44(3):332-335,2003

FORENSIC SCIENCES

Development of a 17-plex Microsatellite Polymerase Chain Reaction Kit for Genotyping

Horses*
Pero Dimsoski
Applied Biosystems, Foster City, Calif, USA

Aim. To describe the development and performance of the new horse genotyping kit.
Methods. Highly discriminatory 17-Plex horse genotyping kit was designed by adding the fifth dye to the StockMarks
kit for genotyping horses and taking advantage of the new instrument platforms. This was accomplished by using a new
set of five fluorescent dyes developed by Applied Biosystems (DS-31), with four of the dyes used to label the forward
amplification primers (6-FAMTM, VIC, NEDTM, and PET) in each primer set.
Results. The new equine kit contained five extra loci (ASB17, LEX3, HMS1, CA425, and ASB23) in addition to the 12
original loci (VHL20, HTG4, AHT4, HMS7, HTG6, HMS6, HTG7, HMS3, AHT5, ASB2, HTG10, and HMS2) recommended by the International Society for Animal Genetics. The kit performed well on different instrument platforms
(ABI PRISM 377, 310, and 3100 instruments) and across wide ranges of DNA template concentrations (1-10 ng).
These 17 loci were combined and amplified in a single polymerase chain reaction (PCR) cycle, which dramatically improved the power of statistical tests for pedigree analysis while reducing the time and work required to perform such
tests. An in-lane size standard labeled with the fifth dye (LIZ) provided accurate size determination for genotyping.
Conclusion. The new 17-Plex horse kit, designed to improve the laboratory efficiency by genotyping more markers in a
shorter time, has 17 primer sets labeled with new fluorescent dyes, which can be amplified in one PCR cycle and genotyped in one run on a high-throughput instruments.
Key words: breeding; genetic markers; genotype; horses; polymerase chain reaction; microsatellite repeats

In the past decade, the DNA forensic field was

primarily concerned with the identification and cataloguing (creating DNA databases) of the human genotypes. These efforts required the development of sophisticated genotyping technologies based on polymerase chain reaction (PCR) (1-3). DNA genotyping
technology, including restriction fragment length
polymorphism (RFLP), short tandem repeats (STR),
amplified fragment length polymorphism (AFLP), and
single nucleotide polymorphism (SNP), was simultaneously developed for animals, plants, and microorganisms. Originally intended for gene mapping, new
genotyping methods were also used to help the classification of the microorganisms and plants, in animal
and plant forensics, and were largely utilized by animal and plant breeding societies for the construction
and verification of large pedigree files and databases.
Modern tools of molecular biology provided
means for fast, accurate, and relatively inexpensive
way for animal genotyping. Applied Biosystems
Stockmarks line of products (Applied Biosystems,
Foster City, CA, USA) has been designed primarily for
*For research use only. Not for use in diagnostic procedures.

332 www.cmj.hr

paternity testing and verification of pedigree. DNA

genotyping by use of commercial kits is the most reliable method for parentage testing. The kits have also
been used for tracking animal products to particular
herd, identification of animals in criminal cases, linkage mapping, and diagnostic studies. The Applied
Biosystems Horse Genotyping Kit (4,5) has been a
great asset to the horse breeding community, helping
to genotype the pedigrees of tens of thousands of
horses as well as to demarcate various horse breeds
(6). Considering that the price of pure breed horse can
easily reach several thousand dollars, the value of accurate and efficient pedigree verification system by
DNA genotyping can be put in a rather significant
economical perspective.
Genotyping laboratories have used the current
Horse Genotyping Kit (4) primarily for pedigree and
parentage verification, with the end-customers being
various horse breeding associations. The current users have been very satisfied with the kit performance.
However, there was a need to update the kit to the
new technology standards. Before PCR, the user had
to mix the primers into two tubes and amplify them
separately. The kit consists of 12 primer sets amplified

