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.

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

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.

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

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.

결핵균 검출을 위한 모세관 중합효소연쇄반응의 적정조건

김재룡,전동석,박헌찬,전효진 啓明大學校 醫科大學 1993 계명의대학술지 Vol.12 No.4

For detection of M. tuberculosis using hot-air capillary-thermocycler, FTC-2000[Daehan Medical System Co, Korea], We amplified 188 base pair IS986 fragment from chromosomal DNA of M. tuberculosis H37Rv strain by two-step nested method, and the concentrations of each component of polymerase chain reaction(PCR) mixture and the temperatures and times of each PCR stage were indivisually optimized. When compared to conventional heating-block method, only one tenth of reaction volume and time were required, i. e. 10ul and 10-30 minute, respectively. Final concentrations of each component at optimal condition were 0.5uM each primer, 0.2mM each dNTP, 50mM Tris-HCI(pH 8.3), 500ug/ml bovine serum albumin(BSA), 4mM MgCl₂and 0.5-1U Taq polymerase per reaction. Optimal temperatures and times of each stage were 94℃ and 1 sec of predenaturation, 45-55℃ and 1 sec of annealing, 72℃ and 10-20 sec of post-elongation, and optimal cycles of each nested-PCR were 30-50. Among various condition of capillary-PCR, BSA, annealing temperature and MgCl₂effect most critically on PCR result. At an annealing temperature of 45℃ with 3.5mM MgCl₂or 55℃ with 3mM MgCl₂, two-step nested-PCR gave greatest yield and highest sensitivity. At these optimal condition of capillary-PCR, 0.5fg of M. tuberculosis H37Rv DNA(same amout of DNA extracted from a single tubercle bacillus) was amplified effectively.

Polymerase Chain Reaction-Reverse Dot Blot Hybridization에 의한 Human Papillomavirus Typing과 Hybrid Capture System의 평가

윤영호,최선주,유기숙,고창원,김영탁 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.3

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 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 reaction 양성 소아 마이코플라스마 폐렴에서 혈청 IgM enzyme-linked immunosorbent assays의 진단적 가치

이혜진 ( Hye Jin Lee ),이윤태 ( Yoon Tae Lee ),김경훈 ( Kyung Hoon Kim ),양은애 ( Eun Ae Yang ),김환수 ( Hwan Soo Kim ),전윤홍 ( Yoon Hong Chun ),윤종서 ( Jong-seo Yoon ),김현희 ( Hyun Hee Kim ),김진택 ( Jin Tack Kim ) 대한천식알레르기학회 2018 Allergy Asthma & Respiratory Disease Vol.6 No.5

Purpose: Mycoplasma pneumoniae (MP) is a common cause of community-acquired pneumonia (CAP) in children. MP serum IgM and polymerase chain reaction (PCR) are the methods that enable early diagnosis in patients with MP pneumonia. The objective of this study was to investigate the clinical value of serum MP-specific IgM antibodies in PCR-positive MP pneumonia for the early diagnosis of MP pneumonia in children with CAP. Methods: Out of 129 patients with lower respiratory tract infection aged over 3 years, 90 CAP children were enrolled in the study. Throat swab MP real-time PCR and serum enzyme-linked immunosorbent assays (ELISA) IgM antibodies were performed. A positive rate of MP PCR and serum IgM, the level of IgM index, clinical features, and laboratory findings were analyzed. Results: MP PCR was positive in 57 cases. Longer fever duration before admission (P<0.001), higher rates of lobar or segmental pneumonia (P=0.048), unilateral infiltration (P=0.038), and extrapulmonary symptoms (P=0.049) were associated with MP PCR-positive pneumonia. Serum IgM index was significantly higher in MP PCR-positive pneumonia them in MP PCR-negative pneumonia (3.9±3.0 vs. 0.8±1.3, P<0.001). Using MP PCR as a gold standard, the sensitivity, specificity, positive predictive value and negative predictive value of serum IgM were 85.5%, 82.1%, 91.4%, and 71.9%, respectively. The area under the curves for serum IgM index was 0.892, and the ROC analysis indicated that an optimal cutoff value of 1.05 for serum IgM provided the highest sensitivity and specificity interestingly (83.9% vs. 85.7%, P<0.001). Conclusion: Serum IgM ELISA has useful diagnostic value in PCR-positive MP pneumonia. Applying an IgM index cutoff of 1.05 improves diagnostic accuracy. (Allergy Asthma Respir Dis 2018;6:248-254)

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.