KR20220158946A - Method for removing DNA contamination in DNA polymerase using nylon membrane - Google Patents
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- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
Abstract
Description
본 발명은 나일론 멤브레인을 이용한 DNA 중합효소의 오염 DNA 제거방법에 관한 것이다.The present invention relates to a method for removing contaminated DNA from a DNA polymerase using a nylon membrane.
중합효소연쇄반응(polymerase chain reaction, PCR)은 원하는 유전자의 일부를 복제 및 증폭시키는 분자생물학적인 기술로, 아주 적은 양의 DNA만으로도 단시간에 특정 부위의 유전자를 기하급수적으로 증폭할 수 있다. 또한, 실험 과정이 단순하고, 전자동 기계를 이용하여 쉽고 빠르게 결과를 얻을 수 있기 때문에 생명공학 분야 연구, 질병 진단, 범죄 수사, 생물의 분류 등에 매우 유용하게 사용되고 있다. 이러한 중합효소연쇄반응을 수행하기 위해서는 주형 DNA(template), 한 쌍의 프라이머(primer), DNA 중합효소(polymerase), 구아닌(G), 아데닌(A), 티민(T), 시토신(C) 네 가지 디옥시뉴클레오티드(dNTPs), 완충액 등이 필요하다. Polymerase chain reaction (PCR) is a molecular biological technique that copies and amplifies a part of a desired gene, and can exponentially amplify a gene at a specific site in a short time using only a very small amount of DNA. In addition, since the experimental process is simple and the results can be obtained easily and quickly using a fully automatic machine, it is very useful for research in the field of biotechnology, disease diagnosis, criminal investigation, and classification of organisms. In order to carry out this polymerase chain reaction, template DNA (template), a pair of primers, DNA polymerase, guanine (G), adenine (A), thymine (T), cytosine (C) Branched deoxynucleotides (dNTPs), buffers, etc. are required.
그 중, DNA 중합효소는 DNA 사슬에 뉴클레오티드를 연결하면서 새로운 DNA를 합성하는 효소로, DNA 복제(DNA replication) 과정에 필수적이다. 새로운 DNA를 합성할 때는 반드시 이미 존재하는 DNA 끝의 하이드록시기(-OH)가 필요하며, 여기에 포스포디에스터 결합(phosphodiester bond)을 통해 상보적인 뉴클레오티드를 하나씩 첨가하는 형태로 합성이 이루어진다. Among them, DNA polymerase is an enzyme that synthesizes new DNA while linking nucleotides to a DNA chain, and is essential for DNA replication. When synthesizing new DNA, a hydroxyl group (-OH) at the end of the already existing DNA is required, and synthesis is performed in the form of adding complementary nucleotides one by one through a phosphodiester bond.
한편, 현재 사용되고 있는 대부분의 DNA 중합효소는 재조합 대장균의 발현 시스템을 이용하여 발현 및 정제 과정을 거쳐 생산되고 있는데, DNA 중합효소는 DNA와 결합하는 단백질이기 때문에 정제하는 과정 중에 대장균의 게놈 DNA(genomic DNA) 및 도입된 재조합 DNA와 결합할 가능성이 아주 높다. 이렇게 내부 및 외부로부터 유래된 DNA가 완전히 제거되지 않는 DNA 중합효소를 사용하여 중합효소연쇄반응을 실시할 경우, 해당 DNA가 증폭되어 위양성(false positive) 결과가 도출될 수 있고, 시퀀싱 결과 오류 등의 문제가 발생할 수 있다. 이에, DNA 중합효소 내 오염 DNA를 제거하기 위해서, 효소(DNase, 제한효소 등) 처리법이나 화학물질(polyethyleneimine 등) 처리법을 이용해왔다. 그러나, 효소 처리법은 시료 내의 환경에 따라 효소의 활성이 달라지고, 살아있는 세포의 생존력에 영향을 주며, 반응 후 효소의 제거가 미흡하면 프라이머가 분해되는 단점이 있다. 또한, 화학물질 처리법은 시료 내 DNA 잔류량에 따라 일정 농도를 맞추어 화학물질을 매번 직접 첨가해야 하는 불편함이 있으며, 반응 후 화학물질을 제거하기 어렵다는 단점이 있다. 