Dimsoski: PCR-based 17-Plex Horse Genotyping Kit

with two PCR reactions, 8-plex and 4-plex. The 8-plex

reaction includes the following loci: VHL20, HTG4,
AHT4, HMS7, HTG6, HMS6, HTG7, and HMS3; and
the 4-plex reaction includes AHT5, ASB2, HTG10,
and HMS2 loci. One of the primers in the primer set is
labeled with a fluorescent dye: 5Fam, Joe, or
Tamra. The products are separated on either ABI
PRISM 377 or ABI PRISM 310 genetic analyzer instruments (Applied Biosystems) and virtual filter set
A is used to differentiate between the dyes' spectral
composition. However, with the introduction of the
additional fluorescent dyes and high throughput instruments, it became necessary to update the kit in accordance with the new technology. For example, filter set A was no longer an option on the new generations of instruments (Applied Biosystems 3100 and
3730). In addition, mixing the primers before PCR
and having two PCR reactions was adding several extra steps to the laboratory procedures, consuming
time and effort.
The goals were to simplify the PCR protocols,
combine all primer sets into one tube, implement
new fluorescent dyes, so that the new kit could be
used on the new instrument platforms, and, if possible, to develop new primer balances, so that all of the
12 primer sets could be amplified in one PCR cycle,
possibly under the same conditions used by the current Equine Paternity Kit. Furthermore, five other
markers, already used by various genotyping laboratories, were included in the new kit to increase the
power of discrimination/inclusion.
Material and Methods
The general procedures used for development of the horse
kit were generally described by Wallin et al (3). All primer sequences used in the kit are publicly available (7). The kit loci,
chromosome location, dye label, and the size range of the amplified products for all markers used in Horse Genotyping Kit are
presented in Table 1.
The following loci are included in the new StockMarks
Equine Genotyping Kit for horses: VHL20, HTG4, AHT4, HMS7,
HTG6, AHT5, HMS6, HTG7, HMS3, AHT5, ASB2, HTG10,
HMS2, ASB17, LEX3, HMS1, and CA425. The International Society of Animal Genetics (8) has recommended nine loci that are
part of the kit. The rest of the loci included in the kit were selected because various service laboratories in Europe and the

Croat Med J 2003;44:332-335

United States used them. There is one primer pair per locus. One
of the primers is labeled with a fluorescent dye and the other is
unlabeled. There are four dyes for primers, 6-FAM, VIC, NED,
and PET. Also, LIZ dye is used for labeling the size standard with
filter set G5 (9). The primers labeled with 6-FAM dye are VHL20,
HTG4, AHT4, and HMS7. Primers labeled with VIC dye are
HTG6, AHT5, HMS6, ASB23, and ASB2, and the primers labeled
with NED dye are HTG10, HTG7, HMS3, and HMS2. The PET
dye is attached to ASB17, LEX3, HMS1, and CA425. Virtual filter
set G5 (9) is used to differentiate between the dyes spectral
composition. The most optimal thermocycling conditions for the
kit are described in Table 2. It is recommended to use
GeneAmp PCR system 9700 in 9600 emulation mode.

Table 2. Thermocycling condition for the horse kit for

GeneAmp PCR system 9700 run in 9600 emulation mode.
Temperature (C)
95
95
60
72
72
4

Time
10 min
30 sec
30 sec
1 min
60 min

Cycles
1
30
1
1

The above cycling conditions are exactly the same as the

conditions for the current Equine Paternity Kit usage, which was
one of the goals during the development of this kit. The kit was
optimized for 15-mL PCR reaction. In addition to the primer-mix,
the kit will contain dNTPs, AmpliTaq Gold Polymerase (Applied Biosystems) and Stockmarks buffer. Customer has to provide only PCR-grade water. Amounts of each of the components
for one PCR cycle are shown in Table 3.

Table 3. Polymerase chain reaction (PCR) components and

the amount for the standard reaction
PCR Component
Stockmarks Buffer
dNTP mix
Amplification primer mix
AmpliTaq Gold Polymerase
DNA Template
Deionized water

Amount (mL)
2.5
4.0
4.0
0.5
1.0
3.0

Results
The kit was optimized for use with a DNA template concentrations ranging form 0.2 ng to 10 ng.
Electropherograms generated on ABI PRISM 3100
Genetic Analyzer instrument by using template con-

Table 1. Loci names, chromosome location, dye label,

and the size range of the amplified products for all
markers used in 17-Plex Horse Genotyping Kit
Locus
VHL20
HTG4
AHT4
HMS7
HTG6
AHT5
HMS6
ASB23
ASB2
HTG10
HTG7
HMS3
HMS2
ASB17
LEX3
HMS1
CA425

Fluorescent
dye
6-FAM
6-FAM
6-FAM
6-FAM
VIC
VIC
VIC
VIC
VIC
NED
NED
NED
NED
PET
PET
PET
PET

Chromosome
location
30
9
24
1
15
8
4
3
15
21
4
9
10
2
X
15
28

Size range
(nucleotides)
83-102
116-137
140-166
167-186
74-103
126-147
154-170
176-212
237-268
83-105
114-126
146-170
215-236
104-116
137-160
166-178
224-247

Figure 1. GeneScan software electropherograms of amplified control horse DNA (1.25 ng) by 17-Plex Horse Genotyping Kit run on ABI PRISM 3100 Genetic Analyzer.

