PlusTM results
of the three stains showed two mixed male/female profiles and one
stain with no result. A Y-profile consistent with the suspect's
profile was obtained in all three stains. The ability of the Y-triplex
to selectively amplify the limited number of male cells in the third
stain demonstrates the sensitivity of the Y-system. The results
indicated that with mixed stains, the use of a small Y-STR multiplex
in addition to autosomal STRs for forensic casework can help to
discriminate multiple suspects and is more useful than using only
DNA mixtures from males and females are frequently encountered in
forensic casework, particularly in rape and sexual assault cases.
Often, these mixed stains are in limited quantity or of poor quality,
thus they pose a challenge in the resolution and interpretation
of their resulting genetic profiles. Complementing existing STR
analysis with validated Y-STR systems appears to be the current
as well as future approach in forensic casework involving mixtures
of body fluids. Besides their ability to track paternal lineages,
Y-STRs are important forensic tools in mixture analysis largely
because of their ability to target and detect male-specific DNA
(Jobling et al. 1997; Kayser et al. 1997; Kloosterman 1999). The
use of Y-multiplexes to detect semen traces arising from multiple
sources could obviate problems in the analysis and interpretation
of complex autosomal profiles. However, the optimal type, number,
and size of loci to be included in Y-STR multiplexes for practical
forensic use are not yet clear.
in forensic laboratories worldwide is reaching for more loci but
is currently centered on a nine-locus core set mentioned in the
Y-STR Haplotype Reference Database (http://www.ystr.org).
The National Institute of Standards and Technology is developing
high-performance Y-STR multiplexes that combine more than 20 loci
However, unlike the autosomal loci, a high number of Y-STR loci
in multiplexes may be of little evidential value. Theoretically,
as the number of loci in a Y-multiplex increases, the confidence
in identifying a Y-chromosome and the ability to exclude increases,
but incremental gains in exclusion power decreases. Additionally,
as multiplexes get more loci, the compromises in amplification conditions
usually lower the sensitivity. Thus, a small triplex may be the
optimum loci size to use in a Y-STR multiplex. Whereas new, larger
multiplexes such as Y-megaplexes certainly have advantages in studying
the evolution of human Y-chromosomes, small Y-multiplexes consisting
of three to five loci might be much more practical and robust. They
may be more sensitive to low template levels and sufficient in discriminatory
power for forensic application if used as an additional system to
clarify and confirm autosomal DNA profiles.
This study investigates
the application of one small Y-multiplex to see what advantages
it may have when used in conjunction with a common autosomal multiplex
such as the Profiler PlusTM. Thirteen
evidentiary stains in two sexual assault cases were used. The Y-triplex
consists of one locus from the core set of Y-STRs, DYS390 (Y-STR
Haplotype Reference Database), and two relatively new Y-STR loci,
DYS438 and DYS439 (Ayub et al. 2000). DYS390 was chosen because
it has complex mutational features (Forster et al. 1998). DYS438
was chosen because it is a pentanucleotide repeat locus and is expected
to produce minimal stutter artifact in the PCR. DYS439 has been
found to exhibit high locus diversity values in populations with
similar ancestry, like the Malaysian ethnic groups (Ayub et al.
2000; Hou et al. 2001).
A group of
men entered a house to rob the occupants. After taking the valuables,
some of the men raped the female family member. Ten stains were
recovered from the crime scene (M1, M2, M3, M4, M5, M6, M7, M8,
M9, and M10) and were submitted for DNA analysis. Reference bloods
were collected from the six suspects involved (S1, S2, S3, S4, S5,
and S6). No vaginal swab from the victim was submitted for analysis.
A naked woman
was found dead in a drain. The case was classified as murder and
a suspect was arrested. Police recovered two stains from the interior
of the suspect's carone from the floor mat (N1) and one from
the seat cushion (N2). A third stain was taken from a clothing item
found inside the car (N3). The three stains were submitted for DNA
For both cases,
genomic DNA was isolated from the reference bloods of suspects and
victims using the Chelex method (Walsh et al. 1991). Because the
ten evidentiary stains in Case 1 were semen traces, they were extracted
using a slightly modified differential lysis procedure. The fabric
materials (~ 0.5cm X 1cm) containing the stains were incubated for
one hour with 700µl of sterile water with frequent vortexing.
