TR201700177A2 - PRODUCTION METHOD OF TAQ DNA POLYMERASE ENZYME - Google Patents
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Abstract
Buluş, tıp, biyoloji, moleküler biyoloji, biyokimya, adli tıp, tarım, biyoteknoloji gibi alanlarda tanı ve bilimsel araştırma amacıyla yaygın olarak kullanılan Polimeraz Zincir Reaksiyonu (PZR) temel bileşeni olan Taq DNA polimeraz enziminin üretim yöntemi ile ilgilidir.The invention relates to a process for the production of Taq DNA polymerase, the main component of Polymerase Chain Reaction (PCR), which is widely used for diagnostic and scientific research in fields such as medicine, biology, molecular biology, biochemistry, forensics, agriculture, biotechnology.
Description
TEKNIK ALAN Bulus, tip, biyoloji, moleküler biyoloji, biyokimya, adli tip, tarim, biyoteknoloji gibi alanlarda tani ve bilimsel arastirma amaciyla yaygin olarak kullanilan Polimeraz Zincir Reaksiyonu (PZR) temel bileseni olan Taq DNA polimeraz enziminin üretim yöntemi ile ilgilidir. TECHNICAL FIELD Invention, type, biology, molecular biology, biochemistry, forensics, agriculture, biotechnology widely used for diagnosis and scientific research in fields such as Taq DNA polymerase, the basic component of Polymerase Chain Reaction (PCR) related to the production method of the enzyme.
TEKNIGIN BILINEN DURUMU Günümüzde, DNA teknolojileri dogal kaynaginda üretilmesi zor ve pahali olan birçok protein ve enzimin bakteri, maya ve memeli hücrelerinde daha kolay ve ucuz üretilmesine olanak vermektedir. Ticari degeri yüksek olan ve genis kullanim alanina sahip Taq DNA polimeraz enzimi de söz konusu enzimi kodlayan plazmitin Escherichia coli bakterisine aktarilmasi ile rekombinant olarak üretilmektedir. KNOWN STATE OF THE TECHNIQUE Today, DNA technologies are difficult and expensive to produce in natural source. in bacteria, yeast and mammalian cells It allows easy and inexpensive production. with high commercial value and Taq DNA polymerase enzyme, which has a wide usage area, is also in question. by transferring the plasmid encoding the enzyme to Escherichia coli bacteria. produced recombinantly.
Mevcut uygulamalarda, Taq DNA polimeraz enziminin üretim teknigi temel olarak hücre Iizatindaki proteinlerin çöktürmesi, diyaliz islemi ile tuzlarin ve küçük moleküllerin uzaklastirilmasi ve kromatografik yöntemler ile saflastirma islemlerini içermektedir. Taq DNA polimeraz enziminin ekstraksiyonunda öncelikle, amonyum sülfat, polietilenimin (PEI) ya da trikloroasetik asit (TCA) ile çöktürme islemi uygulanir. Ardindan da çökelen tuzlardan ve küçük moleküllerden kurtulmak için diyaliz islemine ihtiyaç duyulur. Fakat ortamda bulunabilecek çok düsük düzeylerde polietilenimin (PEl) kontaminasyonu ya da istenmeyen tuzlar (iyonlar) Taq DNA polimeraz enzimini inhibe etmektedir. In current applications, the production technique of Taq DNA polymerase enzyme is basic. Precipitation of proteins in the cell as a result of dialysis process of salts and removal of small molecules and purification by chromatographic methods contains the entries. Extraction of Taq DNA polymerase enzyme primarily, ammonium sulfate, polyethyleneimine (PEI) or trichloroacetic acid (TCA) with the precipitation process is applied. Then from the precipitated salts and small Dialysis is needed to get rid of molecules. But in the environment very low levels of polyethyleneimine (PEI) contamination that may be present or In addition, unwanted salts (ions) inhibit the Taq DNA polymerase enzyme.
