PracticeReport Friday, 29 May 2009
M.K.Dasar-dasar Genetika Ikan
Rani Rehulina Tarigan, , group 4, sift 2.
To know the characteristic of the nature of genetic inmolekuler level which directly show the nature of genotipe owned by the certainorganism can do by DNA analising. Three steps to do it namely: DNA extraction,PCR, and electroforesis. Extraction of DNA is the firs step to do analysis DNA. Next, Polymerase Chain Reaction (PCR)is a method of in vitro which multiply the certain sekuens nukleutida through anetwork incubation at different temperature. The last, electroforesis will showus the visualization of DNA by seeing the DNA ribbon. The results of analysingDNA is to find the DNA ribbon to guess what meat that each group has. Gouramy (Osphronemousgouramy) has DNA ribbon on 0,5 kbp, Tilapia (Oreochromis niloticus)has DNA ribbon on 0,3 and 0,4 kbp, Barbir has DNA ribbon on 0,3 and 0.6 kbp,and Zebra fish has DNA ribbon on 0.3 and 0.6 kbp. The purpose of thisexperiment is to know the method used in analyzing DNA.
Keyword :DNA analyzing, PCR, Extraction, Elecroforesis, results.
Fenotip nature of an individual can be seen through analysisof DNA. To commonly used identifygenetic variations and population distribution get from analisis DNA. The DNAis then sepparated from contaminants such as RNA and proteins (Stine, 1973 inHilwa, 2004). According to Muladno (2002) in Hilwa (2004), to double theammount of DNA molecules on a certain target by sinthesysing new DNA moleculescomplemented with the DNA molecules on that target, an in vitro reaction calledPCR (Polymerase Chain Reaction) is used.
To separate, identify and purify the DNA fragments,the electrophoresis process is used. The purpose of this experiment is to knowthe method used in analyzing DNA. DNA analysis used in the search for a specieskinship relations, Taksonomi of an individual, diagnosis of disease (especiallydisease derivative), and others. DNA analysis is done with some of the DNA thatis ektraksi body from the tissue , the use of PCR on DNA extracts, cuttingretriksi or DNA, and elektroforesis. From this practice we able to conduct DNAanalysis correctly, knowing the benefits and impact, and capable toapplications.
The practice of analysis DNA do at Friday 8 May 07:30– 10:30a.m, Friday 15 May at 07:30 – 10:30 a.m, and Friday 22 May 2009 at 07:30 – 10:30 inlaboratorium of genetic, Department of Aquaculture, fissheris and marine sainsfaculty, Bogor University ofagriculture.
Micropipet, eppendorf tube, microchip, sentrifuge, PCR, and electrophorsismachine are the aquitment thatused. While substance weared is DNAsample, Cell Lysis Solution, Porteinase K, RNAse, Protein of PrecipitationSolution, etanol 70%, DNA rehydration solution, SDW, isopropanol, extaq polymerase,gel agarosa, bromophenol blue, meat of gouramy, tilapia, barber, and zebrafish.
Extraction DNA is the firs atep to do analisis DNA. Inextraction of DNA and than gived cell lysis solution 200 µL+1,5 proteinase K,incubation during overnight (55˚C). Than, RNAse 1,5 ml and incubation again (1hour, 37˚C). For getting sediment, used protein precipitation sollution 100µLand sentrifuse 12.000 rpm (15’) and for getting DNA sediment so needisopropanol 300 µL, sentrifuse 12.000 rpm in 10’. The next is gived cold etanol70% 300 µL, sentrifuse again in 12.000 rpm 10’. After wet, mixed SDW (SterylDestilated Water) 20 µL. The extraction of DNA consist of PCR, denaturation, annealing, and extension.