The fabric was placed in a spin basket, and the tube was spun down
for five minutes to pellet crude DNA. The samples were then incubated
at 56°C with 5µl of proteinase K (10mg/ml) and 100µl
of sterile water for 30 minutes. After spinning down, the supernatant
containing the epithelial fraction was isolated. The sperm pellet
was washed twice with 250µl of sperm wash buffer (10mM Tris-HCl
pH 7.5, 10mM EDTA, 50mM NaCl, 2% SDS) and spun down for ten minutes
after each wash to collect the sperm fraction. The epithelial fraction
was digested with 30µl of 20% Chelex. The sperm fraction was
digested with 100µl of 5% Chelex, 13.5µl Proteinese
K, and 10µl of DTT. The sperm fraction was then spun down
twice to collect the supernatant, then concentrated to a volume
of 40µl using MicroconTM B100
microconcentrators (Amicon, Incorporated, Beverly, Massachusetts)
according to the manufacturer's instructions.
For the three
stains in Case 2, two procedures were performeda differential
procedure (as above) and a nondifferential procedure. In the nondifferential
extraction, the three stains were extracted without the female epithelial
digestion. The stains were saturated directly with 100µl 5%
Chelex, 13.5µl of proteinese K, and 10µl of DTT, then
incubated 56°C for one hour with the subsequent protocols performed
as in Case 1. However, quantitation of the extracts from both cases
was not performed. In this study's validation work on the Y-triplex,
the suitable target level of DNA for this amplification system was
between 0.3-1ng of template DNA (data not shown). The sensitivity
of the Y-triplex was determined as 0.1ng.
Profiler PlusTM kit was used to
perform autosomal STR profiling according to the manufacturer's
instructions. Between 10-15µl of the extracts were used for
amplification on the GeneAmpâ
PCR System 9600 (PE Applied Biosystems, Foster City, California).
The PCR products were analyzed on the ABI PRISMTM
377 DNA Sequencer (PE Applied Biosystems, Foster City, California)
using the internal size standard GSâ-400HD
ROX (PE Applied Biosystems, Foster City, California). Resulting
fragments were sized with GeneScanTM
(PE Applied Biosystems, Foster City, California) using 75 RFU threshold
for making allele calls in GenotyperTM
(PE Applied Biosystems, Foster City, California). A threshold of
35 RFU was used for NR allele.
The three Y-STR
primers from Geneset Oligos (Lismore, Australia) were labeled with
fluorescent dyes (DYS438 with 6-FAM, DYS390 with JOE, and DYS439
with TAMRA). The PCR reaction mix of 15µl contained DYS438
primer pair (each at 0.13µM), DYS390 primer pair (each at
0.10µM), DYS439 primer pair (each at 0.50µM), 1X GeneAmpâ
PCR Buffer II (PE Applied Biosystems, Foster City, California) containing
10mM Tris-HCl pH 8.3, 50mM KCl, 1.5mM MgCl2, 200µM of each
dNTPs (PE GeneAmpâ), 1.2U
AmpliTaq GoldTM polymerase (PE
Applied Biosystems, Foster City, California), and 16mg/ml BSA (Sigma
Chemical Company, St. Louis, Missouri). Amplification was carried
out on the GeneAmpâ PCR
System 9600 with the cycling conditions at 95°C for 11 minutes,
followed by 30 cycles of 94°C for 1 minute; 57°C for 3 minutes;
72°C for 3 minutes, and ending with 72°C for 20 minutes. Between
4-7µl of the extracts were used in the PCR assay. Y-STR alleles
were designated by the variable number of repeat units at the respective
locus according to recommendations by the International Society
of Forensic Haemogenetics. Allele designation was achieved by using
two Y-STR control samples with known repeat numbers, supplied by
M.A. Jobling, University of Leicester, United Kingdom (personal
communication). The PCR products were analyzed on the 377 DNA Sequencer
using the same internal size standard and 35 RFU threshold for allele
sizing in GeneScanTM.
PlusTM results for the sperm fractions
of the ten stains in Case 1 are presented in Table
1. There are five multiple-source stains (M1, M2, M4, M6, and
M7), four single-source stains (M3, M5, M8, and M9), and one stain
with no profile (M10). Interpretation of the five mixed profiles
is limited by partial profiles and shared alleles of some of the
1 Electropherograms of stain M2 in Case
A Profiler PlusTM
results in GenotyperTM. Based
on the five alleles detected at D8 locus, there are at least
three contributors to this complex DNA profile. Suspects S4,
S5, and S6 cannot be excluded from contributing to this mixture.
B Y-triplex results in GeneScanTM.
The Y-profile confirms the three male contributors to the
complex stain M2.
