High yield, high quality genomic DNA from cattle nasal samples using Performagene™

J.O. Niles, C.M. James, E.J. Maclean, and R.M. Iwasiow
DNA Genotek Inc., Ottawa, Canada

Introduction

Results

Genetics has been utilized for decades to improve livestock breeding, as a result of its increased industry adoption,
genetic research and DNA testing is accelerating worldwide. Common forms of genetic testing being utilized by theindustry include: parentage verification, trait characteristics (e.g., coat colour determination of red vs. black [MC1R]),
and various genetic diseases. These applications require a reliable, high quality DNA sample; previous sample sources
such as semen, blood, hair follicle or ear tissue have been inconvenient for the producer to collect or difficult,unreliable and costly to process in the lab. Performagene™ (DNA Genotek, Ottawa) is a DNA collection kit which uses
nasal samples resulting in an easy-to-use, quick and reliable sampling method that provides high quantity, highquality genomic DNA.

Table 1: Comparison of sample workflows

An unreliable sample or difficult sampling method impacts performance in downstream applications such asparentage verification and genotyping for trait characteristics. Commonly used assays for parentage and genotyping
include microsattelite, SNP genotyping and microarray. For these reasons, we show the suitability and reliabilityof nasal samples collected from cattle into Performagene on the ABI PRISM® 3100 Genetic Analyzer (Taqman),Sequenom® MassARRAY and Illumina BovineSNP50™ BeadChip.
Nasal samples were collected into the Performagene kit from 38 beef and dairy cattle with minimum restraint.
To compare sample types from the same animal, additional blood and tail hair samples were collected from 5 cattle.
All samples were purified using the Performagene protocol. Using fluorescent quantification, we show that thePerformagene DNA collection method yields a high quantity and high quality of genomic DNA. This study reportsthat purified DNA is suitable for any downstream application.

DNA source

Nasal(Full purification)

Nasal(Quick to PCR)

2 min
Non-invasive

2 min
Non-invasive

Blood
5 min
Invasive

Processing time per sample
Hands on time per sample

1h 40 min20 min1h 30 min
5 min

28 min17 min1h 31 min
31 minProcessing time per 24 samples
Hands on time per 24 samples

2h 05 min
45 min1h 36 min11 min41 min30 min4h 38 min3h 38 minWorkflow impact
Collection time
Ease of collection

Hair(50 follicles)
7 min
Non-invasive

Table 2: Quantification results for A) nasal and B) paired samples
A)

B)

Sample
ID1
2
3
4
5
6
7
8
9
10
11
12
13
14

Materials and methods
Nasal samples
Figure 1: Nasal sample collection using Performagene

DNA yield
per 1 mL ofsample (µg)
71.64
15.03
7.29
6.79
4.01
11.61
2.75
2.71
3.05
8.73
2.29
21.18
10.12
24.75

SampleID1516
1718
19202122232425262728

DNA yieldper 1 mL ofsample (µg)
11.90
2.70
4.83
4.51
16.85
3.58
3.40
10.58
1.24
9.71
22.02
3.27
37.59
19.97

Sample
ID
2930
31
32
33
34
35
36
37
38
Average
MedianMax
Min

DNA yield per
1 mL ofsample (µg)
19.72
16.57
22.24
10.79
14.33
5.96
38.22
9.94
1.68
12.92
13.07
10.03
71.64
1.24

Sample
ID
175
134
94
353
304
Average
MedianMax
Min

DNA yield per aliquot purified (µg)
Nasal (1 mL of
Blood (200 µL of
Hair (50 follicles)
sample)sample)
10.12
15.66
0.85
4.51
14.00
1.12
4.83
18.09
1.20
2.70
9.21
0.05
21.18
9.14
0.57
8.67
13.22
0.76
4.83
14.00
0.85
21.18
18.09
1.20
2.70
9.14
0.05

Figure 2: A) Sample comparison of nasal (N), blood (B) and hair (H) high molecular weight DNA.
B) Representative gel of 16 nasal samples. 50 ng of total DNA was loaded into a 0.8% agarose gel
and run at 80 volts for 45 minutes.

5 sec

DO NOTSPILL• 38 nasal samples were collected from cattle according to the DNA Genotek protocol PD-PR-099 and stored at room• temperature until processed.
• Samples were collected by 7 diﬀerent people and were collected from the following breeds of cattle:• Holstein, Jersey, Limousin and Charolais.
• 0.5 mL aliquot of the nasal samples was purified according to the DNA Genotek purification protocol PD-PR-083 and• the purified DNA was quantified by fluorescence, evaluated by agarose gel electrophoresis, and analyzed on the• Illumina BovineSNP50 BeadChip.
• 100 µL aliquot of the nasal sample was processed according the DNA Genotek Quick to PCR protocol PD-PR-00220• and analyzed by Sequenom and Taqman assays.

Blood samples• 5 blood samples were collected from Holstein cattle for which nasal samples were also collected (see Table 2).
• Blood was collected from the tail vein using EDTA tubes and kept at 4°C until processed.
• 200 µL of blood sample was purified according to the Qiagen® QIAamp Blood Mini Kit.

Tail hair samples• 5 hair samples were collected from Holstein cattle for which blood and nasal samples were also collected (see Table 2).
• The hairs were pulled from the tail, counted and inspected for clean, intact root bulbs then aligned in a plastic bag untilprocessed.
• For each animal, DNA from 50 hair follicles was purified using an SDS and proteinase K protocol.
• Briefly, hair follicles were placed in 0.1% SDS with 0.2 mg proteinase K and incubated at 50°C for 1 hour. After incubation,
20 μL of 2.5 M of KCl was added and incubated on ice for 10 minutes and then centrifuged at maximum speed for 5 minutes.
The supernatant was then ethanol precipitated and gDNA was recovered.

B)

A)

NN

BB

H
H

23.1 kb

23.1 kb

Table 3: Summary of results from downstream applications:
results from Sequenom, Microsatellite and Illumina arrays

Figure 3: Representative scatterplot from Sequenom data
Averageconcordance (nasalwith hair)

Processing method(Nasal)Average call rate
(Nasal)Averageconcordance (nasalwith blood)

Sequenom® MassARRAY analysis of nasal
Microsatellite analysis ABI PRISM® genetic
analyzer - Taqman assay

Quick to PCR

98.74%

99.49%

95.96%

Quick to PCR

91.73%

100.00%*100.00%*Illumina BovineSNP50™ BeadChip

Full purification

99.63%

100.00%100.00%Assay* no calls not included in concordance calculation

Discussion• Performagene provides a quick and easy method for collecting large
amounts of high quality DNA from cattle using non-invasive methods.
• Performagene oﬀers two DNA preparation methods which oﬀer
decreased hands on time and result in DNA of high quality suitable fordownstream applications.
• DNA collected from nasal samples is the same as DNA from blood as shown
through the high concordance rates on all three platforms tested.

DNA Genotek would like to extend their appreciation to all parties who assisted in the collection of this data, including
but not limited to the Cattlemen who collected the samples, as well as GenServe Labs and DNA Landmarks for providing
DNA analysis services.

Superior Samples
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Performagene™ is a trademark of DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc.
All other brands and names contained herein are the property of their respective owners.
Patent (www.dnagenotek.com/legalnotices)
MK-00193 Issue 1/2013-05