USER GUIDE
KBC-HorseFiling® Kit for Horses
Equine Genotyping Kit
Contents
Chapter 1: Product Information ....................................................................................................3
Background Information .............................................................................................................3
Product Description ......................................................................................................................3
Kit contents and storage .............................................................................................................4
Control DNA.......................................................................................................................................... 4
Primers..................................................................................................................................................... 4
Required materials and equipment (not provided) .............................................................4
Chapter 2: Amplification of Short Tandem Repeat (STR) Loci ..............................................5
Loci Amplification ..........................................................................................................................5
Prepare the 22-plex PCR .................................................................................................................. 5
Chapter 3: Data Analysis ..................................................................................................................7
Prepare Equine PCR Products for Analysis .............................................................................7
Determine the size of amplified equine DNA ........................................................................7
Expected size ranges for HorseFiling® STR Loci ..................................................................... 8
Chapter 4: Interpretation of Results .......................................................................................... 10
Successful amplification using HorseFiling® for Horses ................................................. 10
Allele calling and stutter peaks .................................................................................................... 10
Homozygous individual: 2-bp and 4-bp stutter ................................................................... 10
Heterozygous individuals with the two alleles > 2 bp apart............................................ 11
Heterozygous individuals with the two alleles 2 bp apart ................................................ 12
Troubleshooting ......................................................................................................................... 13
KBC-HorseFiling® kit for Equine Genotyping
2
Product Information
1
Background Information
Throughout the history, human have made efforts to selectively breed
animals including cows, horses, dogs, etc. Animal parentage has
become an important indicator for trades, sports, breeding, etc. Before
the invention of molecular techniques, the method used was based on
physical observation of specific traits in animals. Recently, breeders
have turned to molecular biology techniques and use of DNA STR
markers for rapid and accurate parentage verification.
Short Tandem Repeats (STRs), or microsatellites, are a class of DNA
markers consisting of two to six base pair sequences, tandemly repeated,
spread in chromosomes. Because of their high level of polymorphism,
STRs have become the markers of choice for parentage testing and
individual identification similar to forensic cases in human. Alleles in a
given STR loci are defined by the number of times a given sequence
motif is repeated. The number of repeats can be detected using
Polymerase Chain Reaction (PCR) and separation of the amplified
products by capillary electrophoresis. Each animal has two copies of
any given gene (two alleles at each locus), one derived from the father
and one from the mother. If the father and mother are related, there is a
chance that the two genes in the offspring are both identical copies
contributed by the common ancestor.
Product Description
The HorseFiling® is a kit for horse parentage testing. This kit employs
a multiplex PCR process for amplification of STRs loci using
fluorescently labeled primers. This kit contains 22 microsatellites (STR)
markers, located on 16 chromosomes of equine genome. Nine of these
loci are recommended by the International Society of Animal Genetics
(ISAG) and thirteen additional loci are highly polymorphic markers
which are commonly used for horse parentage testing and identification.
3
KBC-HorseFiling® kit for Equine Genotyping
Kit contents and storage
Component
Storage Conditions
Store at –15 to –25°C.
Protect from light.
Store at –15 to –25°C.
Store at –15 to –25°C.
Store at –15 to –25°C
(for long term storage)
Primer mix
Taq DNA polymerase
PCR mix
Control DNA
Control DNA
A control DNA with known STR profile is included in kit to verify the
correct amplification and detection of the horse-specific loci and proper
genotyping.
Primers
The kit contains primer pair mix. All loci are amplified in a single
multiplex reaction. Samples should load with the GeneScan™ 500 LIZ®
internal size standard into one of the Applied Bio-systems® Genetic
Analyzers (e.g. ABI PRISM® 3130, ABI PRISM® 3500, etc.).
Required materials and equipment (not provided)
1.
2.
3.
4.
5.
6.
7.
8.
9.
Thermocycler
PCR workstation
Vortexer
Microcentrifuge
Disposable gloves
Pipet tips
Pipettors
Microtubes
One of the following genetic analyzers:
 Applied Biosystems® 3130 or 3130XL Genetic Analyzer
 Applied Biosystems® 3500 or 3500XL Genetic Analyzer
10. GeneScan™ 500 LIZ® Size Standard (from Thermo Fisher
Scientific)
KBC-HorseFiling® kit for Equine Genotyping
4
2
Amplification of Short Tandem
Repeat (STR) Loci
The determination of parentage and profile begins with PCR
amplification of the STRs using fluorescently labeled primers. This
chapter describes how to prepare the reactions for PCR amplification
and provides specific conditions for the thermal cycler program.
Loci Amplification
Prepare the 22- The amplification primer mix for the HorseFiling® contains 22 primer
sets, one for each of the STR locus. Prepare the 22-plex PCR
plex PCR
components in a total volume of 17-μl reaction for each DNA to be
tested.
1. Let the tube contents thaw completely at room temperature (RT),
(do not put the contents at RT more than 15 minutes, you can keep
them on ice).
2. Combine the following reagents in a tube (e.g. 1.5 ml) to prepare a
master mix for the 22-plex reactions:
Component
PCR mix
Taq DNA polymerase (5
U/μl)
Primer mix
Deionized water
Total volume
Volume (µL)
for One Sample
5
Volume (µL) for
Ten Samples*
55
2
22
1
11**
11
120
Depended on DNA
concentration
Depended on DNA
concentration
* Includes volume for one additional sample to account for losses that may occur during reagent transfers.
** Depend on DNA concentration, add ddH2O to make the total volume 19 μl
3. Vortex the master mix briefly and centrifuge for 10 seconds.
4. Transfer 19 µL of mix to a separate 0.2 mL PCR tube for testing
each DNA sample.
5. Add 100-200 ng of template DNA to each reaction.
5
KBC-HorseFiling® kit for Equine Genotyping
6. Add deionized water to reach the final volume of the PCR reaction,
20 µL.
7. Centrifuge tubes briefly to bring contents to the bottom of the tubes.
8. Place tubes in a thermal cycler and close the lid.
9. Select and run the recommended protocol:
Initial
Step
95 °C
5 min
Cycling
95 °C
1 min
63 °C
90 sec
30 cycles
70 °C
2 min
Final
Extension
70 °C
17-20 min
Final
Step
4 °C
∞
10. After completion of the thermal cycling protocol, store amplified
samples at –20°C in a light-protected box.
Notes:
 Sample preparation should be done on ice, because this kit
contains DNA polymerase enzyme.