따라서, DNA 중합효소의 활성에는 영향을 주지 않으면서 효소 내 포함된 오염 DNA를 제거하는 효율적인 방법에 대한 연구가 필요하다. On the other hand, most DNA polymerases currently used are produced through expression and purification processes using recombinant E. coli expression systems. Since DNA polymerases are proteins that bind to DNA, during the purification process, genomic DNA DNA) and introduced recombinant DNA. When a polymerase chain reaction is performed using a DNA polymerase that does not completely remove internally and externally derived DNA, the corresponding DNA is amplified, resulting in false positive results, and sequencing errors, etc. Problems can arise. Accordingly, in order to remove contaminating DNA in DNA polymerase, enzyme (DNase, restriction enzyme, etc.) treatment method or chemical substance (polyethyleneimine, etc.) treatment method has been used. However, the enzyme treatment method has disadvantages in that the activity of the enzyme varies depending on the environment in the sample, affects the viability of living cells, and the primer is degraded if the removal of the enzyme is insufficient after the reaction. In addition, the chemical treatment method has the disadvantage of having to directly add chemicals each time to match a certain concentration according to the residual amount of DNA in the sample, and it is difficult to remove the chemicals after the reaction. Therefore, there is a need for research on an efficient method for removing contaminating DNA contained in the enzyme without affecting the activity of the DNA polymerase.
한편, 한국등록특허 제1965409호에는 '중합효소연쇄반응을 이용한 분자진단검사용 중합효소 및 보조효소의 DNA 오염 제거를 위한 정제 방법'이 개시되어 있고, 한국등록특허 제1546358호에는 'Taq DNA 중합효소의 신규한 제조방법'이 개시되어 있으나, 본 발명의 '나일론 멤브레인을 이용한 DNA 중합효소의 오염 DNA 제거방법'에 대해서는 기재된 바가 없다.On the other hand, Korean Patent No. 1965409 discloses 'a purification method for removing DNA contamination of polymerase and coenzyme for molecular diagnostic tests using polymerase chain reaction', and Korean Patent No. 1546358 discloses 'Taq DNA polymerization A 'new method for preparing an enzyme' is disclosed, but the 'method for removing contaminating DNA of a DNA polymerase using a nylon membrane' of the present invention is not described.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 주형 DNA를 첨가하지 않은 조건에서, 나일론 멤브레인과 반응시키지 않은 Taq DNA 중합효소를 이용하여 PCR을 수행한 결과, 비특이적 산물이 증폭되는 것을 확인한 반면, 나일론 멤브레인과 반응시킨 Taq DNA 중합효소를 이용하여 PCR을 수행한 결과, 비특이적 산물이 증폭되지 않는 것을 확인하였으며, 상기 나일론 멤브레인과 반응시킨 Taq DNA 중합효소의 활성에는 변화가 없는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present inventors performed PCR using Taq DNA polymerase that did not react with a nylon membrane under the condition that template DNA was not added, and as a result, non-specific products were amplified. On the other hand, as a result of PCR using Taq DNA polymerase reacted with the nylon membrane, it was confirmed that non-specific products were not amplified, and there was no change in the activity of Taq DNA polymerase reacted with the nylon membrane. , completed the present invention.
상기 과제를 해결하기 위해, 본 발명은 DNA 중합효소를 나일론 멤브레인과 반응시키는 단계를 포함하는 DNA 중합효소 내 오염 DNA를 제거하는 방법을 제공한다.In order to solve the above problems, the present invention provides a method for removing contaminating DNA in DNA polymerase comprising the step of reacting DNA polymerase with a nylon membrane.
또한, 본 발명은 상기 방법에 의해 오염 DNA가 제거된 DNA 중합효소를 포함하는 중합효소 연쇄반응용 반응액을 제공한다.In addition, the present invention provides a reaction solution for polymerase chain reaction containing a DNA polymerase from which contaminating DNA has been removed by the above method.
