Characterization and PCR application of a thermostable DNA polymerase from <i>Thermococcus pacificus</i>

Lee, Jong Il,Cho, Sung Suk,Kil, Eui-Joon,Kwon, Suk-Tae Elsevier 2010 Enzyme and microbial technology Vol.47 No.4

<P><B>Abstract</B></P><P>The biochemical properties of the <I>Thermococcus pacificus</I> (<I>Tpa</I>) DNA polymerase were determined to evaluate its feasibility for use in polymerase chain reaction (PCR) application. The <I>Tpa</I> DNA polymerase gene was expressed under the control of the T7<I>lac</I> promoter in the pET-22b(+) plasmid in <I>Escherichia coli</I> BL21-CodonPlus(DE3)-RIL. The enzyme was then purified by heat treatment followed by two steps of column chromatography after which the optimum pH and temperature of the enzyme were determined to be pH 7.5 and 75°C. The optimal PCR buffer for <I>Tpa</I> DNA polymerase consisted of 50mM Tris–HCl (pH 8.4), 4mM MgCl<SUB>2</SUB>, and 10mM KCl. <I>Tpa</I> DNA polymerase performed significantly more efficiently in PCR amplification than <I>Taq</I> or <I>Pfu</I> DNA polymerase. By fusing the <I>Sulfolobus solfataricus</I> DNA binding protein Sso7d to <I>Tpa</I> DNA polymerase, we obtained a fusion polymerase which exhibits profound advantages over unmodified <I>Tpa</I> DNA polymerase in PCR applications. <I>Tpa</I> DNA polymerase (2.04×10<SUP>−6</SUP>) and <I>Tpa</I>-S DNA polymerase (2.20×10<SUP>−6</SUP>) revealed a 5-fold higher fidelity than <I>Taq</I> DNA polymerase (12.13×10<SUP>−6</SUP>).</P>

Pyrococcus furiosus 유래 고온성 DNA polymerase 유전자의 발현 및 정제

조현국,서동호,정종현,박천석 慶熙大學校 食糧資源開發硏究所 2009 硏究論文集 Vol.28 No.-

현재 Pfu DNA polymerase을 이용한 PCR은 여러 방면에서 많이 사용되는 한 방법으로 자리잡고 있으며 다양한 PCR 방법의 출현으로 다양한 학문에서 널리 쓰이고 있다. 본 연구는 P. furiosus 유래 DNA polymerase를 E. coli에 클로닝 및 발현에 성공하였으며,affinity chromatography를 이용해 손쉽게 Pfu DNA polymerase를 정 제할 수 있었다. 정제된 재조합 Pfu DNA polymerase는 여 러 DNA를 이용해 PCR반응을 수행한 결과,성공적으로 원하는 DNA가 증폭 됨을 확인 되었다. 또한 PCR반응에서 재조합 Pfu DNA polymerase 의 농도가 중요한 factor가 됨을 확인하였다. DNA polymerase gene from Pyrococcus furiosus, an extremely thermophilic archaea, was amplified using PCR. Pyrococcus furiosus DNA polymerase (Pfu) was successfully expressed in E. coli MC1061 and the recombinant enzyme was efficiently purified with Ni-NTA affinity chromatography.Optimal Pfu concentration was studied for the effective PCR reaction.

만성간염 환자의 혈청에서 중합효소연쇄반응을 이용한 HBV DNA 및 HCV RNA 검출소견

박용욱(Yong Uk Park),서강석(Kang Suk Suh),한상우(Sang Woo Han),김신묵(Sin Mook Kim),최성규(Sung Kyu Choi),서순팔(Soon Pal Suh),김세종(Sei Jong Kim) 대한내과학회 1996 대한내과학회지 Vol.50 No.5

