PERFORMAgene LIVESTOCK™

For Bovine DNA Collection

Quality, Yield, Performance and Bacterial Content of gDNA
from cattle nasal samples collected and purified using
Performagene™•LIVESTOCK
R.M. Iwasiow, C.M.P. James, and Jacques Niles
DNA Genotek Inc., Ottawa, ON, Canada
Performagene™•LIVESTOCK is a kit designed for noninvasive collection of genomic DNA from cattle. In
this document, we demonstrate that the average
DNA yield recovered from cattle nasal samples is24.2 ug. The DNA has an average corrected A260/
A280 ratio of 1.7, and a molecular weight > 23kb.
The average bacterial DNA content from thePerformagene•LIVESTOCK samples is 3.3%.

were corrected for minor amounts of turbid material
by subtracting the A320 value. The molecular weightof the DNA was determined by electrophoresis on
a 0.8% agarose gel using a Lambda/HindIII DNAmarker. Purified DNA was shown to be of sufficientquality for real-time PCR by amplifying a portion ofthe bovine thymidylate synthetase gene.
Quick to PCR Assay

Introduction
Quick, reliable and easy-to-use sample collection
methods facilitate and accelerate molecular geneticexperiments.
The Performagene•LIVESTOCK kit is designed forreliable collection of genomic DNA (DNA) fromunrestrained cattle. The product is an all-in-one
system for collection, stabilization, transportation andextraction of DNA from nasal samples. Once the nasal
sample is collected into the Performagene•LIVESTOCK
solution a large amount of DNA is released and stabilized
at room temperature. The solution is bacteriostatic andminimizes the bacterial content in the sample aftercollection. Performagene•LIVESTOCK is ideal for thecollection of large amounts of high quality DNA fromcattle where blood is difficult to obtain and tail hairs or
ear punches are inefficient to process and can provide alow quality and quantity of DNA.

Materials and Methods
Cattle Nasal Sample Collection/Purification
Nasal samples were collected from 225 unrestrained
cattle ranging in age from 4 days to 14 years. During
the field collection, no regard was taken to the lasttime the cattle ate or drank. After collection using
Performagene•LIVESTOCK samples were transported atroom temperature to the lab. Once at the lab the samples
were heated at 50°C for 1 hour in a water bath. Following
the heating step a 500 μL aliquot was purified according
to the Performagene•LIVESTOCK purification protocol(PD-PR-083 Issue 3.0).
DNA Analysis
DNA was quantified by fluorescence following the DNA
Quantification Using Sybr Green I dye and a MicroPlate Reader protocol (PD-PR-075). The absorbance
spectrum between 320nm and 230nm was determinedusing a Tecan Infinite M200. The A260 and A280 values

Performagene•LIVESTOCK samples can also bequickly prepared for use in downstream applications
without the need for full purification. Raw (unpurified)samples were prepared for real-time PCR according
to protocol PD-PR-098. Up to 2.5 μL of the preparedsample was used in each PCR reaction.
Bacterial DNA Analysis
Bacterial DNA was quantified using a real-time
PCR assay (PD-PR-065 Issue 1.0). PCR primers
were chosen from a region of the 16S rRNA genewhich is known to be conserved across a wide
variety of microorganisms. Randomly selected, 68
Performagene•LIVESTOCK samples were tested
for the 16S rRNA gene by quantitative PCR using aRotor-Gene™ 6200 real-time thermal cycler (CorbettResearch). Each reaction used 15 ng of total DNA asthe template. To check the efficiency of each reaction,a second 15 ng aliquot from each sample was spiked
with 5 ng of bacterial control DNA and run alongside
the first sample. Assuming the reactions were perfectly
efficient, the amount of bacterial DNA should be
5 ng plus the amount of the unknown bacterial DNA.
A standard curve was used to quantify the samples.
Purified bacterial control DNA, used to construct
the standard curve, was obtained from Sigma (E.coli,strain B, Cat. #D4889).

Results
DNA Yield & Spectral Quality
A subset of nasal samples collected from 225
unrestrained cattle were used for the experiments.
Following the purification protocol supplied with thePerformagene•LIVESTOCK kit (PD-PR-083 Issue3.0) the DNA was measured using fluorescence withSybr Green I dye according to protocol PD-PR-075.
The average amount of collected bovine DNA was
24.2 μg. A similar amount of DNA was collected from

PERFORMAgene LIVESTOCK™

For Bovine DNA Collection

different breeds (Table 1). A yield scatter plot (Figure 1)
demonstrates the yield distribution based on animal age.
The purity of the recovered DNA was assessed by thecorrected A260/A280 ratio. Absorbance at wavelengths 260nm, 280 nm, and 320 nm was measured. The absorbanceat wavelength of 320 nm which corresponds to presenceof turbid (insoluble) material was subtracted from theA260 and A280 values before calculating the A260/A280 ratio.
The average A260/A280 ratio for 68 randomly selectedbovine samples was 1.7. The quality of the samples
was further assessed by running 10 randomly selectedpurified samples on an agarose gel (Figure 2). Using a
Lamda-HindIII ladder we were able to demonstrate thatthe purified DNA had a molecular weight >23kb.

