가축사료 및 그 원료에 대한 DNA 추출방법의 효율 비교

신공식,우희종,임명호,이진형,친 양,박순기,권택윤,조현석 한국국제농업개발학회 2016 韓國國際農業開發學會誌 Vol.28 No.2

Recently, in Korea various kinds of genetically modified (GM) crops have been imported and used as a raw material to manufacture foods and livestock feeds, but the different social concerns about the benefits and the potential risks of GM crops are being shown with a different reaction from the public. Thus a persistent management is required for the safe utilization of genetically modified organism (GMO). PCR analysis of transgene into crop is generally performed for the efficient post management of GMOs. The most important prerequisite for the application of nucleic acid detections is to decide the effective DNA-extraction methods. Particularly, in the case of processed feeds, the nucleic acids of which may be damaged by heating, high pressure, pH treatments, fermentation, etc. in processing, DNA must be extracted with high sensitivity from the samples to perform the PCR successfully. In this study, seven of DNA-extraction methods used commercially and non-commercially were compared with respect to the yields and quality of DNA extracted from livestock feeds and those crop materials. Amounts of genomic DNA obtained from the extraction methods varies according to feed configurations and crop materials. The DNA yield and uniformity of samples extracted with PG, CTAB, and QF method is greater than that obtained from other extraction methods. In the DNA integrity of the selected extraction methods, PCR analysis showed distinct amplifications and similar patterns in detecting crop endogenous genes and GMO genes. These results would be applicable for the selection of an adequate DNA-extraction method in extracting processed feeds and/or crop materials.

유해로부터의 DNA 추출법에 대한 해외 동향 분석

김유희,정규식,정주연 경찰대학 경찰대학 2024 경찰학연구 Vol.24 No.1

대량 재난 사건이나 범죄 사건이 발생하였을 때, 유해의 뼈나 치아 등 경조직이 유일한 DNA 분석 시료가 되기도 한다. 하지만 경조직은 여전히 DNA 확보가 매우 어려운 시료 중 하나이다. 그 이유는 연조직이나 체액에 비하여 DNA의 양이 적고, 시신이 노출된 환경조건(습도, 온도, pH 및 토양 미생물 등)과 사후 보존 상태에 따라 유해의 분해 정도와 PCR 억제제 포함 정도가 다르며, 칼슘 제거 등 전처리 과정이 요구되기 때문이다. 그러므로 경조직에서 DNA 추출의 효율을 높이기 위해서는 시료의 상태에 따라 최적의 DNA 추출법을 선택하는 것이 중요하다. 본 논문은 DNA 프로파일링의 성공 확률을 높이는데 효과적인 DNA 추출법을 찾기 위하여 2013년에서 2023년까지의 문헌을 기준으로 DNA 추출법 및 전처리 방법에 대한 통계적 분석을 수행하였고, 법의학적 시료와 고고학적 시료를 나누어 제시하였으며, 이와 함께 익사체 및 소사체와 같은 손상도가 높은 시료에서의 DNA 추출 연구 동향을 조사하였다. 본 논문은 DNA 분석이 어려운 법과학 시료에서 최적의 DNA 추출법을 정립하는 데 도움이 될 것으로 사료된다. At the occurrence of a mass disaster or crime, hard tissues such as bones or teeth of the skeletonized remains may be the only available source for DNA analysis. However, recovering DNA from these samples is challenging, owing to the limited quantity of DNA available when compared to samples such as soft tissues or body fluids. Moreover, the degree of decomposition and presence of PCR inhibitors in the sample varies depending on the environmental (humidity, temperature, pH, and soil-derived microorganisms) and preservation conditions. The samples also generally require complex pre-treatment processes including decalcification. To enhance the efficiency of DNA extraction from hard tissues, it is important to select an optimal DNA extraction method based on the condition of the samples. Herein, a statistical analysis of international trends of DNA extraction methods was performed, categorizing them based on literature published between 2013 and 2023. The classification highlighted methods that were applicable to forensic and archaeological samples. Moreover, this study included DNA extraction methods specifically for highly damaged samples, such as drowned bodies and burnt remains. Thus, this study is expected to be beneficial in establishing an optimal DNA extraction method for challenging forensic samples such as skeletal samples.

