Identification of Goat Skin and Pig Skin as the Raw Material of Rambak Using PCR-RFLP Method

The aim of this research was to detect the skin which comes from goat and pig using PCR-RFLP method so the raw material used in rambak product was discovered. The comparison of combination between goat and pig skin was 100, 75:25, 91:9, 94:6, 97:3, and 99:1. PCR-RFLP uses the universal primary from from mitochondria cytochrome b gen which results in the length of fragment of 359 bp. The restriction enzyme used in the DNA cut is BamHI enzyme and BseDI enzyme. The result of the research showed that seven samples have been successfully isolated perfectly so the total bands of genomic DNA have been obtained which is clearly seen and amplification with target of cytochrome b gen results in PCR product of goat and pig of 359 bp. The digestion's result using BamHI enzyme gets fragment size on 359 bp in length on goat samples and length of fragment size of 359, 244, and 115 bp on samples which contain pig. The digestion results of using BseDI results in fragment size of 359 bp in length on goat samples and the fragment size of 359, 228, and 131 bp in length on samples which contain pig. The conclusion of the research is PCR-RFLP using BamHI enzyme and BseDI enzyme can be used to detect the types of pig skin, but can't be used to detect the goat skin.


INTRODUCTION
Food products in human's life were the most important thing to fulfill the nutrient needed by the body. The decent food product which gets to the requirement of being consumer is the healthy, nutritious, and safe food product. This fits the explanation of regulations Number 18 Year 2012 about the food which becomes the most basic need of human and the fulfillment is the part of every Indonesians citizens' rights. Food has to be always be enough, safe, qualified, nutritious, and various with affordable prize for people's purchasing ability and not against their purchasing ability as well as their religion, faith, and culture. In these some couples years, the producers who work in food companies have started increasing the technology of producing animal feed ingredients, one of which using skin that becomes the side result of animal slaughtering.
Skin was used as the food material because it contains high nutrient. The skin nutrient content was protein, calorie, calcium, phosphor, fat, vitamin A, and vitamin B1. The fresh skin content was generally structured from 65% water, 1.5% fat, 0.5% mineral, and 33% protein. The specialty of goat skin and pig skin was that it is possible to be processed into various skin products such as food and also some other things like leather shoes and leather bag (Sarkar, 1982). According to Fauziah (2016), PCR technology using D-Loop mitochondria has high sensitivity in detecting pork DNA with rambak cracker samples so that the PCR method is proposed as an analysis in monitoring meat products.
The type of skin that is usually used for processing rambak is cow's and buffalo's skin. However, due to the technology development and innovation, people have started making rambak out of goat and pig skin as well as any other skin. After the PCR-RFLP is found, the identification process food basic ingredients especially the food, which is made out of skin can be detected.
The main problem faced in method with DNA basis is the ease of DNA isolation process to get the complete genomic DNA since the DNA isolation is the important factor in the success of next process. DNA which has good quality is the requirement which has to be fulfilled in molecular research. The analysis of PCR continued to RFLP is the step to ease the identification of skin types used in rambak raw material. Consequently, there has to be a basic research to figure out the species from various fresh skin types especially goat and pig skin in order to identify the type of skin used in rambak product.

Sample preparation
The fresh goat skin and pig skin are bought from RPH Giwangan, Yogyakarta. The skin is cut into pieces separately to avoid contamination. After that the skin is packed and stored on -20°C temperature until it is used for research. Comparison between goat skin and pig skin sample was showed in Table 1.

DNA Isolation
The DNA extraction and purification method uses the procedure of Sambrooket al.
(1989) which has been modified, which is the ±30 mg are cut and crushedby mortar until smooth. Then, they are entered into a tube with capacity of 1.5 ml. The extraction is started by adding 500 μlbuffer TEN/STE. After that, those are being under vortex as well as the 30 μl proteinase K, 50 µl 10% SDS, then being incubated into the water bath in 42°C temperature for 18 -19 hours. There are 50 µl 5M NaCl, 400 µl phenol, 400 µl CIAA being incubated into waterbath in 37 o C temperature for 1 hour. Then the centrifugation of 3.000 rpm happens on 4°C temperature for 5 minutes. The supernatant is moved to new tube by adding the 50 µl 5M NaCland 800 µl absolute ethanol. Those are shaken by hands until white threads are formed. The incubation takes place in freezer for 1 hour with -20°C temperature. The centrifugation of 8.000 rpm exists for 5 minutes, and thenthe liquid is thrown away. 1000 µl ethanol 70% is added. Another centrifugation of 8000 rpm takes place in 4°C for 5 minutes. The liquid is thrown away and leaked until there is no liquid left. After then, it is dried using densicator for ±19 hours until it is completely dried. Lastly, it is added with 50 µl TE and stored in -20°C temperature until it is used again. Then 12 µl of each sample is taken from DNA isolation and added with 3 µl loading dye then being under electrophoresis on 100 volts of 2% agarose gel for 30 minutes in TBE 1Xbuffer. 100 bpmarkers (thermo scientific) is used as DNA ladder.

