percentage of sample with high concentration and high purity ... their status as vulnerable species needs to be protected. Genetic ... With small animal like seahorse the .... method showed no band, smear or not clear band (figure 1). ... Lane 7, 8: fin tissue samples. ..... Dr. Hoang Tung (Biotechnology School of International.
A DNA Extraction Method Applied for Living Seahorses Nguyen Thi Hue and Nguyen Thi Thanh Tran School of Biotechnology, International University, VNU, HCM City
Abstract –– The DNA extraction is the first step provided DNA samples for further genetic studies. As a small animal the DNA extraction from seahorse means killing the living specimens. This study is investigated to estimate the tissue sample size which can be used for extraction of enough DNA for further studies without killing living specimens and find out the suitable method for extraction of DNA from seahorse. Tissue samples were obtained by taking small tail rings at the end of the tails or the fins of living seahorses. Two methods were applied for extraction of DNA: Phenol method and salt method. The extracted DNA was concentrated by Nanodrop system. The quality of DNA was measured by agarose gel and PCR assay. While DNA from fin tissue gave no result in PCR, all DNA from tail tissue with about 20ng/ul gave the good PCR result even extracted by phenol or salt method. With the tail tissue, high concentration of DNA was achieved in both methods. 41% samples and 57.4% samples gave DNA concentration higher than 20ng/ul by phenol and salt method, respectively. The purity of DNA yield extracted by phenol is lower than by salt method (18% in phenol method compared to 44.5% in salt method). Thus, salt method gave higher efficiency than phenol method. The salt method gave higher percentage of sample with high concentration and high purity of DNA than phenol method. The salt method is considered as the suitable method. With the weight of tail tissue around 20mg total amount of DNA at least is 3µg which adequate for further genetic analysis. Beside the over success of salt method compare to phenol method, the salt method is lower cost and less hazardous chemicals as in salt method phenol, an expensive chemical and toxic, is not required. Keywords –– Seahorse, DNA extraction, Phenol method, salt method.
I.
INTRODUCTION
DNA extraction is a routine essential step in any biological studies about DNA. This step is widely utilized in a variety of genomic and molecular studies as well as other fields of medical examinations, clinical diagnostics, and forensic investigation. Therefore, many methods have been established to extract DNA molecules from biological materials [1]. Various effects can be obtained on DNA extraction by using different methods [2, 3]. An ideal isolation technique should optimize DNA yield, minimize DNA degradation,
and be efficient in terms of cost, time, labor, and supplies. It must also be suitable for extracting multiple samples and generate minimal hazardous waste [3]. The Phenol and Salt methods are commonly used for DNA extraction from diverse organisms. In principal DNA is first released by lysing the cells with an anionic detergent in the presence of a DNA stabilizer. Proteins and other contaminants are removed by salt precipitation. Finally, DNA precipitated with absolute ethanol or isopropanol from its aqueous solution. There are tremendous variations in volume of ethanol or isopropanol (1- 2.5x volume of supernatant with DNA), incubation temperature (-80oC25oC) and time (1- 24 hours) used for DNA precipitation. DNA molecules then stored in sterilized water or TE solution [2, 3]. Seahorses (genus Hippocampus) are in the family Syngnathidae, which also includes pipefish, pipehorses, and seadragons. Seahorses have a worldwide distribution inhabiting both temperate and tropical seas, with 33 species identified to date [4, 5]. Some species are reported in Vietnam: the spotted seahorse (H. kuda), three-spotted seahorse (H. trimaculatus), Japanese seahorse (H. mohnikei), and tiger tail seahorse (H. comes) [5]. Seahorse populations are in decline due to habitat destruction and overexploitation [6]. Monitoring the status of seahorses is possible via an analysis of the mitochondrial (mt)DNA control region. The mtDNA control region is sufficiently variable to use at the population level [7, 8] [9-11]. The results from an mtDNA analysis can subsequently be applied to the development of seahorse conservation and management strategies, such as increasing natural seahorse abundances while avoiding impacts on genetic variations [12]. Hippocampus kuda, also known as spotted seahorse. Spotted seahorse is the main objective in the study due to their status as vulnerable species needs to be protected. Genetic studies in these seahorses are necessary to understand more about the genetic diversity which may support the breeding selection for the new generation. The DNA extraction is the first step provided DNA samples for further genetic studies. With small animal like seahorse the DNA extraction from tissue means killing the living specimens. For the spotted seahorse, an animal need to be protected, genetic studies need to be performed but killing
V. Van Toi et al. (Eds.): 4th International Conference on Biomedical Engineering in Vietnam, IFMBE Proceedings 40, pp. 178–183, 2013. www.springerlink.com
A DNA Extraction Method Applied for Living Seahorses
