• BMB Reports
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• 제40권3호
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• pp.444-447
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• 2007

Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.

• 생명과학회지
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• 제16권5호
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• pp.729-733
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• 2006

크립토스포리디움의 활성 및 감염성 판정을 위해 Direct RT-PCR, 세포배양 후 RT-PCR 및 면역형광염색법을 비교한 결과는 다음과 같다. 1) 크립토스포리디움의 HSP70 gene에 대해 direct RT-PCR한 결과, 민감도가 매우 높아 저농도로 존재하는 환경시료에서의 크립토스포리디움 활성을 모니터링하는데 장점이 있을 것으로 보이나, 감염성의 판정은 알 수 없으며, 정량화가 안되는 단점이 있었다. 2) ${\beta}-tubulin$ gene에 대해 RT-nested PCR을 한 결과 크립 토스포리디움의 난포낭이 $1{\times}10^4$ 세포수정도 되어야 검출이 되는 것으로 나타나 HSP70 gene에 대한 RT-PCR결과와 비교할 때 10,000배 이상 민감도가 떨어지는 것으로 나타났다. 3) 세포배양 후 RT-PCR 또는 면역형광염색법을 이용할 경우에는 민감도가 direct RT-PCR보다 다소 떨어지는 단점이 있었으나 크립토스포리디움의 오염원이나 오염이 심한 지역의 감염성 조사에 적합할 것으로 나타났으나, 정량화가 필요한 경우에는 세포배양 후 면역형광염색법이 효과적일 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1616-1621
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• 2007

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was $1{\times}10^4\;CFU/g$ feces for S. flexneri and $2{\times}10^4\;CFU/g$ feces for S. typhimurium; this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.

• 한국동물위생학회지
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• 제37권4호
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• pp.253-261
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• 2014

Porcine circovirus disease (PCVD) is a major problem of swine industry worldwide, and diagnosis of PCV2, causal agent of PCVD, has been doing in clinical laboratories of pig disease by polymerase chain reaction (PCR) methods. But the PCR analyses have a serious problem of misdiagnosis by contamination of DNA, in particular, from carryover contamination with previously amplified DNA or extracted DNA from field samples. In this study, an uracil DNA glycosylase (UNG)-based direct PCR (udPCR) without DNA extraction process and DNA carryover contamination was developed and evaluated on PCV2 culture and field pig samples. The sensitivity of the udPCR combined with dPCR and uPCR was same or better than that of the commercial PCR (cPCR) kit (Median diagnostics, Korea) on PCV2-positive serum, lymph node and lung samples of the pigs. In addition, the udPCR method confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PCV2 DNA from previous udPCR. In clinical application, 170 pig samples (86 tissues and 84 serum) were analysed by cPCR kit and resulted in 37% (63/170) of positive reaction, while the udPCR was able to detect the PCV2 DNA in 45.3% (77/170) with higher sensitivity than cPCR. In conclusion, the udPCR developed in the study is a time, labor and cost saving method for the detection of PCV2 and providing a preventing effect for DNA carryover contamination that can occurred in PCR process. Therefore, the udPCR assay could be an useful alternative method for the diagnosis of PCV2 in the swine disease diagnostic laboratories.

• Journal of Ginseng Research
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• 제33권1호
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• pp.55-58
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• 2009

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

• Clinical and Experimental Pediatrics
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• 제53권3호
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• pp.397-407
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• 2010