Bu durum söz konusu üretim süreci için handikap olusturmaktadir. Bu islemleri takiben iyon degistirici kolon kromatografisi teknikleri ile saflastirma yapilmaktadir. Ayni sekilde kolondan toplanan fraksiyonlardaki enzimlerin yogunlastirilmasinda da çöktürme ve diyaliz islemleri uygulanmaktadir. This situation creates a handicap for the production process in question. This Purification by ion exchange column chromatography techniques following the procedures is being done. Likewise, the enzymes in the fractions collected from the column Precipitation and dialysis processes are also applied in the condensation process.
Bahsedilen bu islemler hem maliyeti arttirmakta hem de zaman kaybina neden olmaktadir. These processes both increase the cost and cause loss of time. causes.
Sonuç olarak Taq DNA polimeraz enziminin üretiminde gelistirmelere gidilmekte, bu nedenle yukaridaki deginilen dezavantajlari ortadan kaldiracak ve mevcut sistemlere çözüm getirecek yeni yapilanmalara ihtiyaç duyulmaktadir. As a result, improvements have been made in the production of Taq DNA polymerase enzyme. therefore, it will eliminate the disadvantages mentioned above. and there is a need for new structures that will bring solutions to existing systems. is heard.
BULUSUN AMAÇLARI Mevcut bulus, yukarida bahsedilen gereksinimleri karsilayan, tüm dezavantajlari ortadan kaldiran ve ilave bazi avantajlar getiren Taq DNA polimeraz enziminin üretim yöntemi ile ilgilidir. OBJECTS OF THE INVENTION The present invention has all the Taq DNA, which eliminates the disadvantages and brings some additional advantages It is related to the production method of the polymerase enzyme.
Bulusun ana amaci; ticari degeri yüksek olan Taq DNA polimeraz enziminin mevcut uygulamalara kiyasla çok daha düsük maliyetle elde edilmesini saglayan bir yöntem ortaya koymaktir. The main purpose of the invention; of the commercially valuable Taq DNA polymerase enzyme achieved at a much lower cost compared to existing practices. is to present a method that provides
Bulusun bir amaci; diyaliz gibi ekstra maliyet ve zaman kayiplarina sebep olan islem adimlarina gerek olmaksizin Taq DNA polimeraz enzimini üretmektir. Bu sayede maliyet ve zamandan tasarruf saglanmasi amaçlanmaktadir. An aim of the invention is; cause extra costs and time losses such as dialysis Taq DNA polymerase enzyme without the need for processing steps is to produce. In this way, cost and time savings are achieved. is intended.
Bulusun bir diger amaci; DNA sentez aktivitesini kaybetmeden Taq DNA polimeraz enzimini aktif formda elde etmektir. Diyaliz gerektirmeksizin ve kolon kromatografisi ile ileri düzeyde saflastirma yapmaksizin üretime imkân veren enzim üretim yöntemi ile söz konusu enzimin aktif formda eldesi mümkün olmaktadir. Another purpose of the invention; Taq DNA without losing DNA synthesis activity is to obtain the polymerase enzyme in active form. without the need for dialysis and Possibility of production without further purification by column chromatography Obtaining the enzyme in active form by the enzyme production method that gives is possible.
Bulusun bir baska amaci; kolondan yüksek oranda geri kazanim saglanan bir üretim yöntemi elde etmektir. Mevcut uygulamalarda kullanilan iyon degistirici kolon yerine bulusa konu yöntemde jel filtrasyon kolonu kullanilarak elde edilen enzimin % 85-90 oraninda geri kazanildigi saptanmis durumdadir. Another purpose of the invention is; a high recovery from the column production method. Ion exchanger used in current applications obtained by using a gel filtration column in the method of the invention instead of a column. It has been determined that 85-90% of the extracted enzyme is recovered.
Uygulamada PEI gibi iyonik özellikte bir madde kullanilmadigi için, kromatografi doldu maddesi sadece iyon degistirici dolgu maddeleri ile sinirli kalmamaktadir. Since no ionic substance such as PEI is used in the application, chromatography filler is limited to ion exchange fillers only does not exist.