N/A Objectives: It is well-known that chronic hepatitis can be caused by hepatitis viral infection, drugs and toxins, inborn errors of metabolism, and autoimmume disease. Hepatitis B (with or without superimposed hepatitis D) and hepatitis C viral infections are known as the common causes of chronic viral hepatitis. Recently there have been several reports that chronic hepatitis and chronic liver disease can be caused by the superinfection or co-infection of HBV and HCV. We detected HBV DNA and HCV RNA in patients with chronic hepatitis using polymerase chain reaction and compared polymerase chain reaction with enzyme immunoassay in evaluating the presence of a superinfection or co-infection of HBV and HCV. Methods : Using sera from 61patients with histologically proven chronic hepatitis (chronic active hepatitis' 51cases, chronic persistent hepatitis: 10cases), we checked the HBV DNA and HCV RNA using polymerase chain reaction. We also checked the HBV and HCV markers using enzyme immunoassay. Results: 1) Only HBV DNA could be detected in 37patients (60.7%). HBV DNA and HCV RNA were detected in 11patients (18%). Only HCV RNA was dectected in 4patients (6.6%). Neither HBV DNA nor HCV RNA was found in 9patients (14.8%). 2) HBV DNA and HCV RNA were detected in 11patients (18%) whereas HBsAg or anti-HBe and anti-HCV were seropositive in 4patients (6.5%). 3) The positive rates of HBsAg and HBV DNA were 83.6% and 78.7%, respectively, and the positive rates of HBV DNA in HBsAg-positive cases and in HBeAg-positive cases were 90.2% and 93.0%, respectively. 4) The positive rates of anti-HCV and HCV RNA were 11.5% and 24.6%, respectively, and the positive rates of HCV RNA in anti-HCV positive cases and in anti-HCV negative eases were 85.7% and 14.8%, respectively. Conclusion: It has been suggested that hepatitis B viral infection is the most common cause of chronic hepatitis in Korea, and that hepatitis C virus might also play an etiological role. In this study, we found that 18% of chronic hepatitis patients were superinfected or co-infected with HHV and HCV, and that polymerase chain reaction was more sensitive than enzyme immunoassay to detect HBV and HCV infection when superinfection or Qo-infection was suspected.

만성 HBsAg 보유자에서 B 형 간염 바이러스에 의한 말초혈액단핵세포의 감염

조성원(Sung Won Cho),이문성(Moon Sung Lee),김진홍(Jin Hong Kim),심찬섭(Chan Sup Shim),이희발(Hi Bahl Lee) 대한내과학회 1994 대한내과학회지 Vol.46 No.5

N/A Objectives: Hepatitis B virus infection of peripheral blood mononuclear cells has been observed in hepatitis B virus-associated liver disease. We have analyzed 18 chronic HBsAg carriers to investigate the presence of hepatitis B virus in peripheral blood mononuclear cells. Methods: Hepatitis B virus DNA was detected in peripheral blood mononuclear cells by southern blot hybridization and polymerase chain reaction. Southern blots of polymerase chain reaction products were hybridized with cloned hepatitis B virus DNA. Peripheral blood mononucldar cells from carriers were cultured for 48 to 96 hours with or without lipopolysaccharide. Hepatitis B virus DNA was detected in the serum by dot blot and polymerase chain reaction. Results: Southern blot analysis of peripheral blood mononuclear cell DNA showed a faint 3.2 kb full genomic hepatitis B virus DNA in only 1 of 18 carriers studied, even though the sensitivity of our method enabled us to detect as little as 1 pg of cloned hepatitis B virus DNA. Hepatitis B virus DNA in peripheral blood mononuclear cells was detected by polymerase chain reaction in 11 of 12 carriers who were positive for serum hepatitis B virus DNA by dot blot, in 1 of 3 carriers who were negative and positive for serum Hepatitis B virus DNA by dot blot and polymerase chain reaction, respectively. Hepatitis B virus DNA in peripheral blood mononuclear cells was detected in 2 of 3 carriers even without serum hepatitis B virus DNA. In the presence of lipopolysaccharide, the signal derived from the DNA extracted from stimulated peripheral blood mononuclear cells increased. In 2 of 4 carriers in whom hepatitis B virus DNA was absent in unstimulated peripheral blood mononuclear cells, Hepatitis B virus DNA was identified in lipopolysaccharide stimulated peripheral blood mononuclear cells. Thus, 88.8% (16/18) of chronic HBsAg carriers were found to have hepatitis B virus DNA in peripheral blood mononuclear cells. Conclusion: 1) Hepatitis B virus infection of peripheral blood mononuclear cells occurs at very low levels in the majority of chronic HBsAg carriers 2) Hepatitis B virus replication may be stimulated by lipopolysaccharide.