또한, 본 발명은 상기 방법에 의해 오염 DNA가 제거된 DNA 중합효소를 포함하는 중합효소 연쇄반응용 키트를 제공한다.In addition, the present invention provides a kit for polymerase chain reaction comprising a DNA polymerase from which contaminating DNA has been removed by the above method.
또한, 본 발명은 나일론 멤브레인을 DNA 중합효소에 첨가하고 반응시키는 단계를 포함하는 오염 DNA가 제거된 DNA 중합효소의 제조방법을 제공한다. In addition, the present invention provides a method for preparing a DNA polymerase from which contaminant DNA is removed, comprising the step of adding a nylon membrane to the DNA polymerase and reacting.
본 발명에 따른 방법을 이용하면 DNA 중합효소의 활성에는 영향을 주지 않으면서, DNA 중합효소 내 오염 DNA를 효과적으로 제거할 수 있으므로, 중합효소연쇄반응의 효율성, 정확도 및 신뢰도를 향상시킬 수 있어 중합효소연쇄반응을 이용하는 관련 산업 분야에 매우 유용하게 사용할 수 있을 것이다.Since the method according to the present invention can effectively remove contaminating DNA in DNA polymerase without affecting the activity of DNA polymerase, it is possible to improve the efficiency, accuracy and reliability of polymerase chain reaction. It will be very useful in related industries that use chain reactions.
도 1은 나일론 멤브레인 디스크의 처리 개수(0, 1, 2, 3, 4 또는 5개)에 따른 Taq DNA 중합효소 내 오염 DNA 제거 효과를 분석한 결과로, A는 주형 DNA를 첨가하지 않은 조건에서 PCR을 수행한 결과이고, B는 주형 DNA를 첨가한 조건에서 PCR을 수행한 결과이다.
도 2는 나일론 멤브레인 디스크의 처리 시간(0, 3, 6, 12, 18, 24 또는 48시간)에 따른 Taq DNA 중합효소 내 오염 DNA 제거 효과를 분석한 결과로, A는 주형 DNA를 첨가하지 않은 조건에서 PCR을 수행한 결과이고, B는 주형 DNA를 첨가한 조건에서 PCR을 수행한 결과이다.
도 3은 FTA(Flinders Technology Associates) 카드의 처리 개수(0, 1 또는 2개)에 따른 Taq DNA 중합효소 내 오염 DNA 제거 효과를 분석한 결과이다. Figure 1 is the result of analyzing the effect of removing contaminating DNA in Taq DNA polymerase according to the number of nylon membrane disks (0, 1, 2, 3, 4 or 5). It is the result of performing PCR, and B is the result of performing PCR under the condition of adding template DNA.
Figure 2 is a result of analyzing the effect of removing contaminating DNA in Taq DNA polymerase according to the treatment time (0, 3, 6, 12, 18, 24 or 48 hours) of the nylon membrane disk, A is the template DNA is not added This is the result of performing PCR under the conditions, and B is the result of performing PCR under the condition of adding template DNA.
3 is a result of analyzing the effect of removing contaminating DNA in Taq DNA polymerase according to the number (0, 1 or 2) of FTA (Flinders Technology Associates) cards processed.
본 발명의 목적을 달성하기 위하여, 본 발명은 DNA 중합효소를 나일론 멤브레인과 반응시키는 단계를 포함하는 DNA 중합효소 내 오염 DNA를 제거하는 방법을 제공한다. In order to achieve the object of the present invention, the present invention provides a method for removing contaminating DNA in a DNA polymerase comprising the step of reacting the DNA polymerase with a nylon membrane.
본 명세서에서 용어 "오염 DNA"는 DNA 중합효소 내 포함된 DNA를 의미하는 것으로, 오염 DNA가 존재하는 DNA 중합효소를 이용하여 중합효소연쇄반응을 수행하면 위양성(false positive) 결과 및 DNA 중합효소의 활성 감소 등의 문제를 야기할 수 있다. As used herein, the term "contaminating DNA" refers to DNA included in DNA polymerase, and when a polymerase chain reaction is performed using a DNA polymerase containing contaminating DNA, a false positive result and a loss of DNA polymerase It can cause problems such as reduced activity.