목 적 : F9 유전자는 혈우병B의 병인 유전자이다. 기존의 RFLP를 이용한 연관분석은 정보제공율이 55.6%에 불과하였다. 직접 염기서열 분석법은 98%의 돌연변이를 진단할 수 있지만, 고가의 비용이 든다. 본 연구는 F9 유전자를 대상으로 돌연변이의 선별검사로써 mPCR-CSGE를 사용하고, 이후 특정 유전자 부위만을 염기서열 분석하여 mPCR-CSGE의 유용성을 확인하기 위해 고안되었다. 방 법 : 연구대상은 비혈연 관계인 27명의 혈우병B 환자였다. 직접염기서열 분석법은 독립된 다른 기관에서 시행하였고, mPCR-CSGE 선별 후 염기서열 분석법은 본 연구자의 기관에서 시행되었다. 직접 염기서열 분석법의 결과가 참고치가 되어 mPCR-CSGE 선별 후 염기서열 분석법을 정확성, 경제성, 신속성, 편이성 측면에서 비교하였다. 두가지 방법으로 진단이 되지 않는 환자에게는 MLPA를 이용하여 돌연변이를 발견하였다. 결 과 : 직접 염기서열 분석법으로 26명(96.3%)의 환자에서 돌연변이를 확인할 수 있었다. mPCR-CSGE 선별 후 염기서열 분석법으로는 23명(85.2%)의 환자에서 돌연변이를 찾아낼 수 있었다. 1명의 환자는 MLPA로써 돌연변이를 확인할 수 있었다. 27명의 환자에게 21개의 독립적인 돌연변이가 있었다. mPCR-CSGE 선별 후 염기서열 분석법은 직접 염기서열 분석법에 비해 비용은 55.7%로 줄일 수 있었으나, 실험 단계는 더욱 복잡하였고, 시간도 하루가 더 걸렸으며, 세심한 실험상의 주의가 필요하였다. 결 론 : mPCR-CSGE 선별 후 염기서열 분석법은 85.2%의 높은 돌연변이 선별력을 보이고, 직접 염기서열 분석법의 57.7%의 비용만 소모하였으나, 실험과정에 세한 주의가 요구되었으며, 노동집약적이고, 실험 시간도 하루가 더 소요되었다.

• 분석과학
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• 제32권4호
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• pp.155-162
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• 2019

Analysis techniques using DNA profiling are widely used in various fields including forensic science and new technologies such as the Direct PCR amplification method are being developed continuously in order to acquire the DNA profiles efficiently. However, it has a limits such as non-specific amplification according to the quality of crime scene evidence samples. Especially, split peaks caused by excessive DNA samples are one of the important factors that could cause the debate to allow researchers to interpret the DNA profile results. In this study, we confirmed the occurrence rate of split peaks in each STR (short tandem repeats) locus of the $GlobalFiler^{TM}$ kit and investigated the possibility of improving the split peaks using several PCR additives such as DMSO (dimethylsulfoxide), $MgCl_2$, Betaine and Tween-20. As a result, we could make three groups according to the occurrence rate of split peaks in Direct PCR and it was confirmed that the ratio of split peaks could be reduced by DMSO (87.4 %), $MgCl_2$ (84.5 %) and Betaine (86.1 %), respectively. These results indicate that PCR additives such as DMSO, $MgCl_2$ and Betaine can be improve the split peaks in Direct PCR and thereby facilitate subsequently a successful DNA profile results.

• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• 한국균학회지
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• 제49권4호
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• pp.455-467
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• 2021

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH₄)₂SO₄ after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

• 대한화학회지
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• 제47권2호
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• pp.127-132
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• 2003

소의 타일레리아병 진단을 위해 DNA 추출과정 없이 전혈에서 바로 중합효소연쇄반응(PCR)을 통하여 T. buffeli(buffeli/orientalis/sergenti)의 16S rRNA 유전자 단편을 증폭시킨 뒤, 증폭된 DNA를 전기영동법으로 분석하는 방법을 개발하였다. 특이유전자 단편의 증폭을 위해 formamide를 사용하여 혈액세포를 용해시켰으며, 단백질의 응고를 줄이기 위해 낮은 반응온도를 사용하는 FoLT(Formamide Low Temp.) PCR법을 이용하였다. 전혈 100-200 nL를 바로 PCR 증폭에 사용하였으며, PCR 산물(816-bp DNA)은 전기영동법으로 분석하였다. 본 결과는 T. buffeli에 감염된 소의 혈액으로부터 정제된 DNA를 사용하여 얻은 실험결과와 잘 일치하였다.