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*Faculty of Biotechnology and Biomolecular Sciences,

**Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Malaysia,
***Department of Wildlife and National Parks, No 10, Jalan Cheras, Kuala Lumpur, Malaysia


The blood of the Painted Storks (Mycteria leucocephala) and the Milky Storks (M. cinerea) from Malaysia were collected
invasively from the breeding site. The blood was dropped on to FTA® cards and stored at room temperature. DNA was isolated from
the FTA® cards through a modification of the Wizard DNA Purification kit (Promega) procedure and PCR was performed with 11 pairs
of microsatellite primers of the American Wood Stork (M. americana). The collection of a drop of blood onto the card is superior to the
usual practice of collecting about five ml of blood into a vacuum tube as it causes fewer traumas to these sensitive birds. Moreover, this
collection procedure can be adopted for use in various wild animal species which are usually found in the remote areas of Malaysia as
the sample collection cards can be transported back to the laboratory at room temperature. Our procedure allows the typing of several
molecular genetic markers from just a drop of blood collected in the field and stored at room temperature alleviating the need for storage
in expensive deep freezers or liquid nitrogen tanks.

Collection of avian samples is no easy task as the birds are free using the Australian abalone on the quality of the DNA isolated
flying. DNA extraction from these samples is therefore a crucial from various types of the tissues collected on FTA cards and from
step before proceeding to any genetic analysis work. Generally, the same tissues collected in tubes. Two different methods of
samples can be collected invasively, intermediate invasively or DNA isolation were compared for the samples collected on cards.
minimal invasively and non-invasively. Non-invasive method as One was to use direct PCR amplification on the purified FTA disc
defined by Taberlet in 1999, implies a collection of biological and another method was to use the Wizard SV Genomic DNA
samples without the studied animals being aware of the collection Purification kit (Promega). Ten different types of tissues from the
process (Caudron et al. 2007) The method is useful especially for abalone were collected and the extracted DNA tested for PCR.
endangered species and free ranging animals (Horváth et al. 2005, The results showed that 10 microsatellite loci were amplified in
Morin et al. 2001) since the samples can be obtained without all the Promega kit DNA extracted samples and 9 in the 10 direct
catching the animals (Wasko et al. 2003), large numbers can be FTA samples (Carr et al. 2008). The DNA extracts of the tube
obtained (Kim. 2001) and thus help to increase the population collected samples showed the same microsatellite profiles as those
parameters for statistical studies (Parson, 2001) The disadvantage collected on the cards hence showing the reliability of the card
of non-invasive sampling is the low yield and poor DNA quality sample collection method for this aquatic mollusk. Our work is to
(Horváth et al. 2005; Morin, et al. 2001; Parsons 2001) Non- gauge the feasibility of using the card blood collection method for
invasively acquired samples can be shed feathers, shed hair, the genetic studies of Malaysian wild animals by using the Painted
feaces, urine, shed skins and chewed fruit remnants (Morin etal. and the Milky storks as the experimental animals.
2001; Wasko et al. 2003; Bensch et al. 2002; Rohland et al. 2007) These cards are chemically coated papers of small sizes
The intermediate invasive or minimal invasive approach is defined (128mm X 74mm) with four circles on each as is shown in Figure
as being intermediate in terms of impact on the animals, between 1. They were designed to simplify collection, transportation,
biopsy on capture and non-invasive sampling. These can be the archiving and purification of DNA and RNA from a range of
plucked feathers and plucked hairs. The quality of DNA obtained biological sources. (Guitierres-Corchero et al. 2002; Drescher et
is much better than those of the non-invasive samples (Hogan et al. al. 2002; Fujita et al. 2006). The chemicals on these cards have
2007; Rudnick et al. 2005; Harvey et al. 2006; Segelbacher, 2002). antimicrobial properties to prevent contamination, induce lysis
The invasive method is to capture the animals to obtain tissues via of the cells, denature proteins, and protect nucleic acids from
biopsy or to draw blood with needles, both of which cause high nucleases, degradation, oxidation and UV damage (Guitierres-
stress to the animals. Invasively collected samples are usually Corchero et al. 2002; Drescher et al. 2002; Fujita et al. 2006).) The
systematically obtained from individuals with clear identities and nucleic acids are physically entrapped immobilized and stabilized
no contamination.Thus, the extracted DNA quality is of the best. for storage (Fujita et al. 2006) There are a range of proprietary
The flaw is that it has to be processed fresh if not immediately. collection papers available, including IsoCode card (Scheleicher
Conventionally, blood is usually collected in tubes treated with an & Schhuell, Saseel, Germany) Generation Capture system (Biozyn
anticoagulant such as EDTA, heparin or citric acid. It is hard to Diagnostik Gmbh, Hessisch-Oldendoft, Germany) and FTA®
carry tubes in an ice-box to gather blood of free ranging animals cards (Whatman, Kent, UK) (Guitierres-Corchero et al. 2002)
in remote areas and in addition with no equipment to process Blood was drawn using a 5ml syringe from the leg vein of
freshly collected blood. Hence, FTA® cards have been used as painted storks (M. leucocephala) and milky storks (M. cinerea)
an alternative to replace the conventional method to harvest blood by Wildlife Department veterinarians. A droplet of 2ml – 3ml of
samples for studies. Blood is dropped onto the card, dried and kept blood was dropped onto the ring of a FTA card and allowed to dry
in a plastic bag at room temperature. DNA can be extracted later for one hour. The FTA cards were then placed in a zigzag plastic
without compromising its quality. A comparison study was done bag and brought back to the laboratory. These cards were then