Direct Extraction of DNA from Soil for Amplification of 16S rRNA Gene Sequences by Polymerase Chain Reaction

Cho, Jae-Chang,Lee, Dong-Hun,Cheol, Cho-Young,Cho, Jang-Cheon,Kim, Sang-Jong The Microbiological Society of Korea 1996 The journal of microbiology Vol.34 No.3

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

고대 유전자에 대한 두 종류의 DNA 분리 방법의 비교 연구: 실리카 현탁액 방법 및 초원심분리 농축 방법

Lee, Eun-jung,Maixner, Frank,Zink, Albert 한국분석과학회 2018 분석과학 Vol.31 No.2

This study compared two methods for preparing ancient DNA (aDNA) for the construction of successful shotgun libraries that may be applied to massive parallel sequencing. For the comparative analysis, the DNA of prehistoric rib samples from Hungary was extracted using either a manually prepared silica suspension or the Amicon Ultracel-15 10K ultracentrifugal device (Millipore). After the extraction of the same amount of bone powder (about 150 mg) from three samples by each method, the amount of extracted double-stranded DNA and the subsequent degree of construction of the shotgun library were analyzed. The Amicon device method was rapid and easier to perform and resulted in an approximately 11-fold higher DNA recovery than that obtained using the silica suspension. The shotgun library constructed using DNA templates prepared by the Amicon device was more successful than that constructed from templates isolated using the silica suspension. The comparative study of these two aDNA extraction methods showed that the Amicon device has the advantages of saving time, process simplicity, and high efficiency. 이 연구에서는 대규모 병렬형 염기서열 분석 (massively parallel sequencing)에 적용할 샷건 라이브러리 (shotgun library)를 성공적으로 제작하기 위해 두 가지 유형의 고대 DNA (ancient DNA, aDNA) 분리 방법을 비교하였다. 헝가리 선사 시대 늑골 뼈 시료로 실리카 현탁액을 이용한 추출법과 Amicon Ultracel-15 10K 초원심 분리 장치(Millipore)를 이용한 추출법을 비교하였다. 약 150 mg의 뼛가루에서 각각의 방법으로 3 회 반복 추출한 후 이중 가닥 DNA (double stranded DNA, ds DNA)의 양을 측정하였다. 초원심분리 농축 방법은 실리카 현탁액을 사용하는 것보다 더 빠르고, 더 쉬운 공정이며 약 11 배 높은 DNA 회수율을 나타냈다. 또한 초원심 분리 장치로 획득한 DNA 주형은 실리카 현탁액으로 획득한 것보다 샷건 라이브러리가 훨씬 성공적으로 만들어졌다. 두 종류의 aDNA 추출 방법을 비교한 본 연구는 Amicon 장치를 사용하는 분리법이 시간의 절약, 단순한 프로세스 및 높은 효율 등의 장점을 지니고 있음을 보여주었다.

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil

Mathur Nadarajan Kathiravan,김근호,류재원,김평일,이철원,김시욱 한국미생물학회 2015 The journal of microbiology Vol.53 No.11

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 μg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A260/A230 and A260/A280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP–purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

Necessity of purification during bacterial DNA extraction with environmental soils

Hyun Jeong Lim,Jung-Hyun Choi,Ahjeong Son 환경독성보건학회 2017 환경독성보건학회지 Vol.32 No.-

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.

Evaluation and modification of alkaline lysis plasmid preparation method from Lactobacillus spp.

Lee, Deog-Yong,Seo, Yeon-Soo,Kang, Sang-Gyun,Yoo, Han Sang The Korean Society of Veterinary Science 2007 大韓獸醫學會誌 Vol.47 No.2