Bulusun amaçlarini gerçeklestirmek üzere, tip, biyoloji, moleküler biyoloji, biyokimya, adli tip, tarim, biyoteknoloji gibi alanlarda kullanilmak üzere; Taq DNA polimeraz I geni tasiyan plazmitin Escherichia coli Bahsedilen plazmiti tasiyan bakteri kültürünün hazirlanmasi ve sonrasinda kültür ortamindaki bakterilerin çöktürülmesi, Çöktürülen hücrelerin Iizis tamponunda parçalanmasi ile Iizat hazirlanmasi ve Taq enziminin saflastirilmasi, temel islem adimlarini içeren Taq DNA polimeraz enzimi üretim yöntemi olup, özelligi; Bahsedilen lizat içerisinde hücre artiklarinin santrifüjleme ile uzaklastirilmasi ve enzim içeren sivi fazin (hücre Iizati) elde edilmesi, Bahsedilen hücre lizatina , -10 °C ile -30 °C sicaklik araliginda aseton eklenerek Taq DNA polmeraz enziminin çözünürlügünün ortadan kaldirilmasi, Çözünürlügünü kaybeden Taq DNA polmeraz enziminin santrifüjleme ile çöktürülmesi, Bahsedilen Taq DNA polmeraz enzimi çökeltisinin kurutulmasi, Bahsedilen Taq DNA polmeraz enzimi çökeltisinin çözündürülmesi, Çözünmemis halde kalan maddelerin santrifüj edilerek uzaklastirilmasi, Çözünür haldeki kismin saflastirma kolonundan geçirilmesi, Kolondan toplanan ham fraksiyonlarindaki Taq DNA polimeraz aktivitelerinin standart polimeraz zincir reaksiyonu ile saptanmasi, o Aktivite saptanan kolon fraksiyonlarina -10 °C ile -30 °C sicaklik araliginda aseton ekleyerek Taq DNA polmeraz enziminin çözünürlügünün ortadan kaldirilmasi, o Çözünürlügünü kaybeden Taq DNA polmeraz enziminin santrifüjleme ile çöktürülmesi, o Bahsedilen çökeltilerin kurutulmasi ve saklama tamponunda çözündürülmesi, o Hazirlanan enzimin -10 °C -20 °C'de saklanmasi islem adimlarini içermesidir. For the purposes of the invention, type, biology, molecular biology, to be used in fields such as biochemistry, forensic medicine, agriculture, biotechnology; Escherichia coli plasmid carrying the Taq DNA polymerase I gene Preparation of bacterial culture carrying the mentioned plasmid and precipitation of bacteria in the culture medium, Iizat by lysis of the precipitated cells in Iizis buffer preparation and purification of Taq enzyme, Taq DNA polymerase enzyme production, which includes the basic process steps method, its feature is; In said lysate, cell residues are removed by centrifugation. Removal of the enzyme containing liquid phase (cell Iizati) is obtained. to be made, Said cell lysata is stored at temperatures between -10 °C and -30 °C. The solubility of the Taq DNA polymerase enzyme was increased by adding acetone. elimination, Taq DNA polymerase enzyme, which lost its solubility precipitation by centrifugation, Drying of said Taq DNA polymerase enzyme precipitate, The aforementioned Taq DNA polymerase enzyme precipitate to dissolve, Centrifugation of the remaining undissolved substances removal, Passing the soluble fraction through the purification column, Taq DNA polymerase in crude fractions collected from the column by standard polymerase chain reaction. detection, o Temperatures between -10 °C and -30 °C for the column fractions with activity by adding acetone within the range of Taq DNA polymerase removing the resolution o The enzyme Taq DNA polymerase, which has lost its solubility, precipitation by centrifugation, o Drying of said precipitates and storage buffer to dissolve, o Storing the prepared enzyme at -10 °C -20 °C is required. contains the names.