본 발명에 따른 방법에 있어서, 상기 DNA 중합효소는 숙주세포에서 발현시킨 후, 정제된 재조합 DNA 중합효소일 수 있으나, 이에 제한되지 않는다. In the method according to the present invention, the DNA polymerase may be a purified recombinant DNA polymerase after expression in a host cell, but is not limited thereto.
또한, 상기 DNA 중합효소는 바람직하게는 Taq(Thermus aquaticus) DNA 중합효소, 원핵세포 DNA 중합효소 I, 원핵세포 DNA 중합효소 Ⅱ, 원핵세포 DNA 중합효소 Ⅲ, T4 DNA 중합효소, T7 DNA 중합효소, 클레나우 단편(Klenow fragment), Vent DNA 중합효소, Tth(Thermus thermophilus) DNA 중합효소, KOD(Thermococcus kodakaraenis) DNA 중합효소 또는 Pfu(Pyrococcus furiosus) DNA 중합효소일 수 있으나, 이에 제한되지 않는다. In addition, the DNA polymerase is preferably Taq ( Thermus aquaticus ) DNA polymerase, prokaryotic DNA polymerase I, prokaryotic DNA polymerase Ⅱ, prokaryotic DNA polymerase Ⅲ, T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, Vent DNA polymerase, Tth ( Thermus thermophilus ) DNA polymerase, KOD ( Thermococcus kodakaraenis ) DNA polymerase, or Pfu ( Pyrococcus furiosus ) DNA polymerase, but is not limited thereto.
본 발명의 일 구현 예에 따른 방법에 있어서, 상기 반응은 바람직하게는 DNA 중합효소에 나일론 멤브레인 2~5개를 첨가하고 20~50시간 동안 -22~-18℃에서 반응시키는 것일 수 있으며, 더욱 바람직하게는 DNA 중합효소에 나일론 멤브레인 2~3개를 첨가하고 24~48시간 동안 -20℃에서 반응시키는 것일 수 있으나, 이에 제한되지 않는다. In the method according to one embodiment of the present invention, the reaction may preferably be adding 2 to 5 nylon membranes to a DNA polymerase and reacting at -22 to -18 ° C for 20 to 50 hours. Preferably, 2 to 3 nylon membranes may be added to DNA polymerase and reacted at -20 ° C for 24 to 48 hours, but is not limited thereto.
본 발명은 또한, 상기 방법에 의해 오염 DNA가 제거된 DNA 중합효소를 포함하는 중합효소 연쇄반응용 반응액 및 키트를 제공한다.The present invention also provides a reaction solution and kit for polymerase chain reaction containing a DNA polymerase from which contaminating DNA has been removed by the above method.
본 발명에 따른 반응액 및 키트는 dNTPs, 버퍼 등을 포함할 수 있으며, 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다. The reaction solution and kit according to the present invention may include dNTPs, buffers, and the like, and may further include a user guide describing optimal reaction conditions. The guide is a printout explaining how to use the kit, eg, how to prepare the PCR buffer, and the reaction conditions to be presented. The guide includes a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.
본 발명은 또한, 나일론 멤브레인을 DNA 중합효소에 첨가하고 반응시키는 단계를 포함하는 오염 DNA가 제거된 DNA 중합효소의 제조방법을 제공한다.The present invention also provides a method for preparing a DNA polymerase from which contaminating DNA is removed, comprising adding a nylon membrane to the DNA polymerase and reacting the same.
본 발명의 오염 DNA가 제거된 DNA 중합효소의 제조방법에 있어서, 상기 DNA 중합효소 및 반응은 전술한 바와 같다.In the method for preparing a DNA polymerase from which contaminating DNA is removed according to the present invention, the DNA polymerase and reaction are the same as described above.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 이에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.