the seahorses must be avoided [13]. The same situation, with other small animals, extracting DNA from small tissues and do not scarify the living specimens is necessary. This study is investigated in comparison the efficiency of two methods phenol and salt with the limitation of weight of tissue and kind of tissue based on the DNA concentration, purity and cost also. II. MATERIALS AND METHODS A. Materials The spotted seahorse Hippocampus kuda was used as the model sample. Seahorses were collected from Khanh Hoa province, Vietnam in September 2010. Tissue samples were obtained nondestructively by taking small tail rings from the end of the tails or the fins of living seahorses. They were simply taken by a decisive cut down at the tail to get small parts of the tail or fin and did not harm the seahorses. Tissue samples collected from living seahorse were then stored in 20oC freezer for later use. B. Methods DNA extraction. Two methods were applied for extraction of DNA: Phenol method and salt method. Phenol method: Sample tissue is incubated overnight with proteinase K in the buffer TNES pH 8.0, the combination of 1M Tris, 0.5M Na2EDTA, SDS 20% and 2M NaCl. The mixture of phenol: chloroform: isoamylalcohol (25:24:1) is then added to the incubated samples, and then 3M Sodium Acetate is added. The mixture is then centrifuged at 40C, 14000 rpm for 10 mins. The supernatant is then collected for the next precipitation step. Cold absolute ethanol is then added to the collected supernatant for DNA precipitation. The centrifugation at 40C, 14000rpm, for 5 mins is applied after DNA precipitation step to collect the DNA pellet. DNA pellet is then washed two time by ethanol absolute and 70%. The final DNA pellet is then dried at room temperature for at least 1h. Dried DNA pellet is subsequently suspended in 150µl of sterilized distilled water. The DNA solution is then ready for further analysis [14]. Salt method: Sample tissue is treated with proteinase K overnight as the same way with phenol method. The process DNA extraction is performed the same with phenol method except the mixture phenol: chloroform: isoamylalcohol is replaced by chloroform and 6M sodium chloride [14]. DNA quantification. DNA concentration was measured by Nanodrop Spectrophotometer ND- 1000. DNA concentration peak was observed by 260nm light.
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DNA qualitative. The quality of DNA was measure by observing the band of DNA on agarose gel and the successful in PCR amplification using a set of primers (forward primer: 5'-aacggttcttgatggtcagg- 3', reverse primer: 5'- ccgtgtgcactctgaaatgt- 3') to amplify a fragment of 247 nucleotides of mtDNA control region. The PCR assay was performed with total volume of 25µl contained 40ng of genomic DNA, 12.5µl of Toptaq DNA polymerase, 25mM of primers. The PCR profile consisted of an initial denaturation step (3 min at 94oC), followed by 30 cycles of denaturation at 94oC for 30s), then annealing at 61oC for 30s, and elongation at 72oC for 1min, and a final extension step at 72oC for 5min). PCR products were then analyzed by agarose gel 1.5% and visualized by UV and taken photograph by GelDoc- It System from UVP Company. To control the size of PCR product the first well in each gel was loaded by the 100kb marker. III. RESULTS 76 samples with the range of weight from 9.2mg to 68.7mg were colleted from tail rings of seahorse. Table 1 showed the summary of collected samples and the distribution of samples in two DNA extraction methods: phenol and salt. 22 ring samples were extracted by phenol and 54 samples were extracted by salt (table 1). 9 other samples were collected from fin of seahorse. The size of fin sample is range from 0.5mm2 to 2mm2. 4 fin sample were extracted by phenol and 5 samples were extracted by salt method (table 2). Table 1 The collected ring tissue samples with the range of weight and distribution of samples into two DNA extraction methods. Method
Ring tisue sample weight (mg)
No samples n=76
40
Phenol
4
8
7
7
Salt
5
11
26
17
The extracted DNA was checked on agarose gel. Figure 1 showed the presence of DNA in the extracted solution. Most of DNA extracted by salt method showed the clear and bright band on agarose gel while DNA extracted by Phenol method showed no band, smear or not clear band (figure 1). The DNA extracted from fin showed very light smear on agarose gel (figure 1: lane 7, 8).
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Table 2 The collected fin tissue samples with the range of size and distribution of samples into two DNA extraction methods.
Method
Fin tisue sample size (mm2)
No samples n=9
0.5
1
2
Phenol
3
0
2
1
Salt
6
1
3
2
1
2
3
4
5
6
7
8
DNA quantitation was compared between DNA extracted from tail tissue by phenol and salt methods, the result showed that DNA extracted by salt showed the higher purity (44.5%) than by phenol method (18%). Percentage of sample with DNA concentration higher than 20ng/µl in salt method is 57.4% compared to those in phenol method is 41% (table 4). Within the fin tissue samples, 3 samples extracted by phenol give concentration of DNA lower than 10ng/µl with low purity. 2/6 samples extracted by salt method give concentration of DNA at 15 and 16ng/µl with low purity and 4 other samples give low concentration of DNA at lower than 10ng/µl with low purity also. Table 4 Quality and quantity of DNA through UV light measurement. Method
A 1
2
3
4
5
6
7
No Samples
Phenol
22
Salt
54
Purity
DNA Concentration (150µl) C> 20ng/µl
C< 20ng/µl
9 (41%) 31 (57.4%)
13 (59%) 23 (42.6%)
8
(1.7