PCR products can be kept at room temperature (in dark) for
about 24 hours. However, it is better to keep them at 4-8°C.

Long-term storage of amplified samples at 4°C or higher may
produce artifacts.

more than one-week delay between amplification and capillary
electrophoresis cause reduction in the quality of results.

A Positive control and a negative control should be added in
each run.

Amplification and detection instrumentation may vary. You
may need to optimize protocols including cycle number and
injection conditions (or loading volume) for each laboratory
instrument.
Warning: After PCR is complete, tubes should never be opened in the
PCR set up area or beside kit components.
KBC-HorseFiling® kit for Equine Genotyping
6
Data Analysis
3
Prepare Equine PCR Products for Analysis
After the amplification process, first run your PCR products on agarose
gel to be sure that the fragments are amplified correctly. Determine the
size of the dye-labeled PCR products by running them into one of
several Applied Biosystems® genetic analyzers.
1. Combine the following reagents in a 0.2-ml tube:
 1.0 μl of the undiluted 17-plex reaction product
 9.5 μl Hi-Di™ Formamide
 0.5 μl GeneScan™ 500 LIZ® Size Standard
2. Vortex and spin the mixtures briefly.
3. Heat at 95°C for 2 minutes.
4. Place the samples into the Applied Biosystems® Genetic Analyzer.
Use the default module.
Notes:

Be sure that your ABI Data Collection software supports Fivedye fragment analysis.

