Basic Research Workshop : Basic Research Workshop : Polymerase chain reaction (PCR)

( Kyun Hwan Kim ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.2

The polymerase chain reaction (PCR) is a biochemical technology to amplify a few copies of DNA generating thousands to millions of copies of a particular DNA sequence in molecular biology. The PCR was invented in 1983 by Kary Mullis and is now a common and often indispensable technique used for a variety of applications in medical and biological research labs. In 1993, Dr. Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR. Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs, although some techniques allow for amplification of fragments up to 40 kb in size. The application of PCR include: the selective DNA isolation, DNA cloning for sequencing, functional analysis of genes, amplification and quantification DNA, diagnosis of hereditary diseases, identification of genetic fingerprints such as forensic sciences and paternity testing, and the detection and diagnosis of infectious diseases (pathogens). Recently, the real-time polymerase chain reaction (RT-PCR), also called quantitative real time polymerase chain reaction (RT-qPCR) or kinetic polymerase chain reaction is widely used to amplify and simultaneously quantify a targeted DNA molecule. Real Time-PCR enables both detection and quantification. The quantification can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. In this talk, the basic principles, history, and applications of PCR/RT-PCR will presented.

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.

임상가검물과 파라핀 포매 조직에서 PCR법을 이용한 결핵균의 검출

김은중(Eun-Joong Kim),최우순(Woo-Soon Choi),황석연(Seock-Yeon Hwang) 대한의생명과학회 2000 Biomedical Science Letters Vol.6 No.1

PCR을 이용한 임상가검물 중에서 보편화된 객담 이외에 미량의 각종 체액과 임상에서 많이 실시하지 않는 파라핀 포매 조직에서의 결핵균 검출을 실험하여 그 활용 가능성을 규명하고자하였다. 임상가검물인 체액 65예는 항산성 염색과 배양검사, PCR을 실시하였고, 파라핀 포매 조직 50예는 항산성 염색과 병리조직학적 진단, PCR을 실시하여 다음과 같은 결론을 얻었다. 본 실험 검체 중 임상가검물인 체액에서 항산성 염색 음성인 검체 중 12.1%, 배양검사에서 음성인 검체 중 3.7%에서 PCR 양성의 결과를 보였고, 파라핀 포매 조직에서는 항산성 염색 음성인 검체 중 20.0%에서 PCR 양성의 결과를 얻어 PCR이 민감도와 특이도가 높음을 확인할 수 있었다. This study has been carried out to investigate the sensitivity of polymerase chain reaction (PCR) method over conventional acid-fast bacilli (AFB) staining and/culture methods for the detection of Mycobacterium tuberculosis from trace body fluid and paraffin-embedded tissues (PET) specimens. A total of 65 cases were employed for the AFB staining and culture test, and a total of 50 cases were subjected to PCR and histopathological analysis. Among the specimen showing negative reaction to AFB staining, 12.1% were positive to PCR and 3.7% of the specimen representing negative result to culture test showed positive reaction to PCR. In addition, 20.0% of the specimen with AFB negative showed positive reaction to PCR. From these results, it could be concluded that PCR method overwhelms AFB staining and culture tests in sensitivity and specificity to M. tuberculosis detection.