GENETIK Jan 2009 page 20

kept at room temperature in a tightly closed metal tin to avoid the The quality of the DNA isolated was tested on 1% agarose gel. 3-
bites of insects and rats or stored at 4°C to prevent degradation by 5μl of DNA was diluted with 1X TE buffer to give a total volume
the high humidity of our tropical environment. of 10μl; then mix with 4ul of 6X loading dye. Load all the mixture
There are three common ways to isolate DNA from FTA onto agarose gel. The gel was electrophoresed at 65-70V for 30
cards. A 1-3mm disc is punched out with a scientific puncher, then to 45 minutes and then stained with 0.17% ethidium bromide for
washed three times with FTA Purification reagent and three times about 90 minutes in a closed container. Figure 2 is an example of
with TE buffer (Tris-HCl, 10mM pH8, 0.1mM EDTA) (Drescher such a stained gel.
et al. 2002). To strive for a cheaper method, the disc can be washed The PCR reaction contained 1X PCR buffer (Promega),
with NaOH solution three times and then washed with TE buffer 5.5mM MgCl2 (Promega), 0.5mM dNTPmix(Promega), 2.5unit
three times. The PCR mixture is directly added to the washed disc GoTaq(Promega), 0.2uM – 0.8uM of microsatellite primers, 0.5ul
and placed in a thermocycler. The third method which can be to 1ul of stork DNA and totaled up to 15ul volume with ddH2O.
employed is to extract DNA from a disc using a DNA extraction The PCR profile consisted of 94°C pre-denaturation (3mins), 35
kit (King et al. 2005). The short coming of the first two methods cycles of 94°C denaturation(25 seconds), 45-55°C annealing(25
is that each disc can be used for one PCR reaction only. Thus seconds) and 72°C extension(45 seconds) and a final extension of
more blood needs to be sampled for data collection. As for the 72°C(5mins).Eleven pairs of cross species microsatellte primers
scarce Malaysian stork samples, in order to save the blood, whole of American Wood Storks (M. americana) (Bussche et al. 1999)
genomic DNA was extracted from a 3mm disc, re-suspended in successfully amplified the genomic DNA of the painted and milky
50ul of TE buffer. It can then be used for 50-100 PCR reactions storks from Malaysia.
instead of just one PCR reaction. Hence, the fact that a sufficient amount of DNA can be
DNA isolation was performed using the Wizard Genomic isolated from a single drop of blood from an individual bird to allow
DNA Purification Kit ® (Promega) based on the mouse tail the analysis of many microsatellite markers and that the collection
protocol with a few modifications. Punch out a 3mm disc from cards can be transported and stored at room temperature before use
the FTA card using an autoclaved ticket puncher and place in a makes it a useful collection procedure for the treasure house of wild
1.5ml tube. Add 600μl Nucleic Lysis Solution together with 120μl animal species that Malaysia is endowed with. Wild populations
EDTA (0.5M), chill on ice until it was cloudy. Place the disc into of such species, some of which are endangered or rare, are usually
Lysis buffer mixture and add 17.5μl Proteinase K (20mg/ml). The found in the remote areas of Malaysia. This makes the traditional
mixture was vortexed for a few minutes before incubating in a practice of collecting about 10 ml of animal blood into vacuum
55°C water bath overnight. RNA was cleared with 3μl RNase tubes, processing the samples by centrifugation in the field shortly
(4mg/ml) with incubation for 30 minutes at 37°C. The buffer after collection and their transportation back to the laboratory in
mixture was cooled to room temperature for 5 minutes before liquid nitrogen containers tedious and sometimes impossible. By
adding 200μl Protein Precipitation Solution and then chilled collecting the blood onto cards, the problem is solved. Moreover
on ice for 10 minutes. Centrifuge the mixture at 13000rpm for by adopting our DNA extraction procedure as outlined above,
7 minutes. The supernatant was pippetted out with care, not to many PCR based molecular genetic markers can be typed just
touch the precipitated protein and put into a new 1.5ml tube. DNA from as single drop of blood. This, we believe can help expedite
was precipitated with 600ul isopropanol by inverting the tubes research in the molecular population genetics of Malaysian wild
till white spots were formed. The solution was centrifuged for 7 animal species in general and help in the development of a wildlife
minutes at 13000rpm. With care, discard the supernatant; leaving forensics database in particular. The need for such a database is
about 10μl of isopropanol behind to prevent taking out the DNA clearly illustrated by the recent increases in the cases of seizures
which was pelleted at the bottom of the tube. Then, 600μl 70% of wild animals or their body parts in this country. Without strong
absolute ethanol was used to rinse the DNA and centrifuged for evidence that these animals were acquired through illegal means,
4 minutes at 13000rpm. Remove ethanol and air dry the DNA prosecution of such cases in the law courts often fails. Evidence
pellet for 20 – 30 minutes or till all the ethanol had evaporated obtained through the use of an extensive DNA marker based on
completely. Re-suspend the DNA in 50μl of 1XTE buffer (Tris- wild life forensics database should help alleviate this problem in
HCl 10mM, 1mM EDTA, pH8). To dissolve the DNA, incubate the future.
the pellet in a 65°C water bath for 60 minutes or at 4°C overnight.