Calibrate instrument with NED, VIC, 6-FAM, PET and LIZ
before running sample through Applied Biosystems® genetic
analyzers (refer to “Applied Biosystems® genetic analyzer user
guide”).
Determine the size of amplified equine DNA
The expected size ranges for equine-specific loci are shown in the
following table which are based on empirical observations. These allele
sizes are obtained studies on an Applied Biosystems® 3130XL Genetic
Analyzer.
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KBC-HorseFiling® kit for Equine Genotyping
Occasionally, you may observe an allele that falls outside of its size
range and that overlaps with the size range of another locus. Such alleles
may be present at low frequencies in the population.
Expected size
ranges for
HorseFiling®
STR Loci
Locus
Dye
Color
Size Range (bp)*
HTG7
6-FAM™
Blue
105-130
HMS5
6-FAM™
Blue
135-155
VHL20
6-FAM™
Blue
160-195
CA425
6-FAM™
Blue
200-235
HTG10
6-FAM™
Blue
240-275
HMS3
6-FAM™
Blue
280-320
KBC51
6-FAM™
ASB17
AHT5
HMS2
AHT4
KBC61
Blue
330-370
®
Green
120-175
®
Green
180-220
®
Green
240-275
®
Green
285-320
®
Green
325-345
VIC
VIC
VIC
VIC
VIC
HTG6
NED™
Black
105-145
ASB2
NED™
Black
150-185
LEX3
NED™
Black
190-230
HTG4
NED™
Black
240-265
ASB23
NED™
HMS7
ASB25
HMS6
HMS1
KBC71
Black
280-320
PET
®
Red
120-160
PET
®
Red
175-190
PET
®
Red
210-235
PET
®
Red
250-280
PET
®
Red
280-340
* Contents of table are based on ABI PRISM 3130XL. The size range may be from 1 to 6 bp
different depending on type of Genetic Analyzer instrument.
Amplification of the 22 HoresFiling® loci using the equine DNA as the
sample should provide results similar to the one illustrated in Figure 1.
This figure shows plots of GeneMapper® software results when equine
DNA is separated on an Applied Biosystems® 3130XL Genetic
Analyzer.
As described in Chapter 4, “Interpretation of results,” plots of the 22
amplified loci can be compared to GeneMpper® software electropherograms to determine whether the horse tested is homozygous or
heterozygous for each marker.
KBC-HorseFiling® kit for Equine Genotyping
8
Figure 1 GeneMpper® software analysis of PCR amplification products using equine control DNA which
®
detected on the Applied Biosystems 3130XL Genetic Analyzer
9
KBC-HorseFiling® kit for Equine Genotyping
4
Interpretation of Results
By comparing test data to control data presented in this chapter, you can
determine whether the horse tested is homozygous or heterozygous at
each locus. Horses can then be compared to each other to include or
exclude possible parents.
Successful amplification using HorseFiling® for Horses
Allele calling
and stutter
peaks
Successful amplification yields allele peaks with the associated PCR
stutter bands within a maximum range of 8 base pairs from the allele
peak. The number of allele peaks depends on whether the individual
tested is a heterozygote or homozygote. For the HorseFiling®, all loci
are dinucleotide repeat units. Dinucleotide repeats give specific stutter
patterns that are illustrated in Figures 2 through 4 on pages 11 and 12.
When interpreting the results, it is noteworthy that within one locus, the
longer alleles may display lower amplification yields (peak height) than
the shorter alleles. In addition, the stutter peak is normally of much
lower intensity than the true allele peak. Further, within some loci, the
longer alleles may display more significant stuttering than the shorter
alleles.
Typical peak profiles for homozygous individuals, heterozygous
individuals with the two alleles > 2 bp apart and heterozygous
individuals with the two alleles 2 bp apart are shown in Figures 2
through 4 respectively.
The electropherogram of a dinucleotide repeat marker for a
Homozygous
individual: 2-bp homozygous individual is shown in Figure 2.
and 4-bp
stutter
KBC-HorseFiling® kit for Equine Genotyping
10