Lactic acid bacteria (LAB) has been regarded as a useful microorganism and tried to manipulate plasmid DNA for increasing the usefulness. Although several methods have been developed to isolate plasmid DNA from Escherichia coli (E. coli), these methods were not sufficient to apply to LAB with exception of O'Sullivan's lysis method. So, we evaluated plasmid DNA extraction from LAB using general E. coli preparation methods and tried to improve the extraction yield and DNA purity by modifying O'Sullivan's alkaline lysis method. To improve the extraction yield, salt and carrier were added to precipitant and those were incubated at $-70{^{\circ}C}$. Only incubation at $-70{^{\circ}C}$ was the effective method of those modifications. Purity of plasmid DNA was improved by two times of each centrifugation and phenol/chloroform extraction. However, DNA was damaged by twice extraction with phenol/chloroform. Also, exclusion of ethidium bromide showed negative effect to purity. Additionally, it was recommended that improvement of the extraction yield may be due to centrifugation at high speed for more time and to dissolving complete DNA pellet before addition of 7.5 M ammonium acetate. Extraction using this modification produced higher quality of plasmid DNA.

식물로부터 유전물질을 추출하는 방법에 관한 연구 - 브로콜리, 키위 및 바나나를 이용하여 -

박기석 ( Ki-seok Park ),정극일 ( Keuk Il Jung ),전상학 ( Sang-hak Jeon ) 한국현장과학교육학회 2007 현장과학교육 Vol.1 No.2

DNA는 1897년 미셔(Miescher)에 의해서 처음으로 발견되었지만, DNA가 유전물질이라는 것은 1928년 그리피스(Griffith)의 형질전환실험, 1944년 에이베리(Avery) 등의 형질전환 물질에 대한 생화학적인 연구, 그리고 1952년 허쉬(Hershey)와 체이스(Chase)의 방사성 동위원소를 이용한 바이러스의 유전연구를 통해서 규명되었다. 1953년에 왓슨(Watson)과 크릭(Crick)에 의한 DNA 이중나선 구조의 규명은 분자생물학의 발달과 DNA 재조합 시대를 여는 계기가 되었다. DNA 기술은 사람을 포함하는 많은 생물의 유전체연구의 완성을 가져왔고, 형질전환 동식물의 생산, 사람의 질병 예방과 치료, DNA 지문을 이용한 과학적 수사, 친자 확인 등 우리 실생활과 매우 밀접한 연관성을 갖게 되었다. 따라서 DNA를 교육 현장에서 학생들이 직접 뽑아서 눈으로 확인하는 것은 매우 중요한 의미가 있다고 생각된다. 본 연구에서는 구입이 쉽고 저렴한 재료인 브로콜리, 키위 및 바나나를 이용하여 DNA를 추출하는 과정을 연구하였다. 전통적인 복잡한 방법 대신에 세제와 에탄올만으로도 눈으로 확인할 수 있을 만큼 많은 양의 DNA를 나무젓가락으로 감아올릴 수 있었다. 추출된 DNA의 양은 키위에서 실험재료의 단위 무게 당 가장 많은 양의 DNA가 추출되지만 추출액의 색깔이 DNA 덩어리와 비슷하여 관찰이 용이하지 않았으며, 반면에 상대적으로 적은 양이 추출되는 브로콜리는 색깔의 대비로 DNA가 뚜렷하게 관찰되었다. 추출된 DNA를 페놀로 처리하였을 때 단백질이 검출되었기 때문에 추출된 DNA는 단백질도 일부 포함하고 있는 것으로 생각된다. 추출된 DNA는 발암성 물질인 에씨디옴 브로마이드(Ethidium Bromide, EtBr)뿐만 아니라 교육현장에서 이용되는 염색약인 메틸렌블루 용액 및 아세트올세인 용액으로도 염색되었다. 따라서 본 연구 결과는 DNA 추출 및 확인 실험이 중등교육 현장에서도 탐구활동으로 활용가능하며, 향후 새로운 교과과정 개편에서 분자생물학 실험으로 이용될 수 있을 것으로 생각된다. DNA was first discovered by Miescher in 1897, but it was later recognized as genetic material from transformation experiment by F. Griffith in 1928, from biochemical analysis on transformation material by O. Avery in 1944, and finally from study of virus genetics using isotope by A.D. Hershey and M. Chase. J. Watson and F. Crick discovered DNA double helix structure, which opened the era of molecular biology and the DNA recombinant technology. DNA technology has been widely used in producing genetically transformed plant and animals, prevention and treatment of human disease, forensic medicine, paternity test, etc. In this study, we studied the way to extract the genetic materials from broccoli, kiwi and banana. Instead of complex and dangerous traditional way, DNA was extracted just with detergent and ethanol, and then wound up with wood chopsticks. Kiwi gave the most plenty of amount of DNA, but DNA was not able to be easily recognized due to background color. On the while, although brocoli gave the least amount of DNA, but the genetic materals were easily recognized from the green background. Phenol treatment showed that the extracted DNA actually contained protein as well. The extracted DNA was stained with ethidium bromide(EtBr) as well as methylene blue and aceto-orcein dyes. The later two dyes are not harmful. Taken together, we suggest that DNA extraction experiment can be carried out in the secondary school and included in the next version of science textbook.