Polymerase Chain Reaction을 이용한 Mycoplasma synoviae의 진단법 확립

이영주 한국수의공중보건학회 2003 예방수의학회지 Vol.27 No.1

Mycoplasma synoviae만을 특이적으로 동정하기 위한 PCR법을 확립하기 위하여 민감성 및 특이성을 조사한 결과, 아래와 같은 결과를 얻었다. 1 표준균주인 M. synoviae, M. gallisepticum, M. iowae, M. iners, M. pullorum, M. anatis, M. gallinaceum 및 M. glycophilum에 대하여 PCR법을 실시한 결과, M. synoviae에서만 207 bp의 특이적인 증폭산물이 관찰되었을 뿐, 기타 Mycoplasma spp.에 대해서는어떠한 증폭산물도 나타나지 않아 작성된 primer의 특이성이 확인되었다. 2. 국내 발병계의 관절로부터 분리된 M. synoviae 8주에 대하여 PCR법을 적용한 결과, 8주 모두에서207 bp의 특이 band가 관찰되어 신속한 원인체의 동정이 가능한 것으로 나타났다. 3. PCR법의 민감성을 조사하기 위하여 M. synoviae의 genomic DNA양을 1㎍/㎍l에서 10진희석후 PCR법을 실시한 결과, 100 1㎍/㎍l의 DNA농도까지 검출이 가능하였다. 4. PCR법에 의한 증폭산물이M. synoviae의 특이 유전자임을 확인하기위하여 제한효소 WvalI, HphI, NcoI 및 RsaI을 처리한 결과, GenBank의 예상절편부위와 일치하여 정확한 부위에서 증폭이 이루어졌음을 확인할 수 있었다. Mycoplasim synoviae (M. synoviae) causes synovitis and respiratory disease in chickens and turkey. The diagnosis of M. synoviae is currently based on microbiological and serological tests. However, the methods have some problems such as time-consuming, laborious, and cross-reaction. Therefore, a new method has been required for the diagnosis. To establish a sensitive and specific diagnostic method for detection of M. synoviae, synthetic oligonucleotide primers based on the nucleotide sequence of 16s rDNA were synthesized and a polymerase chain reaction (PCR) was established with the primers. The 207 bp DNA fiagments by PCR were only amplified in reference strain and 8 field isolates of M synoviae. However, no PCR product was detected fiom other Mycoplasim spp. Sensitivity of the PCR was up to 100 pg genomic DNA. The identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with AvaI, HphI, NcoI and RsaI This result suggested that the PCR technique was a valuable diagnosis for M synoviae.

Polymerase Chain Reaction을 이용한 FMR1 유전자의 Premutation 빈도에 관한 연구

박원일(Wouil Park),이경자(Kyung Ja Lee),최의열(Eu Yul Choi) 대한소아신경학회 1999 대한소아신경학회지 Vol.7 No.1