Figure 2 A typical pattern for dinucleotide repeat homozygote. The numbers
correspond to the following PCR amplicons: 1. the true allele based on its
complete DNA sequence; 2. the -2 bp stutter peak of the true allele; 3. the -4 bp
stutter peak of the true allele
Heterozygous
individuals with
the two alleles >
2 bp apart
The electropherogram of a dinucleotide repeat marker for a heterozygous individual is shown in Figure 3. Allele sizes differ by 10 bp. The
2-bp stutter peak to the left of each allele peak is always of lower
intensity than the allele peak itself. The larger 188-bp allele peak is of
lower intensity than the smaller 178-bp allele. In heterozygotes, the
higher molecular weight allele often produces a fluorescent signal of
lower intensity than the lower molecular weight peak, suggesting a less
efficient amplification of the larger fragment.
Figure 3 A typical peak profile for a heterozygous individual with the two alleles
> 2 bp apart. The numbers correspond to the following PCR amplicons: 1. The true
alleles based on their complete DNA sequences; 2. The -2 bp stutter peaks of the
true alleles; 3. The -4 bp stutter peaks of the true alleles.
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KBC-HorseFiling® kit for Equine Genotyping
Heterozygous
individuals with
the two alleles 2
bp apart
The electropherogram from a dinucleotide repeat marker of a heterozygous individual is shown in Figure 4. Allele sizes differ by 2 bp. A
dinucleotide repeat marker for a heterozygous individual shows this
typical “triangle pattern” when the alleles differ by 2 bp.
Figure 4 A typical peak profile for a heterozygous individual with the two alleles
2 bp apart. The numbers correspond to the following PCR amplicons: 1. the true
longer allele based on its complete DNA sequence; 2. the true shorter allele and
the -2 bp stutter peak of the longer fragment make the peak of shorter fragment
higher allele which has resulted in the increase of its height; 3. the -4 bp stutter
peak of the true longer allele and the -2 bp stutter peak of the shorter allele.
KBC-HorseFiling® kit for Equine Genotyping
12
Troubleshooting
Problem
Possible explanation
Recommended action
DNA quantity of the test
sample
is
below
the
assay’s level of sensitivity.
Faint or no signals from the test
®
HorseFiling Control DNA.
and add sample DNA to PCR in
the quantity recommended in
the Protocol.
Dilute the sample DNA extract
sample for all loci, but normal
signals for all loci from the
Measure the DNA concentration
into H2O (for example 1:2, 1:5
PCR inhibitor concentra-
and 1:20 dilutions) and repeat
tion of the test sample is
the protocol. Change the DNA
too high.
purification method. Otherwise
use a better DNA extraction and
quantitation.
There has been an error in
Faint or no signals from both
the
test
sample
and
the
®
HorseFiling Control DNA.
the PCR or electrophoresis setup.
Check the setup and repeat the
protocol.
The cycling profile applied is not optimal for the
Check the PCR program.
®
HorseFiling kit.
Measure the DNA concentration
and add sample DNA to PCR in
Overshoot for all or some loci
the quantity recommended in
and occurrence of non-specific
amplification
from
The sample DNA quantity
the sample, but normal signals
added to PCR is too high.
for
all
products
loci
from
the
Sample and DNA extraction,
Alternatively, dilute the sample
DNA
extract
into
H2O
(for
example 1:5, 1:10 and 1:20
®
HorseFiling Control DNA.
dilutions)
and
repeat
the
protocol.
Overshoot for all or some loci
and occurrence of non-specific
amplification
both
the
products
sample
®
from
and
HorseFiling Control DNA.
13
the
There has been an error in
the PCR or electrophoresis setup.
Check the setup and repeat the
protocol.
The cycling profile applied is not optimal for the
Check the PCR program.
®
HorseFiling kit.
KBC-HorseFiling® kit for Equine Genotyping