Amplication of PCR cytochrome b
Amplification of cyt b gen is done in final volume of 25 μl which contains dH2O 2 μl,Green PCR master mix 20 μl (2X hot start PCR buffer, 400 µM dATP, 400 µM dGTP, 400 µM dCTP, 400 µM dTTP, and 4 mM Mg 2+ ) (Thermo Scientific), primary universal reverse 1 µl (20 pmol) (Thermo Scientific), primary universal forward 1 µl (20 pmol) (Thermo Scientific) and 1 µl of extraction result of DNA. Amplification is done by using these programs: Amplification is conducted with these programs: pre denaturation 94°C for 2 minutes, denaturation 95°C for 36 seconds, annealing 53°C for 72 seconds, extension 72°C for 84 seconds, with PCR cycle is repeated for 35 times. When the post extension 72°C has been for 3 minutes, the temperature is decreased to 30°C. After that, 8 µl of each sample is taken from PCR result added by 2 µl loading dye in electrophoresis on 100 volts of 2% agarose gel for 30 minutes in TBE 1x buffer. A 100 bp marker (Thermo Scientific) is used as DNA ladder. The PCR result is stored on -20°C temperature until it is used for the next analysis.

DNA Isolation
DNA isolation uses Sambrook method which has been modified on fresh goat and pig skin samples which result in DNA isolate with good quality. The critical point of DNA isolation modification needs incubation time for ±18 -19 hours in 42°C temperature so it can digest the DNA perfectly and decrease the contaminant in resulted DNA. Result isolation was showed in figure 1.  shows that by using the Sambrook method which has been modified, there will be seen bright and clear DNA bands. This thing shows the DNA which has been successfully isolated perfectly, but there is also smear which marks DNA that has not been isolated perfectly. Smear appears due to the DNA purified phase which can result in the DNA that is free from dirt while the DNA cleaning phase with the addition of ethanol can precipitate the DNA because nucleate acid will precipitate and be difficult to be soluble into ethanol, while the dirt can be soluble into ethanol so the DNA quality result in the electrophoresis will show the existence of smear. The thickness of different band of every sample shows the difference of DNA concentration. Besides, it is influenced by the thickness and thinness of DNA bands formed which show the content or number of DNA that have same molecule weight on the same band position. Even though the sample is not isolated perfectly, through the existence of DNA from samples the isolation result of DNA mitochondria can be used as amplication template of cytochrome b at PCR process. This result is based on research of Ong et al. (2007) that bands with high intention while being in electrophoresis proves that DNA has been enough extracted and has good quality to be used of PCR product of mitochondria cyt b.

Amplification of Cytochrome BGen
The reduplication of DNA chains using Polymerase Chain Reaction (PCR) technique on DNA template is obtained from isolation result. The PCR program used has 35 cycles consisting of pre denaturation phase with 95°C temperature for 2 minutes, denaturation of 95°C temperature for 36 seconds, annealing of 53°C temperature for 1 minute and 13 seconds, extension of 72°C temperature for 1 minute and 24 seconds and post extension of 72°C temperature for 3 seconds. The PCR cycles are 35 times. This is done in order to obtain molecules of double DNA chains which are the result of polymeration in large numbers comparing to the total of DNA used. This shows that the sample are used in the next analysis step. According to Watson et al. (2009) DNA polymerase with primary has function to start the synthesis from single chain. The primary used completes the target DNA from 5' to 3'. The speed of arranging the DNA depends on the DNA molecule target which is wished.
Based on the optimization result of PCR towards the skin samples as seen in above figures, it is the electrophoresis result with 2% agarose. The template sample of DNA has been successfully amplificated by forming the DNA bands with fragment size of 359 bp. This fits the research of Erwanto et al (2011) which results in DNA fragment size of 359 bp on cyt b out of pork, and Jonasi (2006) also gets cyt b out of chicken with 359 bp fragment size. Under the smear, there is a very bright band (with high intention). The band which is in this part is the RNA contaminant. This is due to the addition of rnaAse during the DNA isolation time. The use of universal primary indeed strengthens the pig's DNA fragment. The detection result with PCR of universal primary shows the specification and sensitivity in detecting the pig skin as well as predicting the number of pig skins which have been mixed.

Restriction Fragment Length Polymorphism (RFLP) by Using Bam HI Enzyme and Bse DI Enzyme
Restriction Fragment Length Polymorphism (RFLP) is a technique used to identify the variety of genetics between species or interspecies. The use of restriction enzymes to identify the difference of genetics from various species can result in different fragments (Erwanto et al., 2011). The type of PCR-RFLP has a system to multiply a certain DNA fragment to figure out the difference of a fragmen restriction site on fragment among individuals in certain population or family (Grifiiths et al., 1998).  Figure 3 shows the RFLP result of goat skin and pig skin samples by using 37°C temperature and 4 hours of incubation time with 3% of agarose resulting in some fragment pieces in one line. There is one band on goat skin sample (line 1) with the length of fragment size is 359 bp. On the pig skin (line 2), there are two bands with the length of fragment is 244 and 115 bp and also for the combination of goat skin and pig skin (line 3, 4, 5, 6, and 7) there are length of fragments which are 359, 244, and 115 bp. However, the template of goat skin DNA on the combination is uncut. The cut part is only the DNA of pig skin.
This shows the success of pig skin identification. The success of RFLP is very influential for the DNA isolation result. The length of Bam HI enzyme piece is the same as the research Trijoko et al. (2012) Trijoko et al. (2012) and Murugaiah (2009) who use the identification of pork in every food processing which result in length of fragments of 228 and 131 bp on pork amplicon. This shows the success of pig skin identification. The good quality of DNA is very influential towards the success level of RFLP, the phase for reaching the good quality of DNA started from DNA isolation step.

CONCLUSION
The method of PCR-RFLP using the enzyme restriction of BamHI and restriction enzyme of BseDI on the result of mitochondria amplicon of cytochrome bgen can be used to identify the raw skin material of processing rambak. The use universal primary on cytochrome bgen results in the length of fragment size which is ± 359 bp. The DNA template of goat skin can be cut using BamHI and BseDI enzymes but the DNA template of pig skin cannot be cut.