Bulusun yapisal ve karakteristik özellikleri ve tüm avantajlari asagida verilen sekiller ve bu sekillere atiflar yapilmak suretiyle yazilan detayli açiklama sayesinde daha net olarak anlasilacaktir. Bu nedenle degerlendirmenin de bu sekiller ve detayli açiklama göz önüne alinarak yapilmasi gerekmektedir. The structural and characteristic features of the invention and all its advantages are given below. Figures and a detailed explanation with references to these figures will be understood more clearly. Therefore, this assessment Figures and detailed explanation should be taken into consideration.
BULUSUN ANLASILMASINA YARDIMCI OLACAK SEKILLER Mevcut bulusun yapilanmasi ve ek elemanlarla birlikte avantajlarinin en iyi sekilde anlasilabilmesi için asagida açiklamasi yapilan sekiller ile birlikte degerlendirilmesi gerekir. FIGURES TO HELP UNDERSTAND THE INVENTION The embodiment of the present invention and the best of its advantages with additional elements together with the figures explained below so that it can be understood must be evaluated.
Sekil-1; Bulusta bahsedilen yeni yöntemle elde edilen Taq DNA polimeraz enziminin aktivitesini (DNA sentez etme kapasitesini) göstermektedir. Figure 1; Taq DNA polymerase obtained by the new method mentioned in the invention It shows the activity (capacity to synthesize DNA) of the enzyme.
Sekil-2; Bulusta bahsedilen yeni yöntem kullanilarak jel filtrasyon kolon kromatografisi ile elde edilen Taq DNA polimeraz enziminin aktivitesini (DNA sentez etme kapasitesini) göstermektedir. Figure-2; Gel filtration column using the new method mentioned in the invention. The activity of the Taq DNA polymerase enzyme (DNA) obtained by chromatography its synthesis capacity).
BULUSUN DETAYLI AÇIKLANMASI Bu detayli açiklamada, bulus konusu Taq DNA polimeraz enziminin üretiminin tercih edilen bir metodu, sadece konunun daha iyi anlasilmasina yönelik olarak ve hiçbir sinirlayici etki olusturmayacak sekilde açiklanmaktadir. DETAILED DESCRIPTION OF THE INVENTION In this detailed description, the enzyme of the invention Taq DNA polymerase a preferred method of producing intended and without any restrictive effect is explained.
Bulusa konu olan yöntem, ticari degeri yüksek olan Taq DNA polimeraz enziminin düsük maliyetle eldesine imkan vermekte, zaman ve maliyet kayiplarini minimuma indirmektedir. Söz konusu yöntem, Taq DNA polimeraz enzimini kodlayan genin Escherichia coli bakterisine aktarilmasi, söz konusu Escherichia coli bakterilerinden istenilen ölçeklerde hazirlanan kültür ortaminin enzim izolasyonunda kullanilmasi islem adimlarini içermektedir. The method of the invention is the commercially valuable Taq DNA polymerase. It allows the enzyme to be obtained at low cost, time and cost minimizes its losses. The method in question is Taq DNA polymerase. transferring the gene encoding the enzyme to Escherichia coli bacteria, Culture prepared from Escherichia coli bacteria in desired scales. The use of the medium for enzyme isolation includes the process steps.
Söz konusu enzim izolasyonunda hücreler Iizis tamponuyla parçalanmakta ve mevcut uygulamada çesitli tuz ve kimyasallar kullanilarak Taq DNA polimeraz enzimi çöktürülmektedir. Bulusun ortaya koydugu yöntemin mevcut uygulamalardan en önemli farkli, parçalanmis hücresel artiklardan santrifüjleme ile arindirilan sivi fazdaki (Iizat) Taq DNA polimeraz enziminin lizat:aseton orani 12, 1:3 ya da 1:4 olacak sekilde soguk aseton katilarak çöktürme yapilmasidir. Bu asamada amonyum sülfat, TCA ya da PEI kullanilmadigi için enzim çökeltisi iyon ve PEI kontaminasyonu içermemektedir. Bu sayede diyaliz gerektirmeksizin, kolon kromatografisi ile ileri düzeyde saflastirma yapilmaksizin yüksek aktiflik derecesinde Taq DNA polimeraz enzimi elde edilmektedir. In this enzyme isolation, cells are lysed with Iizis buffer. and Taq DNA using various salts and chemicals in current practice polymerase enzyme is precipitated. The method of the invention is available The most important difference from applications is from fragmented cellular residues. Taq DNA polymerase enzyme in liquid phase (Iizat) purified by centrifugation lysate:acetone ratio 12, 1:3 or 1:4 by adding cold acetone precipitation is not done. At this stage, ammonium sulfate, TCA or PEI Enzyme precipitate ion and PEI contamination because it is not used does not include. In this way, without the need for dialysis, with column chromatography Taq DNA in high activity without further purification polymerase enzyme is obtained.