실시예 1. 나일론 멤브레인 디스크 처리 개수에 따른 효과 분석Example 1. Effect analysis according to the number of nylon membrane disks treated
마이크로 펀치를 이용하여 직경 2 mm의 나일론 멤브레인(nylon membrane, Cat.# RPN203B, GE Healthcare) 디스크를 제조하여 실험에 사용하였다. 1.5 ㎖ 튜브에 Taq DNA 중합효소(시판중인 A사 제품, 500 units/㎕) 10 ㎕를 넣고 0, 1, 2, 3, 4 또는 5개의 나일론 멤브레인 디스크를 각각 첨가하여 잘 섞어주고 24시간 동안 -20℃에서 반응시켰다. 이후, 암피실린 저항성 유전자 bla(beta-lactamases) 및 16s rRNA 유전자 검출용 프라이머(표 1)를 이용하여 하기 표 2와 같은 조건으로 PCR을 수행하였다. 주형 DNA는 암피실린 저항성 유전자를 포함하고 있는 pGEM®-T EASY Vector(Promega)에 16S rRNA 유전자(서열번호 5)를 삽입하여 사용하였다. A nylon membrane (Cat.# RPN203B, GE Healthcare) disk with a diameter of 2 mm was prepared using a micro punch and used in the experiment. Add 10 μl of Taq DNA polymerase (commercially available from company A, 500 units/μl) to a 1.5 ml tube, add 0, 1, 2, 3, 4 or 5 nylon membrane discs, mix well, and incubate for 24 hours - Reacted at 20°C. Subsequently, PCR was performed under the conditions shown in Table 2 below using primers for detecting ampicillin resistance gene bla (beta-lactamases) and 16s rRNA gene (Table 1). The template DNA was used by inserting the 16S rRNA gene (SEQ ID NO: 5) into the pGEM ® -T EASY Vector (Promega) containing the ampicillin resistance gene.
주형 DNA를 첨가하지 않은 조건에서, Taq DNA 중합효소에 나일론 멤브레인 디스크를 처리하지 않고 PCR을 수행한 결과, bla 및 16s rRNA 유전자가 검출되는 것을 확인함으로써, Taq DNA 중합효소 내 오염 DNA가 존재하는 것을 알 수 있었다(도 1A "0" 레인). 이에, 주형 DNA를 첨가하지 않은 조건에서, Taq DNA 중합효소에 나일론 멤브레인 디스크 1, 2, 3, 4 또는 5개를 각각 처리하고 PCR을 수행한 결과, bla 유전자는 나일론 멤브레인 디스크를 3개 이상 처리했을 때부터 증폭 산물이 검출되지 않았고, 16s rRNA 유전자는 나일론 멤브레인 디스크를 2개 이상 처리했을 때부터 증폭 산물이 검출되지 않는 것을 확인하였다(도 1A). 상기 결과를 통해, 나일론 멤브레인을 이용하여 Taq DNA 중합효소 내 오염 DNA를 제거할 수 있음을 알 수 있었다. Under the condition that template DNA was not added, PCR was performed without treating the nylon membrane disk with Taq DNA polymerase, and it was confirmed that bla and 16s rRNA genes were detected, thereby confirming the presence of contaminating DNA in Taq DNA polymerase. (FIG. 1A "0" lane). Therefore, under the condition that no template DNA was added, 1, 2, 3, 4 or 5 nylon membrane disks were treated with Taq DNA polymerase, respectively, and PCR was performed. As a result, the bla gene was treated with 3 or more nylon membrane disks. It was confirmed that no amplification product was detected from the time of treatment, and no amplification product of the 16s rRNA gene was detected from the treatment of two or more nylon membrane disks (FIG. 1A). Through the above results, it was found that contaminating DNA in Taq DNA polymerase can be removed using a nylon membrane.
또한, 주형 DNA를 첨가한 조건에서, Taq DNA 중합효소에 나일론 멤브레인 디스크 1, 2, 3, 4 또는 5개를 각각 처리한 후 PCR을 수행한 결과, 나일론 멤브레인 디스크의 개수에 따른 bla 및 16s rRNA 유전자의 검출양에는 변화가 없는 것을 확인함으로써, 나일론 멤브레인 디스크의 처리 개수가 Taq DNA 중합효소의 활성에는 영향을 주지 않는 것을 알 수 있었다(도 1B). In addition, under the condition of adding template DNA, PCR was performed after treating 1, 2, 3, 4 or 5 nylon membrane disks with Taq DNA polymerase, respectively. As a result, bla and 16s rRNA according to the number of nylon membrane disks By confirming that there was no change in the detected amount of the gene, it was found that the number of treated nylon membrane disks did not affect the activity of Taq DNA polymerase (FIG. 1B).