목 적 : 취약 X 증후군은 정신지체의 흔한 원인질환의 하나로 이의 예방을 위해서는 돌연변이 전 단계의 CGG 삼핵산 반복을 갖는 보인자의 색출이 필요하다. 본 연구는 중합효소 연쇄반응법을 이용해 정상 신생아에서 돌연변이 전 단계 CGG 삼핵산 반복의 발생빈도를 조사하고자 하였다. 방 법 : 1996년 3월부터 8월까지 춘천성심병원 소아과에 신생아 대사검사를 위해 의뢰한 Guthrie paper 혈흔에서 DNA를 분리 후, PCR로 CGG 삼핵산 반복 서열을 포함하는 FMR1 유전자 일부를 증폭시켜 agarose gel에서 분석하였고 PCR based agarose gel 분석에서 비정상적인 예를 chemiluminescent detection을 통해 확인하였다. 결 과 : 669례에서 PCR based agarose gel 분석이 가능하였고 이들 중 4례에서 55회 이상의 삼핵산 반복이 관찰되었으며 chemiluminescent desection 방법에서 4례 중 3례의 이상을 보여 일치율은 75%이었으며, 정상신생아의 233명당 1례에서 돌연변이 전단계의 삼핵산 반복서열을 관찰할 있었다. 결 론 : 취약 X 증후군은 우리나라에서도 다른 나라와 비슷한 유병률을 보일 것으로 추정되며 정확한 유병률을 측정하기 위해서는 정신지체를 갖는 환아를 대상으로 보다 많은 연구가 필요할 것으로 생각된다. 또한 이를 위해 효과적이며 간편한 방법의 개발이 필요할 것으로 생각된다. Background : Fragile X syndrome is one of the most common causes of mental retardation. For its prevention, detection of premutation range CGG repeat in FMR1 gene is necessary. The aim of our study was to determine the prevalence of premutation range of CGG repeat in neonate, and to evaluate the possibility of screening test. Methods : DNA were extracted from Guthrie paper blood spot, referred for neonatal metabolic screening test, collected during the period of March 1996 through August 1996, at Chunchon Sacred Heart Hospital. Then FMR1 gene involving CGG repeat was amplified by polymerase chain reaction, and then abnormal expansion of CGG were analyzed by agarose gel electrophoresis and digoxigenin labelled hemiluminescent detection method. Results : Four cases among 669 PCR product were appeared to have abnormal CGG expansion and 3 out of the 4 cases were confirmed to have abnormal CGG repeat by chemiluminescent detection method. Conclusion : We found 3 premutation range CGG expansion with a prevalence trance of 1/233 in neonate. Although PCR based agarose gel electrophoresis alone is not suitable for screening test, it could be a useful tool for fragile X screening test in combination with chemiluminescent detection method.

Polymerase chain reaction을 이용한 실험적 감염 돼지의 혈액과 조직으로부터 Toxoplasma gondii 검출

신명득,신기욱,Suh, Myung-deuk,Shin, Gee-wook 대한수의학회 2001 大韓獸醫學會誌 Vol.41 No.1

This study was conducted to detect the toxoplasma specific-DNA in circulating blood and organs collected from slaughtered pigs at slaughtering house and experimentally infected pigs with Toxoplasma gondii tachyzoites by polymerase chain reaction(PCR), and also PCR was applied to diagnose for acute phase of swine toxoplasmosis as a newly developed diagnostic test. The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with Tp parasite detection by mouse inoculation(MI). On the other hand, latex agglutination test(LAT) was conducted to detect the serum antibodies comparing with the detection of toxoplasma by PCR and MI. The results obtained were summarized as follows. PCR was able to determine at the lowest level of $10^0/ml$ T. gondii in blood samples which were blended with a serial diluted T gondii in vitro. On the other hand, $10^2/5g$ of T gondii could detect from a variety of tissues including lung, diaphragm, liver, heart, spleen and brain in vitro. The primer was proved to specifically determine T gondii in blood and tissues in vitro but it did not detect Neospora caninum used as a negative control. DNA of T. gondii was effectively extracted by freezing, thawing and grinding twice both tissues mixed with T gondii in vitro and in experimentally infected pig's tissues. PCR detected specific DNA in the blood of experimentally infected pigs at 108 hrs and 120 hrs post-infection, it was the same time that the pigs showed fever and parasitaemia. In case of tissue, specific DNA was, however, detected only lung from experimentally infected pigs. Even though the duration of acute phase was from 3 to 7 days post-infection, but the latex agglutination test (LAT) results appeared from 8 days post-infection. A comparison of sensitivity in determining T gondii in blood samples between PCR and MI, PCR positive rate ranged from 25 to 33.3%, but that of MI covered from 75 to 100%.