Bulusta bahsedilen yöntemde, saflastirma amacina uygun olarak iyon degistirici kolon dolgu maddelerine göre daha ucuz olan jel filtrasyon kolonu dolgu maddesi kullanilarak kromatografi uygulanmaktadir. Kolondan toplanan fraksiyonlarda yine amonyum sülfat, TCA ya da PEl yerine soguk aseton ile enzim çöktürülmekte, diyaliz yöntemi uygulanmadan aktif Taq enzimi elde edilmekte ve ham kolon fraksiyonlarindaki total enzim aktiviteleri ve bulusa konu yöntemde elde edilen fraksiyonlardan aseton ile çöktürme sonucu elde edilen enzim aktiviteleri karsilastirildiginda, ham fraksiyondaki enzimin % 85- 90 nispetinde geri kazanildigi saptanmaktadir. In the method mentioned in the invention, ion is used for purification purposes. Gel filtration column that is cheaper than exchanger column fillers Chromatography is applied using filler material. collected from the column with cold acetone instead of ammonium sulfate, TCA or PEI. enzyme is precipitated, active Taq enzyme is obtained without dialysis method. The total enzyme activities and the findings in the crude column fractions are analyzed. obtained as a result of precipitation with acetone from the fractions obtained in the subject method. When the enzyme activities obtained are compared, the enzyme in the crude fraction is 85%- It is determined that it is recovered at the rate of 90.
Bulus konusu olan Taq DNA polimeraz enziminin üretim yöntemi su adimlari içermektedir. Öncelikle, Taq DNA polimeraz l geni tasiyan plazmit vektör Eschechia coli hücrelerine transfer (transformasyon) edilir. Burada söz konusu plazmit vektör, genetik materyal olup, transfer edildigi bakteride (konak bakteri) Taq DNA polimeraz enzimini kodlayacak ana unsurdur. Eschechia coli (konak bakteri) ise enzimi üretecek olan organizmadir. Bahsedilen transfer isleminden sonra 25-5000 ml doygun bakteri kültürü hazirlanir ve bahsedilen kültür ortaminda, bakteriler oda sicakliginda 5 dakika 8000 rpm'de santrifüj ile çöktürülür. Olusan hücre çözeltisi, uygun hacimde tampon A (50 mM Tris, 50 mM Dekstroz, 1 mM EDTA, pH:8) içerisinde süspanse edilir. Söz konusu tampon A çözeltisi ile esit hacimde Iizis tamponu (10 mM Tris, 50 mM KCI, 1 mM EDTA, %O.5 Tween-20, 2 mM PMSF, pH:8) mevcut çözeltiye ilave edilir. Production method of Taq DNA polymerase enzyme, which is the subject of the invention, water steps contains. First, the plasmid vector Eschechia coli carrying the Taq DNA polymerase I gene transferred (transformation) into cells. Here the plasmid in question vector is the genetic material that is Taq in the bacterium (host bacterium) to which it is transferred. It is the main element that will encode the DNA polymerase enzyme. Eschechia coli (host bacteria) is the organism that will produce the enzyme. The said transfer After the process, 25-5000 ml of saturated bacterial culture is prepared and the mentioned In culture medium, bacteria are centrifuged at 8000 rpm for 5 minutes at room temperature. with precipitate. The resulting cell solution is diluted with an appropriate volume of buffer A (50 mM Tris, It is suspended in 50 mM Dextrose, 1 mM EDTA, pH:8). Aforementioned buffer A solution and an equal volume of Iizis buffer (10 mM Tris, 50 mM KCl, 1 mM EDTA, 0.5% Tween-20, 2 mM PMSF, pH:8) is added to the existing solution.