DYS439 system in GeneScanTM.
to enlarge image.
were drawn based on just the Profiler PlusTM
- S4, S5, and
S6 could not be excluded from contributing to stain M2
- There were
five alleles at D8 that are not stutter bands based on their peak
heights (Figure 1A).
- S4 and S6
could not be excluded as the main contributors to stain M4
- There were
five alleles at VWA where one was an NR allele (VWA*18= 40 RFU).
The presence of a very minor third contributor could not be established.
- Both S4 and
S5 could not be excluded from contributing to stain M7.
- Stain M1
could not exclude S6 and showed a partial profile of S4.
- S5 could
not be excluded from stains M3 and M8.
- S1 could
not be excluded from stains M5 and M9.
- Stain M6
only showed a partial profile of S1 and S4.
- Stain M10
did not yield any profile.
results (Table 2) confirmed that
stains M2 and M4 have at least three contributors, whereas stains
M1 and M7 have at least two contributors. Stains M3, M5, M8, and
M9 have only one male contributor. The Y-triplex was optimized and
tested on 96 female samples. The three loci were found to be male-specific
with no Y-STR alleles detected (data not shown). Allele and haplotype
frequencies of the three Y-STRs were determined in 338 unrelated
individuals in the Malaysian population, and the three loci were
found to show high locus diversity values. Additionally, DYS438
was found to show a strong ethnic affiliation between the Caucasian
and the non-Caucasian groups studied (Chang et al. 2002).
The six suspects
in Case 1 are Malays and have the same unique DYS438-allele 10,
common in the Malaysian Malay population group (data not shown).
DYS390 alone provided a firm exclusion of two of the suspects, S2
and S3, whose DYS390 alleles were not seen in any of the stain profiles
(Table 2). DYS439 was the most
discriminative among the three loci. The presence of three different
DYS439 alleles in stain M2 confirmed that there were at least three
male contributors to this stain, whereas the presence of two different
DYS439 alleles in stain M7 confirmed that there were at least two
male contributors to this stain. Based on the Y-profile alone, having
the same haplotype, S1 and S4 could not be differentiated. The frequency
of this shared haplotype in the Malaysian Malay population is 0.0885
(data not shown). However, both suspects could be differentiated
based on their autosomal profiles. This indicates that both multiplexes
when combined give a uniquely powerful and discriminatory DNA analysis
that cannot be achieved using either multiplex alone.
Electropherograms of the stains in Case 2.
Profiler PlusTM results (in
GenotyperTM) of stain N1
in the suspect's car. The DNA profile is a mixture in which
the major component is from the victim. The suspect is not
excluded as the source of the minor component (detailed results
in Table 4).
Y-triplex results in GeneScanTM
of the three stains: N1, N2, and N3. Each Y-profile depicts
three alleles: DY390 is on the left, DYS438 is in the middle,
and DYS439 is on the right.
that the RFU in the third stain is very weak, and the broad
peak at the far right is the dye artifact from TAMRA in the
DYS439 system. This stain failed to give any autosomal profile
with the Profiler PlusTM
Click to enlarge image.
Case 2, using the differential extraction protocol, stains N1, N2,
and N3 gave no autosomal STR or
Y-STR results. This could not be attributed to the inefficiency
of the differential extraction protocol because the same procedure
gave positive results in all but Case 1, stain M10. However, using
a nondifferential extraction protocol followed by a Microcon step,
a mixed male/female autosomal profile was observed in both stains
N1 and N2, whereas stain N3 still failed to yield any profile
(Table 3). A large imbalance
between the X-allele and Y-allele at the amelogenin locus was observed
in the two mixed profiles, confirming the presence of only a small
amount of the perpetrator's DNA (Figure 2A). The suspect and victim
could not be excluded from contributing to both stains N1 and N2
based on their autosomal profiles. It should also be noted that
the Profiler PlusTM process resulted
in the two stains N1 and N2 showing the presence of an another allele
(D3*16), which was present in neither the suspect's nor the victim's
The three nondifferential
extracts amplified with the Y-triplex yielded a Y-profile consistent
with the suspect's profile (Table
4). Even the third stain, which failed to yield an autosomal
profile, produced a weak Y-profile (Figure 2B). The frequency of
finding the haplotype in stains N1 and N2 in the Malaysian population
is 0.0088 (data not shown). Although the victim's profile is the
major component in the two stains N1 and N2, the presence of her
blood in the suspect's car should be probative even without his
alleles being present. However, because the stains were retrieved
from the suspect's car, one can argue that there is a possibility
of detecting a trace amount of his own DNA in his car. The fact
that no sperm cell was visibly detected from the Christmas Tree
Staining Test and that the results from the differential extraction
protocol were negative, the Y-STR results obtained from stains N1
and N2 could have originated from underlying traces of his epithelial,
saliva, or other cells. It is possible that the male DNA contribution
was unrelated to the crime under investigation; therefore, the DNA
result could not conclude that the suspect had committed the crime,
but only that the victim had bled in his car.