실시예 2. 나일론 멤브레인 디스크 처리 시간에 따른 효과 분석Example 2. Effect Analysis of Nylon Membrane Disc Treatment Time
1.5 ㎖ 튜브에 Taq DNA 중합효소(시판중인 A사 제품, 500 units/㎕) 10 ㎕를 넣고 직경 2 mm의 나일론 멤브레인 디스크 3개를 첨가하여 잘 섞어주고 0, 3, 6, 12, 18, 24 또는 48시간 동안 -20℃에서 각각 반응시켰다. 이후, 암피실린 저항성 유전자 bla 및 16s rRNA 유전자에 대한 PCR을 실시예 1과 동일한 조건으로 수행하였다. Add 10 μl of Taq DNA polymerase (commercially available from company A, 500 units/μl) to a 1.5 ml tube, add 3 nylon membrane disks with a diameter of 2 mm, mix well, and mix 0, 3, 6, 12, 18, 24 or at -20°C for 48 hours, respectively. Thereafter, PCR for the ampicillin resistance gene bla and the 16s rRNA gene was performed under the same conditions as in Example 1.
주형 DNA를 첨가하지 않은 조건에서, Taq DNA 중합효소에 나일론 멤브레인 디스크를 3, 6, 12, 18, 24 또는 48시간 동안 각각 처리한 후 PCR을 수행한 결과, bla 유전자는 24시간 처리했을 때부터 증폭 산물이 검출되지 않았고, 16s rRNA 유전자는 18시간 처리했을 때부터 증폭 산물이 검출되지 않는 것을 확인하였다(도 2A). Under the condition that no template DNA was added, PCR was performed after treating nylon membrane disks with Taq DNA polymerase for 3, 6, 12, 18, 24, or 48 hours, respectively. No amplification product was detected, and it was confirmed that no amplification product was detected from the treatment of the 16s rRNA gene for 18 hours (FIG. 2A).
또한, 주형 DNA를 첨가한 조건에서, Taq DNA 중합효소에 나일론 멤브레인 디스크를 3, 6, 12, 18, 24 또는 48시간 동안 각각 처리한 후 PCR을 수행한 결과, 나일론 멤브레인 디스크의 처리 시간에 따른 bla 및 16s rRNA 유전자의 검출양에는 변화가 없는 것을 확인함으로써, 나일론 멤브레인 디스크의 처리 시간이 Taq DNA 중합효소의 활성에는 영향을 주지 않는 것을 알 수 있었다(도 2B). In addition, under the condition of adding template DNA, PCR was performed after treating the nylon membrane disk with Taq DNA polymerase for 3, 6, 12, 18, 24, or 48 hours, respectively. By confirming that there was no change in the detected amounts of bla and 16s rRNA genes, it was found that the treatment time of the nylon membrane disk did not affect the activity of Taq DNA polymerase (FIG. 2B).
실시예 3. FTA 카드 처리에 따른 효과 분석Example 3. Effect analysis according to FTA card processing
나일론 멤브레인이 아닌 다른 멤브레인의 Taq DNA 중합효소 내 오염 DNA 제거 효과를 분석하기 위하여 FTA(Flinders Technology Associates, Cat.# 16972638, WhatmanTM) 카드를 이용하여 실험을 수행하였다. FTA 카드는 DNA 장기 보관, 표본 DNA 제작 등에 주로 사용되는 면 기반(cotton-based)의 셀룰로스 멤브레인이다.In order to analyze the effect of removing contaminating DNA in Taq DNA polymerase of membranes other than nylon membranes, experiments were performed using FTA (Flinders Technology Associates, Cat.# 16972638, Whatman TM ) cards. FTA cards are cotton-based cellulose membranes that are mainly used for long-term DNA storage and preparation of sample DNA.