Polymerase Chain Reaction에 의한 Lactobacillus casei 및 돌연변이 균주들의 비교 분석

남진식,이정준,신명수,나석환,백영진,유민 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.6

PCR 반응에 따른 RAPD를 산업적으로 유용한 유산균주를 신속하고 정확하게 분리하기 위한 방법의 하나로 사용할 수 있는지 가능성을 조사하기 위하여 L. casei의 4개 strain과 이들의 돌연변이 균주 2 종류를 비교하였다. 또한 L. casei로부터 농도와 순도면에서 PCR template에 적합한 chromosomal DNA를 3시간만에 추출할 수 있는 방법을 확립하였다. RAPD를 위하여는 degenerated primer를 이용하였는데 PCR 결과로 볼 때 분석에 적합한 수와 크기의 random product들이 증폭되었다. 본 실험에 사용된 조건과 primer들은 L. casei의 대상 균주들과 돌연변이균주들을 정확하고 특이적으로 구별할 수 있었는데 경우에 따라서는 적절히 primer들을 혼합하여 사용할 때 더욱 확실한 구별이 가능하였다. 본 실험의 결과는 반복성이 매우 우수한 것으로 확인됨으로서 장차 산업적으로 유용한 L. casei 균주들을 신속, 정확하게 분리, 동정하는 데에 이용 가치가 높을 것으로 사료된다. To classify Lactobacillus casei strains on the basis of difference in their chromosomal DNA sequence, we have performed polymerase chain reactions on their chromosomal DNA by using random primers, and followed by analyzing randomly amplified polymorphic DNA fragments. We also developed a mini-preparative method to isolate PCR-grade chromosomal DNA from Lactobacillus casei strains within 3 hours. Based on RAPD patterns by polymerase chain reactions with degenerated random primers, 4 Lactobacillus casei strains and 2 mutant strains were successfully discriminated. Results were very sensitive, strain-specific and reproducible. It was also reliable. These results suggest that RAPD may be applied efficiently for the identification of several Lactobacillus casei strains.

Polymerase Chain Reaction을 이용한 결핵성 뇌막염의 조기진단

김재우,이상건,하경민,박웅양 동아대학교의과대학 1992 동아의대학술지 Vol.4 No.1

중합효소 연쇄반응을 이용한 첨규 콘딜롬의 원인 바이러스에 대한 연구

구본길(Bon Gil Koo),이증훈(Jeung Hoon Lee),박장규(Jang Kyu Park),최대경(Tae Kyung Choi),백태현(Tae Hyun Paik),박정규(Jeong Kyu Park),이정용(Joung Young Lee) 대한피부과학회 1992 대한피부과학회지 Vol.30 No.4

Polymerase chain reaction was performed to determine he types of human papillomavirus (HPV) causing condylomata acuminata in frozen tissues and paraffinembedded tissues of condylomata acuminata. HPV DNA was detected in 31 of 32 patients with condylomata aciminata. HPV 6 and/or 11, low-risk types in genital carcinogenesis, were present in all cases in whieh HPV was detected. Both types were present in 5 cases. It is, therefore, supposed that there is not much risk of this disease to transform to the invasive cancer in Korean and polymerase chain reaction can be used to deteet HPV and identify its type from paraffin-embedded tissues. (Kor J Dermatol 1992;30(4):439-445)