to detect a male profile in stain N3 clearly indicates the sensitivity
of the Y-triplex and demonstrates the usefulness of this simple
Y-system in analyzing stains that are too weak to give autosomal
profiles. For a variety of technical reasons, the low number of
loci in a triplex potentially enables a higher sensitivity compared
to a larger multiplex such as the Profiler PlusTM
system. Four significant reasons for the higher sensitivity of the
Y-triplex could be:
- The cycling
conditions for each locus is never the same because the match
of more than one is always a compromise that becomes more significant
as more loci are included in the compromise.
- The choice
of exact sequence for the primers loses degrees of freedom as
the primer numbers increase because of the need to avoid inadvertent
cross-homology with resultant consumption of the PCR amplification
by cross-locus primer-dimers. This progressive restriction of
freedom on the choice of primer sequences as multiplexes increase
in size reduces the freedom to optimize the overall amplification.
- Factors 1
and 2 combined largely determine the number of productive PCR
cycles possible. The number of PCR cycles is 30 for the Y-triplex
and 28 for the Profiler PlusTM.
- The ratio
of primer to template DNA is another possible reason. With the
same amount of total DNA, the Y-linked primers have multiples
of one copy at the haploid locus to bind to, whereas the autosomal
primers have the same multiples of two copies at a heterozygous
locus upon which to bind.
The higher sensitivity
of Y-STR analysis compared to autosomal STR analysis was observed
by Betz et al. (2001) when a male profile was successfully detected
from the vaginal epithelial cells in a rape case that failed to
yield any autosomal profile. A number of studies on the application
and validation of small Y-multiplexes with only three to six loci
have been reported in forensic casework (Corash et al. 2001; Dekairelle
and Hoste 2001; Prinz et al. 1999). This author's experience suggests
that Y-multiplexes should be constructed from a choice of highly
informative Y-STRs optimized for a particular population group.
Y-STRs with high locus diversity values in populations from similar
ancestry should be chosen.
and interpretation of mixtures from autosomal STRs have generally
relied on the observation of the number of alleles at each locus
and the relative allele peak heights or peak areas (Clayton et al.
1998; Evett et al. 1990; Gill et al. 1998). The degree of contribution
by multiple suspects in a mixed profile is usually based on the
ratio of the allele peak heights or peak areas. However, this estimation
could become difficult for very weak mixed profiles when there is
interference from stutter and artifact peaks. For stain M2 in Case
1, the peak heights of the DYS439 alleles in the Y-profile directly
indicated the contribution of the three suspects to the mixture
(Figure 1C). It should be noted that a comparison of the peak height
and peak area between the two systems might provide useful information
in assisting autosomal mixture interpretation. To explore whether
the Y-STR estimations relate directly to autosomal peak heights
will take further testing on more casework mixtures. With no size
overlap between DYS438 and DYS439 alleles, the peak height balance
of the triplex could be improved by labeling DYS439 with 6-FAM.
This will also eliminate the presence of dye artifacts from the
TAMRA system. However, in the Y-triplex, these dye artifacts do
not fall within the allelic range of the three loci (Figure 2B).
stains from only two cases were tested, the Y-STR results indicate
that using a small Y-multiplex is practical in a forensic environment.
It should also be noted that with the Y-chromosome, the discriminatory
power did not translate into evidential values as it did with the
autosomes. Because the three chosen loci have alleles sized between
190-260bp, they were ideal for analyzing DNA with environmentally
challenged quality and quantity.
The Y-STR triplex
is useful to establish the minimum number of male contributors in
multiple-source stains and to confirm the multiple suspects who
contributed to complex DNA profiles. The system is also able to
detect and characterize very limited numbers of male cells in a
stain that is too weak to yield any autosomal result. Perhaps the
most important use of the system is the ability to exclude individuals
from complex mixtures. Both the Y-linked loci DYS390 and DYS439
are suitable markers to add to existing STRs in forensic casework.
It is suggested that another Y-STR that is more polymorphic than
DYS438 could be included to enhance the discrimination power of
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