Francisco et al.

| 13

CHAPTER III

MATERIALS AND METHODS

3.1 Sample Collection

Twenty seven samples of market sold crabs were obtained from wet markets in

Buguey, Aparri, Sta. Ana, Cagayan province, Philippines. All collected samples were

chilled on ice prior to tissue sampling. Tissue samples for DNA extraction were obtained

from the crab’s right cheliped. 100 grams of muscle tissue was dissected, preserved in

absolute ethanol and stored at -4 °C prior to DNA extraction.

Figure 2 Map ofAparri, Buguey, and Santa Ana, Cagayan.

 Francisco et al. | 14

3.2 Morphological Identification

The checklist of Motoh & Kuronuma (1980) and Carpenter & Niem (1998) served

as a basis for initial morphological identification of market-sold crabs. It was based on the

given characters, by which the species was determined or identified and provided the key

characters of a crab generally covering the external features such as structure of body

concerning the shape of carapace, abdomen, number of anterolateral margin, color of

cheliped, color of the species and the sex.

3. 3 DNA Extraction

The salting-out method modified from Bruford et al (1998) was used for genomic

DNA extraction. DNA extracts were obtained exclusively from the muscles of crab’s right

cheliped. 0.5–1cm of tissue sample was added to a 1.5 ml microcentrifuge tube with 120µl

of 0.5 M EDTA solution (pH 8.0) and 500 µl of lysis buffer and were chilled on ice

afterwards.

Followed by a 600 µl of EDTA/lysis buffer from the first procedure to the tube. 17

µl of Proteinase K was added to the solution and incubated for 3 hours at 550C. After

incubation, samples were vortexed and 200 µl of 7.5 M ammonium acetate was added to

the samples and were placed on a vortex for 20 seconds. The samples were chilled for 5

minutes. Subsequently, the samples were centrifuged for 4 minutes at 13,000 rpm. The

precipitated protein formed a tight pellet and the supernatant containing the DNA was

carefully removed, but some residual liquid was left in the original tube to avoid DNA

solution contamination with the precipitated protein. Then, it was transferred to a clean 1.5

ml micro centrifuge tube.

 Francisco et al. | 15

A 600µl of isopropanol was added to the sample. The solution was mixed gently

by inversion until the white thread-like strands of DNA form visible mass. Then it was

centrifuged for 5 minutes at 13,000 rpm. The DNA appeared as a small white pellet. The

supernatant was carefully transferred. Then, 600 µl of 70% ethanol was added and was

placed in a centrifuge for 5 minutes at 13,000 rpm. The DNA pellet was very loose at that

point so the ethanol was carefully decanted.

The tube was inverted on clean absorbent paper and allowed the pellet to air-dry.

100 µl of 1X TE buffer was mixed. Periodically, the solution was mixed by gently tapping

the tube. Alternatively, the DNA was rehydrated by incubating the solution overnight at

room temperature or at 4°C. The DNA was stored at 4°C.

3.4 Polymerase Chain reaction

Approximately 600 base pairs of the Cytochrome Oxidase subunit 1 gene region

was amplified by polymerase chain reaction (PCR) using the primer LCO1490: (5'-

GGTCAACAAATCATAAAGATATTGG-3') and HC02198: (5'-

TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al.1994). The total volume of

the reaction mixture was 24 µl; consisting of 13.5 µl of nuclease-free water, 2.5 µl of 10x

PCR buffer, 2.5 µl of 10 mMdNTP, 2.0 µl of 25 mM MgCl2, 1.0 µl of 5x BSA, 1.25 µl of

10mM of both primers, and 0.125 µl of Taq polymerase and 1 µL DNA.

Thermal cycling conditions consisted of an initial denaturation of 940C for 10 min

followed by 38 cycles of DNA denaturing at 940C for 30/s, primer annealing at 450C for

45/s, and sequence extension at 720C for 45/s, ending with a final extension of 720C for 10

min (Chow S, 1998).

 Francisco et al. | 16

3.5 Data Analysis

Amplified products were sent to Macrogen, Inc. Korea for DNA sequencing.

Sequences were assembled in Geneious v6.0.6 (Biomatters, 2012) and was aligned using

MUSCLE v3.8.31 (Edgar, 2004). Representative of CO1 sequence of outgroup were

obtained from Genbank. (Genbank accession number: KC200562, KC200563, HM750949,

HM751078, EF661877, EF661976). The phylogenetic analysis was constructed using

neighbour-joining (NJ) criteria and Kimura-2-Parameter (K2P) that were inferred in

MEGA version 6(Tamura et al, 2014).