Comparison of Two Methods to Extract DNA from Formalin-Fixed, Paraffin-Embedded Tissues and their Impact on EGFR Mutation Detection in Non-small Cell Lung Carcinoma

Hu, Yu-Chang,Zhang, Qian,Huang, Yan-Hua,Liu, Yu-Fei,Chen, Hong-Lei Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.6

Objective: Molecular pathology tests are often carried for clinicopathological diagnosis and pathologists have established large collections of formalin-fixed, paraffin-embedded tissue (FFPE) banks. However, extraction of DNA from FFPE is a laborious and challenging for researchers in clinical laboratories. The aim of this study was to compare two widely used DNA extraction methods: using a QIAamp DNA FFPE kit from Qiagen and a Cobas Sample Preparation Kit from Roche, and evaluated the effect of the DNA quality on molecular diagnostics. Methods: DNA from FFPE non-small cell lung carcinoma tissues including biopsy and surgical specimens was extracted with both QIAamp DNA FFPE and Cobas Sample Preparation Kits and EGFR mutations of non-small cell lung carcinomas were detected by real-time quantitative PCR using the extracted DNA. Results and Conclusion: Our results showed that DNA extracted by QIAamp and Cobas methods were both suitable to detect downstream EGFR mutation in surgical specimens. Howover, Cobas method could yield more DNA from biopsy specimens, and gain much better EGFR mutation results.

Rapid Detection of Pathogens Associated with Dental Caries and Periodontitis by PCR Using a Modified DNA Extraction Method

김재환,김미아,이대우,백병주,양연미,김재곤 大韓小兒齒科學會 2014 大韓小兒齒科學會誌 Vol.41 No.4

DNA extraction is a prerequisite for the identification of pathogens in clinical samples. Commercial DNA extractionkits generally involve time-consuming and laborious multi-step procedures. In the present study, our modified DNA isolation method for saliva samples allows for the quick detection ofpathogens associated with dental caries or periodontitis by PCR within 1 h. To release DNA from the bacteria, 1min of boiling was adequate, and the resulting isolated DNA can be used many times and is suitable for longterm storage of at least 13 months at 4℃, and even longer at -20℃. In conclusion, our modified DNA extraction method is simple, rapid, and cost-effective, and suitable forpreparing DNA from clinical samples for PCR for the rapid detection of oral pathogens from saliva. 구강 병원체의 검출 방법은 여러 가지가 있지만 그 중 PCR을 이용한 검출이 확실하고 빠른 방법으로 알려져 있다. PCR을위한 많은 DNA 추출법이 사용되고 있으나 상업적인 DNA 추출 kit들은 일반적으로 가격이 비싸고 절차가 여러 단계로 되어있으며, 그 외의 방법은 페놀과 클로로포름과 같은 유해한 화학물질을 써야하는 등의 단점이 있다. 이 연구에서 NaOH 용액을 이용한 개선된 DNA 추출 방법은 치아우식증, 치주염과 관련된 병원체를 빠르고 간단하며 비용-효율적으로 검출하였다. 세균으로부터 DNA를 추출하기 위한 boiling은 기존의 10분이 아닌 1분으로 충분하였고 4℃에서 최소 13개월 이상 DNA의 보관이 가능하였으며 sonication 유무에 따른 차이는 없었다. 따라서, 이 방법은 상업적인 kit나 유해한 화학물질을 쓰지 않고서도 타액 표본으로부터 직접적으로 빠른 시간 내에 DNA를 추출하여 병원체의 유무 결과를 확인하는데 매우 적합할 것으로 생각한다.