Ortam 70-75 °C'de 1 saat bekletilip hücrelerin parçalanmasi ve Taq enziminin serbest kalmasi saglandiktan sonra tekrar santrifüjden geçirilerek (15000 rpm'de oda sicakliginda 20 dakika) hücre artiklari çöktürülerek uzaklastirilir. Çökelti üzerinde kalan sivi 1:2, 1:3 ya da 1:4 oraninda (sivi:aseton) soguk aseton ile karistirilir ve -10°C ila -30°C sicaklik araliginda -30 dakika bekletilir. Aseton kullanilarak hücre Iizatinda bulunan Taq DNA polimeraz enziminin çözünürlügü degistirilerek ortamdan ayrismasi saglanir. The medium was incubated at 70-75 °C for 1 hour and the cells were lysed and Taq After the enzyme is released, it is centrifuged again. (20 minutes at room temperature at 15000 rpm) cell debris was precipitated. is removed. The liquid remaining on the precipitate is 1:2, 1:3 or 1:4. (liquid:acetone) mixed with cold acetone and used at a temperature range of -10°C to -30°C. -30 minutes to wait. Taq DNA found in cell strain using acetone By changing the solubility of the polymerase enzyme, it is separated from the environment.
Bekleme safhasindan sonra tekrar santrifüjleme (15000 rpm'de 4 °C'de 20 dakika) uygulanarak Taq DNA polimeraz enzimi çöktürülür. Taq enzimi böylece Iiziz tamponunda bulunan deterjan ve tuzlardan da arindirilmaktadir. Çökelti 2-5 dakikada oda sicakliginda kurutulur ve söz konusu çökelti uygun hacimde tampon A'da ya da saklama tamponunda (50 mM tris, 50 mM KCI, 1 mM EDTA, 1 mM DTT, 2 mM % 25 gliserol, pH:8) çözdürülür. Kurutulup tamponla çözdürülerek hazirlanan örnekler 70-75°C'de 2-5 dakika bekletilir. Çözünmemis halde kalan maddeler santrifüj (15000 rpm, 4 °C'da 10 dakika) edilerek uzaklastirilir. Hazirlanan örneklerde Taq enzimi aktivitesi standart PZR (Polimeraz Zincir Reaksiyonu) ile belirlenir. Tampon A içerisinde çözündürülen örnekler ise hareketli faz olarak tampon A kullanilarak jel filtrasyon kolonundan geçirilir ve elde edilen ham kolon fraksiyonlarindaki Taq DNA polimeraz aktiviteleri PZR (Polimeraz Zincir Reaksiyonu) ile saptanir. Aktivite saptanan kolon fraksiyonlarinda Taq polimeraz enziminin soguk aseton (-10 °C ile -30 °C sicaklik araliginda aseton) ile çöktürülür ve söz konusu çökeltiler oda sicakliginda 2-5 dakika kurumaya birakilir. Bu sayede kolon kromatografi sirasinda seyreltik hale gelen Taq enzimi çöktürülerek yeniden yogunlastirilmasi ve elüsyon tamponda bulunan tuzlarin uzaklastirilmasi mümkün olmaktadir. Kuruyan çökelti uygun hacimde saklama tamponunda çözdürülür ve PZR deneylerinde kullanilmak üzere -20 °C'de saklanir. Re-centrifugation after the holding phase (15000 rpm at 4 °C 20 minutes) to precipitate Taq DNA polymerase enzyme. Taq enzyme thus, it is purified from the detergents and salts in the Iiziz buffer. The precipitate is dried at room temperature in 2-5 minutes and the precipitate in question is suitable volume in buffer A or storage buffer (50 mM tris, 50 mM KCl, 1 Dissolve mM EDTA, 1 mM DTT, 2 mM 25% glycerol, pH:8). dried up Samples prepared by thawing with buffer are kept at 70-75°C for 2-5 minutes. Undissolved substances are centrifuged (10 minutes at 15000 rpm, 4 °C) is removed by. The Taq enzyme activity in the prepared samples is standard. Determined by PCR (Polymerase Chain Reaction). In buffer A The dissolved samples are gelled using buffer A as the mobile phase. is passed through the filtration column and the resulting crude column fractions Taq DNA polymerase activities by PCR (Polymerase Chain Reaction) detected. Taq polymerase enzyme activity was detected in the colon fractions. precipitated with cold acetone (temperature range of -10 °C to -30 °C) and the said precipitates are left to dry for 2-5 minutes at room temperature. This Thus, the Taq enzyme, which becomes diluted during column chromatography, reconcentration by precipitation and the salts in the elution buffer possible to be removed. Dried precipitate in suitable volume It is dissolved in storage buffer and -20 to be used in PCR experiments. It is stored at °C.
