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DNA extraction protocols for molecular studies of Asiatic black bearsAuthor(s): Safia Janjua, Maliha Shahid, Fakhar-i-Abbas and Afsar MianSource: Ursus, 25(1):78-81. 2014.Published By: International Association for Bear Research and ManagementDOI: http://dx.doi.org/10.2192/URSUS-D-13-00013.1URL: http://www.bioone.org/doi/full/10.2192/URSUS-D-13-00013.1

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DNA extraction protocols for molecular studies of Asiatic black bears

Safia Janjua1,3, Maliha Shahid2,Fakhar-i-Abbas1, and Afsar Mian2

1Bioresource Research Centre (BRC) Islamabad,

Pakistan2Institute of Natural and Management Sciences (INAM),

Rawalpindi, Pakistan

Abstract: Optimization of DNA extraction and

amplification techniques increases the reliability of

genetic studies used to estimate population size and

assess genetic diversity, particularly for non-invasively

collected samples. We evaluated the effectiveness of 4

DNA extraction procedures—the Roboscreen and

Qiagen kit methods and the Organic and Chelex

methods, as applied to invasively and non-invasively

collected samples from Asiatic black bears (Ursus

thibetanus). We assessed effectiveness based on

quantity and purity of the extracts by method, and

their ability to amplify genomic DNA via polymerase

chain reaction (PCR) amplification of a single simple

sequence repeat (SSR) marker. For each method, the

greatest average DNA quantity and purity index

(86.25%) was from blood, while muscle showed

greatest DNA quantity and purity index using the

Qiagen DNA extraction method. The Roboscreen kit

and Chelex method provided a 57% and 58% PCR

success rate, respectively, for hairs, demonstrating the

value of such samples for molecular studies of Asiatic

black bears.

Key words: Asiatic black bear, DNA extraction, hair

sample, microsatellite/SSR markers, PCR ampli-

fication, Ursus thibetanus

Ursus 25(1):78–81 (2014)

Advances in molecular biology techniques have

provided useful tools for forensics, diagnostics, genetic

mapping, sexing, population genetics, genetic diversi-

ty, and molecular ecology investigations (Taberlet

et al. 1993, Paetkau and Strobeck 1994). Results

obtained by molecular biology techniques depend on

the DNA extraction procedures, polymerase chain

reaction (PCR) protocols, and primers (Schander and

Kenneth 2003, Suenaga and Nakamura 2005). Ideally,

DNA extraction techniques should be optimized for

the species or species group to obtain the optimal yield

and quality of DNA; this facilitates downstream

processing and analysis of genetic variability.

In genetic studies, DNA analysis is frequently

conducted using blood, feces, or hair samples

(Taberlet et al. 1993, Paetkau and Strobeck 1994).

Extracting DNA from blood and muscle tissuesgenerally provides greater success in PCR amplifi-

cation compared with non-invasively collected DNA

sources such as hairs and feces (Takasaki and

Takenaka 1991, Taberlet and Bouvet 1992, Morin

et al. 1993, Taberlet et al. 1993, Paetkau 2003,

Proctor et al. 2005). Nevertheless, given the difficulty

of safely trapping and handling rare and secretive

animals such as bears, non-invasive techniques couldprovide a practical alternative source of DNA.

Although field sampling of non-invasive samples

such as hairs and feces may require less field

expertise, time, and money than the collection,

storage, and transportation of blood or biopsy

samples, it is important to verify that non-invasively

collected samples provide DNA of sufficiently high

quality and quantity to yield accurate genotypingresults. Further, it is clear that although certain field

expertise, such as trapping or biopsy-darting of

bears, would not be required in studies that gather

non-invasively collected samples, the experience of

laboratory staff is a critical factor in the quality of

results from genetic studies employing non-inva-

sively collected samples (Paetkau 2003). We tested

the quality and quantity of DNA using 4 differentextraction procedures on 4 different DNA sources

collected from Asiatic black bears (Ursus thibetanus),

and by assessing the success rate of PCR amplifica-

tion of a single simple sequence repeat (SSR) marker.

We obtained blood, hair, and fecal samples (n 5

20) from live Asiatic black bears housed at the

Bioresource Research Centre bear sanctuary. We

obtained 10 additional muscle samples from bears

that had died due to natural mortality. We stored

blood samples in tubes containing the anticoagulant

ethylenediaminetetraacetic acid (EDTA), hair sam-

ples in plastic re-sealable bags, muscle samples inethanol, and fecal samples (all collected within 48 hr

of defecation) in sterilized glass bottles. We stored all

samples at 4uC until further experimentation. All

extraction procedures were performed by individuals3email: safiajanjua@hotmail.com

SHORT COMMUNICATIONS N Janjua et al.

78

possessing about 3 years of experience extracting

DNA from various sample types.

We lysed about 3 mm of hair follicles at 45uCovernight with lysis buffer (50mM Tris, pH 8;

200 mM NaCl; 50 mM EDTA; 1%) and Proteinase

K (2 mg/mL). We applied similar lysis procedures toblood (200 mL), muscle (100 mg), and fecal samples

(200 mg); the outer layer of fecal samples was prefer-

entially sampled to maximize inclusion of epidermal

lining debris.

