• 한국식물병리학회지
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• 제14권6호
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• pp.636-641
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• 1998

Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

• 제어로봇시스템학회:학술대회논문집
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• 제어로봇시스템학회 2004년도 ICCAS
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• pp.196-201
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• 2004

In biometric and biomedical applications, a special transporting mechanism must be designed for the ${\mu}$TAS (micro total analysis system) to move samples and reagents through the microchannels that connect the unit procedure components in the system. An important issue for this miniaturization and integration is microfluid management technique, i.e., microfluid transportation, metering, and mixing. In view of this, this study presents an optimal fuzzy sliding-mode control (OFSMC) design based on the 8051 microprocessor and implementation of a complete microfluidic manipulated system implementation of biochip system with a pneumatic pumping actuator, a feedback-signal photodiodes and flowmeter. The new microfluid management technique successfully improved the efficiency of molecular biology reaction by increasing the velocity of the target nucleic acid molecules, which increases the effective collision into the probe molecules as the target molecules flow back and forth. Therefore, this hybridization chip was able to increase hybridization signal 6-fold and reduce non-specific target-probe binding and background noises within 30 minutes, as compared to conventional hybridization methods, which may take from 4 hours to overnight. In addition, the new technique was also used in DNA extraction. When serum existed in the fluid, the extraction efficiency of immobilized beads with solution flowing back and forth was 88-fold higher than that of free-beads.

• 한국산학기술학회논문지
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• 제9권2호
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• pp.550-555
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• 2008

세포벽 분해 효소와 단백질 분해 효소를 이용하여 스피루리나 추출물을 효율적으로 생산할 수 있는 방법을 조사하였다. 특히 단백질 분해 효소의 처리 조건을 최적화하여 효율적인 스피루리나 추출물의 제조공정을 제시하였다. 세포벽 분해 효소인 Tunicase는 스피루리나의 중량 기준으로 2%를 사용하였고 2시간 동안 반응시켰다. 상업용 단백질 분해 효소로는 Alcalase를 사용하였다. 이때, Alcalase의 최적 사용량은 1%이었으며, 효소 반응 시간은 2시간이 적절하였다. Tunicase와 Alcalase의 처리 방법에서 Tunicase를 먼저 사용한 후 Alcalase를 사용하는 순차적으로 처리하는 것이 고형분 회수율과 spirulina extraction (SE) index를 최대로 증가시킬 수 있는 효과적인 방법이었다. 두 효소를 순차적으로 반응시키면 단순 열수 추출보다 고형분 회수율은 약 56%($45.2%\;{\rightarrow}\;70.7%$), SE index는 약 100%($11.4%\;{\rightarrow}\;22.8%$) 증가하였다.

• Parasites, Hosts and Diseases
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• 제52권3호
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• pp.263-271
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• 2014

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. $QIAamp^{(R)}$ DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume ($50-100{\mu}l$) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ${\approx}2$ oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

• 생명과학회지
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• 제9권1호
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• pp.15-21
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• 1999

PCR 기법을 응용한 RAPD 방법은 대상 생물의 유전정보를 전혀 모르는 상태에도 arbitrary primer를 이용하여 증폭함으로써 생물 종간의 유전적 표식자를 확인할 수 있는 간편하고 유익한 유전분석 방법이다. 이같은 RAPD 방법을 이용하여 해조류 방사무늬 김, 미역, 구멍갈파래에 대하여 DNA를 추출하지 않고 직접 생 엽체 및 생 사상체, 생 배우체를 PCR template로 사용하여 PCR product를 생산하였다. 그러나 specific primer를 이용한 nulear r DNA의 internal trancribed spacer (ITS) 부위는 생성물을 만들지 못하였다. 간편한 LiCl 방법에 의하여 추출된 DNA를 사용하였을 때는 IST 및 RAPD 모두 PCR product를 생산하였다. 방사무늬 김의 엽체 (haploid)와 사상체 (dip-loid)로 부터의 ITS 생성물은 동일하였으나 RAPD 생성물은 사용한 arbitrary primer에 따라 36-50$\%$의 다른 band가 만들어졌다. 또한 직접 생 조직을 사용하였을때와 추출 DNA를 사용하였을 때도 53-57$\%$의 다른 band가 만들어 줬다. 그러므로 RAPD 방법으로 유전분석 실험에는 동일한 ploidy의 조직을 template로서 사용하는 것이 중요하다.