Sekil 1'de bulus ile ortaya koyulan yeni yöntemle elde edilen Taq DNA polimeraz enziminin aktivitesini (DNA sentez etme kapasitesini) gösterilmektedir. Söz konusu Sekil 1 üzerinde yer alan 1 numara ile hazirlanan kolon kromatografisi ile saflastirilmamis Taq DNA polimeraz enziminin sentez ettigi DNA bandi gösterilmektedir. 2 ila 5 numara arasinda hazirlanan, kolon kromatografisi ile saflastirilmamis Taq DNA polimeraz enziminin aktivitesi verilmektedir. 6 numara ticari olarak satilan bir Taq DNA polimeraz enzimi ile sentez edilen DNA bandini göstermektedir. M ise molekül agirligi belli olan bir standart DNA bantlarini (k-EcoRl+Hindlll) göstermektedir. Taq DNA obtained by the new method revealed by the invention in Figure 1 activity of polymerase enzyme (capacity to synthesize DNA) is shown. with the number 1 on the Figure 1 in question. non-purified Taq DNA polymerase by prepared column chromatography The DNA band synthesized by the enzyme is shown. between 2 and 5 numbers prepared, not purified by column chromatography, Taq DNA polymerase activity of the enzyme is given. #6 is a commercially available Taq DNA shows the DNA band synthesized by the polymerase enzyme. if M a standard DNA band with a certain molecular weight (k-EcoRl+Hindlll) shows.
Sekil 2'de bulusta bahsedilen yöntem kullanilarak jel filtrasyon kolon kromatografisi ile elde edilen Taq DNA polimeraz enziminin aktivitesini (DNA sentez etme kapasitesini) gösterilmektedir. A, Ham enzim ekstresinin jel filtrasyon kolonundan geçirilmesi ile elde edilen fraksiyonlarin absorbans profilini gösterirken, B kolondan toplanan fraksiyonlardaki Taq DNA polimeraz enzimi ile sentez edilen DNA bantlarini göstermektedir. C harfi ile kolon fraksiyonlarindan aseton ile çöktürülerek elde edilen Taq DNA polimeraz enzimi ile sentez edilen DNA bantlarinin görünümü verilmektedir. In Figure 2, gel filtration column using the method mentioned in the invention The activity of the Taq DNA polymerase enzyme (DNA) obtained by chromatography capacity to synthesize). A, Gel of crude enzyme extract absorbance of the fractions obtained by passing through the filtration column. Taq DNA in fractions collected from column B. shows the DNA bands synthesized by the polymerase enzyme. with the letter C Taq DNA obtained from column fractions by precipitation with acetone The appearance of the DNA bands synthesized by the polymerase enzyme is given.
D ise mevcut yöntemler ile elde edilen ticari bir Taq DNA polimeraz enziminin DNA sentez kapasitesini göstermektedir. D is a commercial Taq DNA polymerase enzyme obtained by existing methods. It shows the DNA synthesis capacity.
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