상기 실시예 1과 동일 조건으로 나일론 멤브레인 대신 직경 2 mm의 FTA 카드 디스크 0, 1 또는 2개를 각각 첨가하여 잘 섞어주고 48시간 동안 -20℃에서 반응시켰다. 이후, 16s rRNA 유전자에 대한 PCR을 수행하였다. Under the same conditions as in Example 1, instead of the nylon membrane, 0, 1 or 2 FTA card disks having a diameter of 2 mm were added, mixed well, and reacted at -20 ° C for 48 hours. Then, PCR was performed on the 16s rRNA gene.
주형 DNA를 첨가하지 않은 조건에서, Taq DNA 중합효소에 FTA 카드 디스크 0, 1 또는 2개를 각각 처리한 후 PCR을 수행한 결과, 16s rRNA 유전자는 FTA 카드 디스크를 2개 처리했을 때 검출되지 않는 것을 확인하였다. 상기 결과를 통해, FTA 카드를 이용하여 Taq DNA 중합효소 내 오염 DNA를 제거할 수 있음을 알 수 있었다. 그러나, 주형 DNA를 첨가한 조건에서, Taq DNA 중합효소에 FTA 카드 디스크 0, 1 또는 2개를 각각 처리한 후 PCR을 수행한 결과, FTA 카드 디스크의 처리 개수에 따라 16s rRNA 유전자의 검출양이 감소하는 것을 확인함으로써, FTA 카드 디스크의 처리가 Taq DNA 중합효소의 활성을 감소시키는 것을 알 수 있었다(도 3). Under the condition that no template DNA was added, PCR was performed after treating 0, 1, or 2 FTA card disks with Taq DNA polymerase, respectively. As a result, the 16s rRNA gene was not detected when two FTA card disks were treated. confirmed that Through the above results, it was found that contaminating DNA in Taq DNA polymerase can be removed using the FTA card. However, under the condition of adding template DNA, PCR was performed after 0, 1 or 2 FTA card disks were treated with Taq DNA polymerase, respectively. By confirming the decrease, it was found that the treatment of the FTA card disk reduced the activity of Taq DNA polymerase (FIG. 3).
<110> Jeonju University Office of Industry-University Cooperation <120> Method for removing DNA contamination in DNA polymerase using nylon membrane <130> PN21112 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tatccgcctc catccagtct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggaaccggag ctgaatgagg 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggactaccag gctatctaat cct 23 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agcagccgcg gtaata 16 <210> 5 <211> 287 <212> DNA <213> Unknown <220> <223> Bacterium BS2145 <400> 5 ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgagcctca gtgtcagtaa 60 cagaccagaa tgtcgccttc gccactggtg ttcttccata tatctacgca ttccaccgct 120 acacatggag ttccacattc ctcttctgta ctcaagtttc ccagtttcca atgaccctcc 180 acggttaagc cgtgggcttt cacatcagac ttaagaaacc acctgcgctc cctttacgcc 240 caataaatcc ggacaacgct tgccacctac gtattaccgc ggctgct 287 <110> Jeonju University Office of Industry-University Cooperation <120> Method for removing DNA contamination in DNA polymerase using nylon membrane <130> PN21112 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 1 tatccgcctc catccagtct 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 2 ggaaccggag ctgaatgagg 20 <210> 3 <211> 23 <212> DNA <213> artificial sequence <220> <223> primer <400> 3 ggactaccag gctatctaat cct 23 <210> 4 <211> 16 <212> DNA <213> artificial sequence <220> <223> primer <400> 4 agcagccgcg gtaata 16 <210> 5 <211> 287 <212> DNA <213> unknown <220> <223> Bacterium BS2145 <400> 5 ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgagcctca gtgtcagtaa 60 cagaccagaa tgtcgccttc gccactggtg ttcttccata tatctacgca ttccaccgct 120 acacatggag ttccacattc ctcttctgta ctcaagtttc ccagtttcca atgaccctcc 180 acggttaagc cgtgggcttt cacatcagac ttaagaaacc acctgcgctc cctttacgcc 240 caataaatcc ggacaacgct tgccacctac gtattaccgc ggctgct 287
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