• Applied Biological Chemistry
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• 제50권4호
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• pp.304-307
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• 2007

세포벽 분해 효소와 단백질 분해 효소를 이용하여 스피루리나 추출물을 효율적으로 생산할 수 있는 방법을 조사하였다. 세포벽 분해 효소인 Tunicase의 사용 농도는 2%가 적당하였다. 고형분 회수율과 핵산 관련 성분의 함량을 나타내는 spiruina extraction(SE) index를 기준으로 상업용 단백질 분해 효소를 선별하였다. 일곱 종류의 효소를 조사한 결과, Esperase가 가장 우수하였으며, 최적 사용량은 2%이었다. Tunicase와 Esperase를 순차전으로 반응시키거나 동시에 반응시켜도 고형분 회수율과 SE index는 매우 유사하였으며 동시에 사용하는 것이 반응 시간을 단축시킬 수 있었다. 두 효소를 동시에 반응시키면 단순 열수 추출보다 고형분 회수율은 약 45%$(45.2%{\rightarrow}65.3%)$, SE index는 약 75%$(11.4{\rightarrow}20.0)$ 증가하였다.

• Journal of Nutrition and Health
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• 제1권2호
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• pp.107-115
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• 1968

Number of aspects, not only nutritional but social as well as political involved in human starvation pose nowadays global problems. In order to help establish the minimum nutritional requirements in the daily life of a man and to free people as well from either undernourishment, malnutrition or even starvation many workers have devoted themselves so far on the research programs to know what and how number of metabolic events take place in animals in vivo. It is the purpose of the present paper to examine in effect to what extent both of the protein and nucleic acids (DNA & RNA) together with an enzyme, guanine deaminase, which converts guanine into xanthine and in turn ends up to uric acid as an end product, undergo changes, quantitatively during acute starvation, using the mouse as an experimental animal. The mouse was strictly inhibited from taking foods except drinking water ad libitum and was sacriflced 24, 48, and 72 hours following starvation thus acutely induced. The animals consisted of two experimental groups, one control and another starvation groups, each being consisted of 6-24 mice of whose body weights ranged in the vicinity of 10 g. The animals were sacriflced by a blow on the head, followed by immediate excision of their livers into ice-cold distilled water, washing adherent blood and other contaminant tissues. The liver was minced foramin, by an all-glass homogenizer immersing it in an ice-bath, followed by subsequent fractionatin of the homogenate (10% W/V in 0.25M sucrose solution made up with 0.05M phosphate buffer of pH 7.4). For the liver protein and guanine deaminase assay, the 10% homogenate was centrifuged at 600 x g for 10 minutes to eliminate the nuclear fraction; and for the estimation of DNA and RNA, the homogenate was prepared by the addition of 10% trichloroacetic acid in order to free the homogenate from the acid-soluble fraction, the remaining residue being delipidate by the addition of alcohol and dried in vacuo for later KOH (IN) hydrolysis. The changes in body and liver wegihts during acute starvation were checked gravimetrically. Protein contents in the liver were monitored by the method of Lowry et al; and guanine deaminase activities were followed by the assay of liberated ammonia from the substrate utilizing the Caraway's colorimetry. The extraction of both DNA and RNA was performed by the Schmidt-Thannhauser's method, which was followed by Marmur's method of purification for DNA and by Chargaff's method of purification for RNA. The determinations of both DNA and RNA were carried out by the diphenylamine reaction for the former and by the orcinol reaction for the latter. The following resume was the results of the present work. 1. It was observed that the body as well as liver weights fall abruptly during starvation, and that the loss of body weight showed no statistical correlation with the decreases in the content of liver protein. 2. The content of liver protein and activity of liver guanine deaminase activity as well decline dramatically, and the specific activities of the enzyme (activity/protein), however, decreased gradually as starvation proceeded. 3. Both of the nucleic acids, DNA and RNA, showed decrements in the liver of mouse during acute starvation; the latter, however, being more striking in the decline as compared to the former. 4. The decreases in the liver protein content as resulted from the acute starvation had no statistically significant correlation with the decrements of DNA in the same tissue, but had regressed with a significant statistical correlation with the fall of RNA in the tissue. 5. The decrease in the activity of guanine deaminase in the liver of mouse during acute starvation was functionally more proportional to the decrease in RNA than DNA, and moreover correlated with the changes in the content of the liver protein. 6. The possible mechanisms involved during in this acute starvation as bring the decreases in the contents of DNA, protein, and guanine deaminase were discussed briefly.