The purified DNA was shown to be of sufficientquality for use in real-time PCR assays by amplifying
a section of the bovine thymidylate synthetase gene.
Both calf and cow samples successfully amplified
100% of the time (Figure 3).

1.2

1.0

Norm. Fluro.

0.8

0.6

0.4

0.2
Threshold

Breed of Cattle

Average amount of Total DNA (µg)

Holstein

24.0

Hereford

31.2Brown Swiss

15.9

Simmental

21.9

0.0
2

4

68

10

1214

16

18

20

22

24

26

28

30

32

34

36

38

40

Cycle
1.0

Norm. Fluro.

0.8

Table 1: Average amount of DNA collected from 4 different bovine
breeds.

0.60.40.2
Threshold0.02468101214161820222426283032343638

40μg of DNA collected from cattle nasal samples

Cycle

Figure 3: Real-time PCR analysis of cattle samples. Curves represent
successful amplification of the bovine thymidylate synthetase gene.
Blue lines represent DNA collected from calves < 6 month old. Greenlines represent DNA collected from cattle > 6 month old. Black lines
are no template controls.

Quick to PCR Assay

Calves < 6 Months

Adults > 6 Months

All Ages of Cattle

Figure 1: Scatter plot of DNA yield from 225 cattle (85 calves and 140adults). The average amount of DNA is shown by the red line,
calves =31 μg , adults =20.1 μg , total = 24.2 μg.

23.1 kb

Figure 2: Agarose gel electrophoresis (0.8% agarose, 90 V, 60 min)of DNA from 10 bovine samples purified within 72 hours of sample
collection. A Lambda-HindIII digest was used as a marker in Lane 1.

In order to streamline sample processing,
Performagene•LIVESTOCK samples can be quickly
prepared for downstream applications such as realtime PCR through the use of Performagene Direct
reagent (PG-L2). An aliquot of the same samples
that were purified in the above experiments were also
processed using reagent PG-L2 according to protocol
PD-PR-098. The prepared samples were used in areal-time PCR assay which amplified a portion of thebovine thymidylate synthetase gene. All of the testedsamples amplified successfully (Figure 4).

PERFORMAgene LIVESTOCK™

For Bovine DNA Collection

1.2

1.0%Bacterial DNA

Norm. Fluro.

0.8

0.6

0.4

0.2
Threshold0.0246810121416182022242628303234363840Cycle

1.2

1.0Norm. Fluro.

0.8

0.6

0.4

Figure 5: Bacterial DNA as a percentage of total DNA in 68 bovine
Performagene™•LIVESTOCK samples. The horizontal red line represents
the median value (3.3%).

0.2
Threshold

0.02

4

68

10

12

14

16

18

20

222426

28

30

32

34

36

3840Cycle

Figure 4: Real-time PCR analysis of bovine samples after quick toPCR protocol. Curves represent successful amplification of the bovine
thymidylate synthetase gene. Blue lines represent DNA collected fromcalves < 6 month old. Green lines represent DNA collected from cattle >6 month old. Black lines are no template controls.

Bacterial DNA Content
Bacterial DNA content was assessed using a realtime PCR assay with universal bacterial primers. Theuniversal primers against a highly conserved region of
the 16S rRNA gene allow for an accurate assessment of
total bacterial DNA content in bovine samples. Using
this method bacterial DNA has been identified as theminor component of total DNA in bovine nasal samples
with an average of 3.3%, ranging between 0.4% and14.2%.

© 2009 DNA GenotekPD-WP-015 Issue 1.0

Conclusion
Performagene•LIVESTOCK is a non-invasive
collection kit that stabilizes DNA from bovine nasal
samples at room temperature. The resulting purified
DNA is of high quality as assessed by the A260/A280
ratio (1.7) and high molecular weight (>23 kb) as
assessed by agarose gel electrophoresis. For the 225
collected samples, we observed an average DNA
yield of 24.2 μg. Bovine nasal samples contain very
little bacterial DNA, with an average bacterial DNA
content of only 3.3%. Performagene•LIVESTOCK
contains potent antibacterial agents which preventthe growth of bacteria between the time of collection
and the time of DNA purification. In summary, thePerformagene•LIVESTOCK kit is an easy and reliable
method for collecting high quality bovine
genomic DNA.

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