We compared 4 extraction methods, including 2

kits (the Roboscreen [AJ Roboscreen GmbH, Leip-

zig, Germany] and Qiagen [Hilden, Germany] kits),

the Organic extraction method of Higuchi et al.

(1988), and the Chelex method of Walsh et al. (1991),to assess yield and quality of extracted DNA. We

measured the quantity and quality of extracted DNA

by spectrophotometer (Barbas III et al. 2007). To

demonstrate that DNA extracted using each method

might be suitable for molecular studies of the Asiatic

black bear, we targeted a single SSR marker (MSUT-3;

Kitahara et al. 2000) for PCR amplification using the

products of each extraction protocol.

Among sample types, blood and muscle tissues

yielded the largest quantities of DNA (i.e., 2,000 ng/

mL; Fig. 1). The greatest yield from blood samples

was obtained using the Qiagen extraction method;

the greatest yield from muscle samples was obtained

using the Roboscreen or Qiagen extraction methods

(i.e., approx. 2,000 ng/mL). These quantities were

sufficient for further PCR analysis. Quantities of

DNA extracted from hair and feces were lower than

those from blood and muscle (,500 ng/mL). DNA

extracted from muscle samples using the Organic

method gave the greatest purity ratio (A260/A280 5

1.6; Fig. 1). DNA extracted from blood and muscle

samples using the Qiagen method showed second

highest purity ratio (A260/A280 5 1.5; Fig. 1). Among

Fig. 1. Quantity and purity index of genomic DNA of Asiatic black bear samples as influenced by differentmethods and sample types. Each point in the chart is the mean of 3 replicates. Error bars represent values ofdifference at P , 0.050. B = blood sample, T = muscle tissue sample, H = hair sample, F = fecal sample, R =Roboscreen method, Q = Qiagen method, O = Organic method, and C = Chelex method.

Table 1. Percentage success of 4 methods used for DNA extraction using 4 sample types from Asiaticblack bears.

Methods Blood Muscle tissue Hair Fecal Average

Roboscreen 100 80 60 55 73.75

Qiagen 100 100 65 70 83.75

Organic 100 90 55 55 75.00

Chelex 45 60 70 45 55.00

Average 86.25 82.50 62.50 56.25

SHORT COMMUNICATIONS N Janjua et al. 79

Ursus 25(1):78–81 (2014)

the 4 different DNA extraction methods, the Qiagen

method gave the highest average percentage success

(83.75%; Table 1).

The PCR success rate for each sample DNA ex-

tracted using the different protocols was greatest

(85%) for blood sample while lowest (3%) for fecal

samples (Table 2). Overall, the Roboscreen extraction

method was the most effective, averaging 60.50% PCR

success.

Reliability, feasibility, and reproducibility of results

in molecular genetics studies depend on high molec-

ular weight and high-quality genomic DNA with low

levels of fragmentation and DNA efficiently and

accurately amplifiable by PCR. In this work, we

obtained genomic DNA with moderate to high purity

and low to high yield from different sample types

collected from U. thibetanus. As expected, as per

number of the cells available for DNA extraction,

blood and muscle tissues samples yielded a higher

quantity and better quality of the DNA relative to

hair and feces. However, as suggested previously

(Takasaki and Takenaka 1991, Taberlet and Bouvet

1992, Taberlet et al. 1993, Paetkau and Strobeck

1994), hair and fecal samples are more easily available

for such studies and at times may be the only type of

sample feasibly obtained for certain species, such as

Asiatic black bears (Zhang 1996).

Although the quantity and quality of the DNA

extracted from hair and fecal samples are low, such

samples are often effectively employed in ecological

studies. Although all methods provided adequate

quality and quantity of extractions from blood and

muscle samples, for hair samples, the highest

quantity of DNA was obtained using the Chelex

extraction method. Although the Chelex method has

some advantages over the other extraction proce-

dures (e.g., is faster and simpler and less expensive

than other methods, and does not employ toxic

chemicals), problems associated with degradation

have been reported for Chelex extractions. For

example, Greenspoon et al. (1998) reported a faster

rate of degradation of DNA in extracts containing

unbuffered Chelex beads stored at 220uC.

The PCR can generate multiple copies of a target

gene, to some extent mitigating the problem of low

quality and quantity of DNA extractable from hair

and feces (Navidi et al. 1992, Taberlet et al. 1996).

However, PCR product amplified from DNA

extracted from noninvasively collected sources such

as hairs and feces can still be subject to PCR artifacts

such as false alleles and allelic dropout, resulting in

genotyping error that can compromise the power

and accuracy of the resulting data. Careful selection

of loci for use in genotyping from these types of

samples, and use of post-amplification assessment of

genotype reliability (e.g., Miller et al. 2002) can

mitigate some of these issues. Although here we

report results generated from only a single locus to

assess PCR success, as in other studies (Taberlet

et al. 1996, Oka and Takenaka 2001, Wasser et al.

2003), this study suggests that hairs are more

efficiently employed than are feces in DNA typing

studies involving bears.

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Received: 5 June 2013Accepted: 15 March 2014Associate Editor: S. Talbot

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