• 한국식품영양과학회지
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• 제21권5호
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• pp.573-579
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• 1992

숙시닐화가 균체 단백질(Aspergillus fumigatus)의 여러가지 기능적 특성에 미치는 영향을 검토하였다. 균체 단백질은 succinic anhydride 2.5와 10% 첨가에 20.7 과 85.3%가 숙시닐화되었다. 숙시닐화된 균체 단백질은 흡광도, 핵산 및 탄수화물의 양은 감소하였으나 단백질소, 단백질 추출율은 증가되었다. 숙시닐화된 균체 단백질의 질소용해도는 milk casein과 soy flour 보다 32와 51% 증가되었다. 유화활성도와 유화 안정성은 균체 단백질의 숙시닐화 비율에 따라서 증가 되었으며, 80% 숙시닐화된 균체 단백질은 약 8.4배 증가율을 나타내어 숙시닐화를 증가시킴에 따라 유화활성도와 유화안정성이 증가 되었다. 숙시닐화 시킨 균체 단백질은 숙시닐화 시키지 않는 균체 단백질, milk casein 및 soy flour에 비하여 향상된 기능적 특성을 나타내었다.

• ALGAE
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• 제21권2호
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• pp.261-266
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• 2006

The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.

• 식물병연구
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• 제12권2호
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• pp.139-143
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• 2006

VC/RT-PCR은 바이러스의 단백질과 PCR 튜브의 재질적 특성을 이용하여 바이러스 이병주의 즙액으로부터 핵산을 지니고 있는 바이러스를 polypropylene에 부착시키고 그 외 PCR 활성을 저해하는 식물 즙액은 PBST로 제거해 주므로써 정확하고 깨끗한 바이러스 진단이 가능하다. 이 방법은 바이러스에 대한 항혈청이 전혀 필요 없으며 포장 시료를 직접 이용해 30 분 이내에 바이러스 핵산을 획득할 수 있어 바이러스 진단이 빠르고 간편하며 경제적이고 감도 높은 실용적인 방법이다. TSWV 에 대한 VC/RT-PCR을 위해 다양한 마쇄 완충액을 시험한 결과 0.5%의 Sodium sulphite를 포함한 0.01M Potassium phosphate (pH 7.0)가 가장 안정적이었으며 VC/RT-PCR을 이용하여 TSWV에 대한 최적의 처리 과정을 확립하였다. TSWV에 최적인 완충액에 마쇄한 바이러스 감염 잎의 조즙액을 이용한 한계 희석 농도는 $10^{-5}$으로 높은 감도를 보여 낮은 농도의 바이러스를 보유한 기주로부터 정확하 게 바이러스를 감지해 낼 수 있게 되었다. 두가지 이상의 바이러스에 감염된 기주로부터 두 가지 이상의 프라이머를 이용해 바이러스를 감지해 내는 다중 진단을 위한 실험 중 식물체의 종류와 바이러스의 Primer의 종류가 진단결과의 정확도에 큰 영향을 미치며 이러한 결과는 식물체 즙액을 바로 이용하는 VC/RT-PCR 방법은 물론 식물체로부터 Total RNA를 따로 분리하여 RT-PCR을 이용한 결과에서도 동일하게 나타났다. 따라서 편리하고 실용 적인 VC/RT-PCR법의 정확성을 최대화시키기 위해서는 다중 진단을 저해하는 식물체적 원인과 Primer 사이의 간섭 현상에 대한 연구가 더 진행될 것이며 이러한 원인을 충분히 제거해 줄 수 있는 마쇄 완충액을 개발하고 프라이머 제작을 더욱 신중히 해야 할 것이다.