333

Dimsoski: PCR-based 17-Plex Horse Genotyping Kit

Croat Med J 2003;44:332-335

Figure 2. GeneScan software electropherogram of amplified control horse DNA (5 ng) by 17-Plex Horse Genotyping
Kit run on ABI PRISM 3100 instrument.
Table 4. Peak heights, run time, peak area and data point of
electropherograms from Figure 1
Dye/
sample peak
9B, 15
9B, 16
9B, 21
9B, 22
9B, 26
9B, 31
9B, 35
9B, 40
9G, 6
9G, 10
9G, 14
9G, 17
9G, 21
9G, 25
9G, 32
9G, 36
9G, 41
9Y, 10
9Y, 14
9Y, 18
9Y, 23
9Y, 26
9Y, 29
9Y, 30
9R, 3
9R, 4
9R, 7
9R, 9
9R, 11
9R, 15

Minutes
7.95
8.02
9.09
9.16
9.66
10.11
10.63
10.97
7.43
7.99
9.19
9.48
10.08
10.42
11.17
11.70
13.16
7.66
8.30
9.06
10.07
10.28
12.19
12.25
8.49
8.57
9.55
9.68
10.62
12.79

Size
93.80
95.95
126.11
128.22
143.35
157.98
174.09
184.38
79.18
94.95
129.08
137.62
156.97
167.67
190.43
206.55
252.58
85.65
103.77
125.25
156.89
163.27
221.72
223.65
109.07
111.08
139.87
144.05
173.61
240.81

Peak
height
3520
2300
2852
1851
2371
1347
2206
1596
836
360
574
602
1445
759
1459
1288
519
520
201
1346
1157
770
866
691
581
358
447
416
715
616

Peak
area
37102
23687
33405
24668
22189
12425
22745
11636
8076
3762
5906
6128
15338
7968
15159
13417
7638
5290
1864
12742
11821
7947
8476
6864
7524
4647
4928
5983
8754
7157

Data
point
2981
3009
3409
3436
3621
3791
3988
4115
2785
2996
3447
3554
3779
3909
4190
4389
4935
2873
3113
3398
3778
3855
4572
4595
3185
3212
3581
3629
3982
4798

centrations of 1.25 and 5 ng are presented in Figures 1

and 2, and Table 4, respectively. This kit was designed to perform well with low template concentrations, close to 1 ng (Table 4), where peaks are above
200 relative fluorescent units (rfu). The peak height
and the color balance are good across all loci for a
DNA template concentration of 1.25, 2.5, and 5 ng.
However, 3100 instrument shows more pull-up peaks
at the higher template concentrations (>10 ng, data
not shown) because of its sensitivity as well as the sensitivity of the kit. Diluting the PCR product before
loading it to the instrument can reduce the number of
pull-up peaks. Since the sensitivity among the instruments varies and the exact template concentration is
not always known, there is no strict recommendation

334

about the dilution factors of the PCR products. Instead, more general recommendations should be applied. For example, if the template DNA concentration is between 1 and 10 ng, the PCR products should
not be diluted before being run. However, if there are
lots of pull-up peaks and the template concentration
is above 10 ng, the dilution factor should be adjusted
to the instrument sensitivity. Therefore, each user
should adjust the running conditions of the kit depending on the instrument sensitivity and the DNA
template concentration. The new 17-Plex Horse Genotyping kit also performs well on the ABI PRISM 377
Prism instrument (Fig. 3). In addition, the kit performs
well if run on ABI PRISM 3100 instrument with regular module (GeneScan36_POP4DefaultModule, Fig.
4) and with new module that enhances the color balance between different dyes (GeneScan36vb_ POP4
Default Module, Fig. 5). The last module (Gene
Scan36vb_ POP4DefaultModule) is the recommended module for Horses Genotyping Kit when used on
ABI 3100 instrument.

Figure 3. GeneScan software electropherogram of amplified control horse DNA (1.25 ng) by 17-Plex Horse Genotyping Kit run on ABI PRISM 377 instrument.