Figure 1. FTA® card

Left – FTA card with cover.
Right – FTA card with 4 circles
with a blood drop in one of

GENETIK Jan 2009 page 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

10 000bp

Figure 2.
Lane (1-8) Milky Stork DNA.
Lane (9-15) Painted Stork DNA.
M is the DNA ladder.
5ul DNA + 5ul 1xTE + 4ul 6x dye
1% agarose gel
70V (30-45mins)


Bensch. S and Pearson. D(2002)The Large-billed Reed Warbler Horváth. M.B., Martinez-Cruz. B, Negro. J.J., Kalmàr. L and
Acrocephalus orinus Revisited. Ibis 144: 259-267 Godoy. J.A (2005) An overlook DNA source for non-
invasive genetic analysis in birds. Journal of Avian Biology
Bussche. R.A.V.D., Harmon. S.A., Baker. R.J., Bryan. A.L., 36:84-88
Rodgers. Jr. A.J, Harris. Jr. M.J and Brisbin. Jr. I.L(1999)
Low Levels of genetic variability in North American King. T.L., Eackles. M.S., Henderson. A.P., Bocetti. C.I., Currie.
population of the wood stork(Mycteria americana) The Auk D and Wunderle. Jrs. J.M. (2005) Microsatellite DNA
116: 1083-1092 markers for declining population structure and kinship among
the endangered Kirtland’s warbler (Dendroica kirtlandii)
Carr. N.A. and Appleyard. S.A.(2008) Using FTA Elute Molecular Ecology Notes 5: 569 – 571.
Microcards to address biosecurity and DNA quality issues in
abalone aquaculture Aquaculture Reseacrh 39: 1799-1802 Morin. P.A., Chambers. K.E., Boesch. C and Vigilant. L. (2001)
Quantitative polymerase chain reaction analysis of DNA
Caudron. A.K., Negro. S.S., Muller. C.G., Boren. L.J. and from non-invasive samples for accurate microsatellite
Gemmel. N.J.(2007) Hair sampling and genotyping from genotyping of wild chimpanzees (Pan trogloolytes verus)
hair follicles: A minimally-invasive alternative for genetic Molecular Ecology 10: 1835-1844
studies in small, mobile pinnipeds and other mammals
Marine Mammal Science 23:184-192 Parsons. K.M. (2001) Reliable microsatellite genotyping of
dolphin DNA from faeces. Molecular Ecology Notes 1: 341-
Drescher. A and Graner. A (2002) PCR-genotyping of barley 344
seedlings using FTA samples from tissue prints Plant
Breeding 121: 228-231 Rohland. N and Hofreiter. M (2007) Comparison and optimisation
of ancient DNA extraction. BioTechniques 42: 343-352
Fujita.Y and Kubo. S-inchi (2006) Application of FTA technology
to extraction of sperm DNA from mixed body fluids Rudnick. J.A., Katzner. T.E., Bragin. E.A., Rhodes. Jr .O.E and
containing semen. Legal Medicine 8: 43-46 Dewoody. J.A. (2005) Using Naturally shed feathers for
individual identification, genetic parentage analyses, and
Gutierrez-Corchero. F, Arruga. M.V., Sanz. L, Garcia. C, population monitoring in an endangered Eastern imperial
Hernandez. M.A and Campos. F (2002) Using FTA cards eagle (Aquila heliaca) population from Kazakhstan.
to store avian blood samples for genetic studies. Their Molecular Ecology 14: 2959-2967
application in sex determination. Molecular Ecology Notes
2:75-77 Segelbacher. G (2002) Noninvasive genetic analysis in birds:
testing reliability of feather samples. Molecular Ecology
Harvey. M.G., Bonter. D.N., Stenzler. L.M and Lovette. I.J.(2006) Notes 2: 367-369
A comparison of plucked feathers verses blood samples as
DNA sources for molecular sexing. Journal of Field Ornitbol, Wasko. A.P., Martins. C, Oliveira. C and Foresti. F.(2003) Non-
77: 136-140 destructive genetic sampling in fish. An improved method
for DNA extraction from fish fins and scales. Hereditas 138:
Hogan. F.E., Cooke. R, Burridges. C.P. and Norman. J.A.(2007) 161-165
Optimizing the use of shed feathers for genetic analysis.
Molecular Ecology Notes 7: 1471-8286
GENETIK Jan 2009 page 22