The second steps ai PCR, after DNA extraction is PCR(Polymerase Chain Reaction).In this step we have to mix SDW, primer F actine,primer ß actine, dNTPs, Buffer Ex Taq, and Ex Taq. Bactery enzyme which usedfor Ex Taq is taken from Thermosaquaticus. The condensation premix have to be made first. As mush as8 µl FBPA primer added with 8 µl RBP primer, 8 µl DNTP, 8 µl buffer ex taq, 8µl ex taq, 8 µl SDW. The premix shared into 8 tube each as mush 9 µl.Hereinafter as much 1 µl DNA sample added on the microtube. Each microtubeputted into the PCR machine which programme for predenaturation at 94 ºC for 3minutes, denaturation at 94 ºC for 30 seconds and moved to annealing process at59 ºC for 30 seconds then to extension process at 72 ºC for 30 seconds andfinal extension at 72 ºC for 3 minutes. After the PCR process finished, PCRmachine was turned off and the result of PCR putted on freezer with temperature-20 ºC.
The last in practice analisis DNA. Is electrophoresis.It is started made gel agarosa consisted of the result of 0,8-1%DNA extraction,result PCR 1%, and result of restriction 1,5-2%. Way of making of gel agarosathat is agarosa powder dissolved in condensation of pregnant Tris Borate EDTA (TBE)of Ethidium Bromide. Condensation heated in microwave of during 1.5 minute oruntil condensation become the transparent. Condensation let by a warmness islater and then infused by a printing;mould which have been equiped by comb aswell elektroforesis. Gel was let till freezing then putted inti a basinelectroforesis which consisted electroforesis buffer condensation. Usage Elektroforesis of about 3 µl of sample volume to be checkedto be mixed with 0,5 µl pregnant loading buffer of substance of DNA selfincriminating and colourant. Mixture entered into wells elektroforesis. Basinclosed and electrics conducted with the tension 250 volt and current strength50-80mA. So, DNA have migration fromnegative pole to positive pole reach ¾ part of gel length, hence process theelektroforesis can be discontinued. Gel lifted from basin and discharged fromprinting;mould is henceforth perceived by using ultraviolet transluminator withthe short wavelength 280nm.
DNA is a polymer chain composed of monomer units thatis nukleotida. Nukleotida consists of purin or pirimidin basa, deoksiribosasugar, and phosphate group. Individual basa associated with sugar deoksiribosathrough kovalen bond between the carbon number of basa pirimidin carbon or nineof basa purin with carbon number one of deoksiribosa sugar, phosphate group,while the number five carbon sugar deoksiribosa's the one with the carbonnumber of three sugar deoksiribosa the other. Basa-basa purin namely adenin andguanin, while basa pirimidin is citocin and timin. Basa basa adenin berikatanwith timin, and basa basa guanin berikatan with citocin. Ties that connect thehydrogen bond. This ties the two chains form the structure of the DNA doubleHelix (ganda twisting) (Flanders, 1981 in Yonatan, 1998).
From the pictureabove, we see that almost all of sample shown the DNA ribbon. This piture asresult of PCR and electrophoresis inpractice analysis DNA.
Picture 1. Result of Polymerase Chain Reaction (PCR)
Picture 2.Result of Electroforesis
The first ribbon is showing a gouramy meat whichribbon stay in 0,5 kbp. The second is a tilapia appearance of ribbon DNA in 0.3and 0.4 kbp. The third sample is barbir in 0.3 and 0.6 kbp. The fourth is zebra in 0.3 and 0.6kbp. The fifth sample is a mix meat between gouramy and tilapia, sample numbersix is a mix meat between tilapia and Barbir fish. The last sample is a mixmeat between barbir and zebra fish.
Cell destroyingstage and nucleic acid isolation stage are two of stages in DNA extraction . Thedestruction of the cell nucleus loosens the DNA so that the viscosity of thesubstance increases and the DNA molecule looks more real. The DNA extractionprocess in this experiment starts by isolating the DNA genome from the mothercell. Lewin (1994) states that the forst step in DNA analysis is by breakingthe DNA genome to specific smaller fragments.
Polymerase ChainReaction (PCR) use to making a DNA copy. PCR allows a number of small DNAsequences to be copied (millions of times) to be multiplied (so it can beanalyzed) or modified in a certain way. PCR can also be used to detect theposition of certain DNA sequences in the sample. (Anonimous, 2009). Generally,there are three major stages of denaturation: to separate DNA and form a singlestrand, annealing (the process of primary binding), and the extension of thenew DNA (Erlich, 1989 in Hilwa, 2004).