Figure 4. GeneScan software electropherogram of amplified horse DNA (2.5 ng) by 17-Plex Horse Genotyping Kit
run on 3100 instrument by using GeneScan36_POP4DefaultModule.

Dimsoski: PCR-based 17-Plex Horse Genotyping Kit

Croat Med J 2003;44:332-335

larly canine and bovine, were used in high-profile

court cases (10). Considering the number of pet animals in the United States, it should be expected for
animal forensics only to expand in the future. In addition, the same technology has been used for animal
tracking (11). From these perspectives, the horse
genotyping kit, described above, should be a welcome tool to forensic scientists and animal-theft
investigators.
References

Figure 5. GeneScan software electropherogram of amplified horse DNA (2.5 ng) by 17-Plex Horse Kit genotyped on
3100 instrument run by using GeneScan36vb_POP4DefaultModule.

The kit has been tested in-house and off-site on

horse DNA samples originating from various horse
breeds. All of the primers amplified well, with the exception of HTG10, which sometimes exhibited low
peak heights not suitable for automated scoring.
Discussion
Breeding societies are primarily concerned with
the improvement and propagation of different breeds
of livestock. A fast and accurate way to construct a
pedigree is by knowing the genotype of parents and
progeny. Therefore, there is a constant need to genotype all commercially available animals. In practice,
horse breeders provide a horse parentage data to
breeding societies, which enter the data into the registry to generate pedigrees. Even though this method
has been working well most of the time, it is not very
reliable for pedigree verification because it is prone to
human errors at a several levels, e.g., data collection
and transfer. The most reliable and efficient method
for pedigree construction and analysis is the one that
employs the DNA genotyping technology. With the
decrease in price of reagents and instruments, the
DNA genotyping has become the most cost-effective
method for pedigree maintenance of large populations of animals. The 17-Plex horse genotyping kit has
been designed to provide high discrimination power,
with minimum time spent on sample preparation and
minimum use of reagents. Therefore, the service laboratories that have high-volume contracts with horse
breeding societies will be the main users of the horse
genotyping kit.
Another interesting development has occurred in
the area of animal forensics. In the past few years, the
PCR-based methods for genotyping animals, particu-

1 Wallin JM, Buoncristiani MR, Lazaruk KD, Fildes N,

Holt CL, Walsh PS. TWGDAM validation of the
AmpFISTR blue PCR amplification kit for forensic casework analysis. J Forensic Sci 1998;43:854-70.
2 Lazaruk K, Walsh PS, Oaks F, Gilbert D, Rosenblum
BB, Menchen S, et al. Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a
capillary electrophoresis instrument. Electrophoresis
1998;19:86-93.
3 Wallin JM, Holt CL, Lazaruk KD, Nguyen TH, Walsh
PS. Constructing universal multiplex PCR systems for
comparative genotyping. J Forensic Sci 2002;47:52-65.
4 Bozzini M, Fantin D, Ziegle J, van Haeringen H, Jacobs
W, Ketchum M, et al. Automated equine paternity testing. Anim Genet 1996;27:32.
5 Marklund S, Ellegren H, Eriksson S, Sandberg K,
Andersson L. Parentage testing and linkage analysis in
the horse using a set of highly polymorphic microsatellites. Anim Genet 1994;25:19-23.
6 Bjornstad G, Roed KH Breed demarcation and potential
for breed allocation of horses assessed by microsatellite
markers. Anim Genet 2001;32:59-65.
7 INRA Biotechnology Laboratories Home Page. Horsemap database. Available from: http://locus.jouy.inra.fr/.
Accessed: May 15, 2003.
8 International Society for Animal Genetics. Available
from: http://www.isag.org.uk/. Accessed: May 15, 2003.
9 Applied Biosystems. Available from: http://www.applied biosystems.com/support/software/310/modules.c
fm. Accessed: May 11, 2003.
10 QuestGen Forensics. Available from: http://www.anim
alforensics.com. Accessed: May 11, 2003.
11 Animal Improvement Institute. Animal forensics. Available from: http://www.arc.agric.za/institutes/aii/main/
divisions/animalbreedgen/animalgen/anfor1.htm. Accessed: May 13, 2003.
Received: May 27, 2003
Accepted: June 5, 2003
Correspondence to:
Pero Dimsoski
850 Lincoln Centre Drive
Foster City, CA 94404, USA
dimsospn@appliedbiosystems.com

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