임상가검물과 파라핀 포매 조직에서 PCR법을 이용한 결핵균의 검출

김은중,최우순,황석연 대한의생명과학회 2000 Journal of biomedical laboratory sciences Vol.6 No.1

PCR을 이용한 임상가검물 중에서 보편화된 객담 이외에 미량의 각종 체액과 임상에서 많이 실시하지 않는 파라핀 포매 조직에서의 결핵균 검출을 실험하여 그 활용 가능성을 규명하고자 하였다. 임상가검물인 체액 65예는 항산성 염색과 배양검사, PCR을 실시하였고, 파라핀 포매 조직 50예는 항산성 염색과 병리조직학적 진단, PCR을 실시하여 다음과 같은 결론을 얻었다. 본 실험 검체 중 임상가검물인 체액에서 항산성 염색 음성인 검체 중 12.1%,배양검사에서 음성인 검체 중 3.7%에서 PCR양성의 결과를 보였고, 파라핀 포매 조직에서는 항산성 염색 음성인 검체 중 20.0%에서 PCR양성의 결과를 얻어 PCR이 민감도와 특이도가 높음을 확인할 수 있었다. This study has been carried out to investigate the sensitivity of polymerase chain reaction (PCR) method over conventional acid-fast bacilli (AFB) staining and/culture methods for the detection of Mycobacterium tuberculosis from trace body fluid and paraffin-embedded tissues (PET) specimens. A total of 65 cases were employed for the AFB staining and culture test, and a total of 50 cases were subjected to PCR and histopathological analysis. Among the specimen showing negative reaction to AFB staining, 12.1% were positive to PCR and 3.7% of the specimen representing negative result to culture test showed positive reaction to PCR. In addition, 20.0% of the specimen with AFB negative showed positive reaction to PCR. From these results, it could be concluded that PCR method overwhelms AFB staining and culture tests in sensitivity and specificity to .M. specificity to tuberculosis detection.

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>

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

Ppyun, H.,Kim, S.H.,Youn, M.H.,Cho, S.S.,Kwon, K.M.,Kweon, D.H.,Kwon, S.T. IPC Science and Technology Press ; Elsevier Scienc 2016 Enzyme and microbial technology Vol.82 No.-

<P>We previously reported that Neq A523RDNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 x 10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8 x 10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2 x 10(-5)), Neq A523R DNA polymerase (13.1 x 10(-5)), and Neq N540R DNA polymerase (37.7 x 10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity. (C) 2015 Elsevier Inc. All rights reserved.</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.

DNA 교차오염 방지기능이 있는 single-tube nested reverse transcription-polymerase chain reaction을 이용한 돼지생식기호흡기증후군바이러스 유전형 감별진단

정필수 ( Pil Soo Jeong ),박수진 ( Su Jin Park ),김은미 ( Eun Mi Kim ),박지영 ( Ji Young Park ),박유리 ( Yu Ri Park ),강대영 ( Dae Young Kang ),차현욱 ( Hyun Ouk Cha ),이경기 ( Kyoung Ki Lee ),김성희 ( Seong Hee Kim ),박최규 ( Choi 한국동물위생학회 2016 한국동물위생학회지 (KOJVS) Vol.39 No.2

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 TCID50/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

Sensitive and Specific Detection of Mycoplasma species by Consensus Polymerase Chain Reaction and Dot Blot Hybridization

Sunhwa Hong,Hyun-A Lee,Sang-Ho Park,Okjin Kim 한국실험동물학회 2011 Laboratory Animal Research Vol.27 No.2

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species of mycoplasmas are important pathogens that cause respiratory infection in laboratory animals and that are known to affect experimental results obtained with contaminated animals. The aim of the present study was to develop a sensitive and specific assay for the detection of mycoplasma species. To this end, we developed a polymerase chain reaction and dot blot hybridization assay (PCR/DBH) for detecting mycoplasma DNA and evaluated it for its sensitivity and specificity. Mycoplasma consensus primer pairswere used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 10² pg of mycoplasma purified DNA. For DBH, the amplified DNA was labeled by incorporation of digoxigenin (DIG). This DIG-labeled probe was capable of detecting 10⁴ pg of purified mycoplasma DNA by DBH. PCR/DBH was more sensitive than PCR or DBH alone and was also very specific. Our PCR/DBH assay can be applied efficiently to confirm the presence of mycoplasma species on clinical samples and to differentiate between mycoplasma species infection and other bacterial infections.

A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR)

Anong Bintvihok,Supitchaya Treebonmuang,Kitiya Srisakwattana,Wisut Nuanchun,Koranis Patthanachai,Sungworn Usawang 한국독성학회 2016 Toxicological Research Vol.32 No.1

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxinproducing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was 65oC. The optimized template and primer concentration were 1.5 μL (50 ng/μL) and 3 μL (10 μM/μL) respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at 88.0oC, 87.5oC, 83.5oC, and 89.5oC respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.