If in PCR ribbonDNA can not seen. It can be caused by many factor as long as we practice andthe main reason is DNA ribbon is not taken when extraction DNA conducted. Italso can be happened because we have lost the time which is needed toelektroforesis too old so that DNA have passed. Migration velocity of DNAdetermined by some factor, one of them is size measure its molecule. Biggerfairish molecule DNA migration slower than the smaller fairish migration. (Hsu, 1996). Another factor which make the ribbon DNA not seen is because theprimer DNA for barbir fish is not specific. According to Sambrook ( 1989 ) PCRwill very inefficient when primary concentration limited. It makes the resultsof PCR is not seen.
According toAusubel (1990) in Hilwa (2004), every process in the PCR cycle needs acertain period of time to work effectively. The denaturation stage needs astandard time of 90 seconds on a temperature of 94oC. the annealingstage, which is the complementary primary stage with one single strand, thetemperature and time used depends on the GC composition. It’s best to increasethe temperature if there is an increase in the primary GC composition. In theextension stage, the time needed depends on the sequence length which will be amplified.The temperature used is 72oC, which is the optimum temperature forthe Tag Polymerase enzyme (Gelfand. 1989 inHilwa, 2004). Lisdiyati (1997) in Arifin(2005) states that to prevent a low end result, usually 25-40 cycles are done.
The end stage isthe electrophoresis. Electrophoresis gel is one major technique in themolecular biology. The basic principle of this principle is that the DNA, RNAor proteins can be separated by an electric field. The displacement velocitydepends on the size of the molecules related. Electrophoresis gel is usuallyused to analytical purposes. To separate proteins or small nucleic acids (DNA,RNA, or oligonucleotyde), the gel used is purified agarose (from seaweedextract). The molecule sample is placed in the well of the gel placed in thebuffer, and electricity is transferred. The DNA can migrate from the negativepole to the positive pole in the electrophoresis because the agarose gelcontains buffers and the tub is flow by electricity. Etidium bromide, silver, orCoomassie blue can be used for this. The bands on different lanes of the gelwill be visible after the staining process. One lane is the moving directin ofthe well. The bands which have the same distance from the gel well on the endof electrophoresis contains molecules that move in the gel with the same velocityduring electrophoresis. This is an indicator that the molecules have the samesize. The marker used to determine the size of molecules in the band samples byelectrophoresis the marker on the lane in the gel parallel to the sample. Thebands on the marker lane can be compared with the sample bands to determine thesize. The distance of the band from the gel well proportionate inverted to thelogarithm of the molecule size (Anonimous, 2009).
Each spescies of fish have different DNA and thispraktice is done to know the characteristics of the genetic characters on amolecular level which directly reflects the ghenotype character of an organism.Gouramy meat appearance ribbon DNA in 0,5 kbp, tilapia 0.3 and 0.4 kbp, Barbir0.3 and 0.6 kbp, zebra 0.3 and 0.6 kbp, mixing between gouramy and tilapia 0.3,0.4, and 0.5 kbp, mixing meat between tilapia and barbir 0.3, 0.4, 0.6 kbp, andmixing meat between barber and zebra 0,3 and 0.6 kbp.The electrophoresisprocess in this experiment has a good enough result with clear bands.
Anonimous. 2009. Polymerasechain reaction DNA Extraction www.wikipwedia.org [May, 25th 2009].
Hilwa, Z. 2004.Karakterisasi Genotipe Ikan Lele Sangkuriang dengan Metode PCR-RFLP ADNMitokondria. Skripsi. Departemen Budidaya Perairan, Fakultas Perikanandan Ilmu Kelautan, Institut Pertanian Bogor.
Yonatan, HH. 1998.Pemanfaatan Teknik RAPD untuk Analisi DNA Pisang Nangka dan Barangan dalamKultur In Vitro. Skripsi. Departemen Kimia, Fakultas Matematikadan Ilmu Pengetahuan Alam, Institut Pertanian Bogor.
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