• 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.

• The Plant Pathology Journal
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• 제20권2호
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Advances in Traditional Medicine
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• 제6권2호
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• pp.134-143
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• 2006

The capacities of bacterial DNA, extracted from Salmonella typhimurium, and lipopolysaccharide (LPS), extracted from Salmonella minnesota, to activate mouse peritoneal macrophages in vitro were compared. Activation was assessed by estimating e levels of 3 cytokines, IL-10, IL-12, and $IL-1{\beta}$, at time intervals of 3, 6, 9, and 24 h after addition of LPS and/or DNA to macrophage cultures. Cytokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA levels were estimated based on band intensity in cultured cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Results obtained demonstrated the ability of DNA and LPS to elicit increased production of all 3 cytokines as compared to controls. In the amount tested, LPS appeared to be a more potent inducer of IL-12, and $IL-1{\beta}$, whereas DNA induced higher levels of IL-10. DNA and LPS, used in combination, exhibited neither an additive nor a synergistic effect. Rather, an antagonist effect appeared to occur. RT-PCR results correlated well with ELISA.

• Asian-Australasian Journal of Animal Sciences
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• 제17권9호
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• pp.1223-1226
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• 2004

Previously candidate gene approach revealed estrogen receptor (ESR) locus was associated with increased litter size. In this study, PvuII polymorphisms of ESR gene was detected by PCR-RFLP, and ESR locus was evaluated for its association with reproductive tracts components in the Large $White{\times}Meishan$ ($LW{\times}M$) F2 offspring. Ninety seven gilts with reproductive tracts components records and 136 offspring with performance traits records were genotyped and the results were used to estimate allele substitution effects. The results showed that two alleles (A and B) were identified, and 121 bp fragments were observed for the AA genotype and 65 bp and 56 bp fragments for the BB genotype; the length of uterine body (LUB) of BB gilts were significantly shorter than AA gilts', the additive effect was -1.762 cm; the uterine weight (UW) of AB gilts were significantly lighter than AA gilts' with the additive effect -18.058 g; no significant associations of ESR alleles with ovulation rate (OR), length of uterine horn (LUH), length of uterine cervix (LUC), weight of two ovaries (OW), volume of uterine lumen (VUL), length of oviduct (LO) were observed. BB genotypes gilts need significantly less days to 100 kg ($D_{100kg}$) than AA genotypes (p<0.01), the additive effect was per copy of B allele. Allele B is also favorable for average daily gain (ADG), with additive effect 0.015 kg/d (p<0.05). There was no difference between genotypes for backfat thickness at the 13th rib (SF13), loin meat height (ELMH), and loin meat percentage was estimated (ELMP), individual birth weight (IBW) and teat number (TN).

• 대한소아치과학회지
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• 제42권4호
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• pp.312-318
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• 2015

본 연구의 목적은 치과분야에서 줄기세포 공급원으로써 과잉치의 활용 가능성을 알아보고자 Real Time Quantitative Reverse Transcription Polymerase Chain Reaction (Real Time qRT-PCR)법을 이용하여 발치된 과잉치 치수 유래 줄기세포 (supernumerary dental pulp stem cells, sDPSCs)로부터 상아모세포로의 분화여부를 관찰해 보는 것이었다. 이를 위해 상아모세포의 대표적인 발현인자로 알려진 alkaline phosphatase (ALP), osteocalcin (OC), osteonectin (ON), dentin matrix acidic phosphoprotein 1 (DMP-1) 그리고 dentin sialophosphoprotein(DSPP)의 발현을 분화제 처리 후 0일, 8일 그리고 14일째에 각각 Real Time qRT-PCR 법을 통해 상대적인 mRNA의 발현 양을 비교하여 변화 양상을 알아보았다. 또한 Alizarin-red solution 의 염색을 통해 sDPSCs가 분화제 처리 7일, 14일, 21일 그리고 28일째에 석회화 결절을 형성하는 정도를 시각적으로 확인해 보았다. Real Time qRT-PCR 결과 분화제 처리 8일째에 가장 높은 발현 양을 보이다가 14일째에 감소하는 추세를 나타내었으며, Alizarin-red solution 염색 결과는 7일째부터 흐리게 나타나다가 14일째에는 배지 전반에 걸쳐 진하게 염색되는 소견을 보였다. 따라서, sDPSCs를 이용한 연구에서 Real Time qRT-PCR법을 위한 분화제의 처리 기간은 8일 정도가 적절하며, Alizarin-red solution 염색은 14일이 적당한 것으로 사료된다.

• 대한소아치과학회지
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• 제48권3호
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• pp.291-301
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• 2021

이 연구의 목적은 발거 된 매복 상악 과잉치에서 얻은 치수유래 줄기세포의 초기 계대와 후기 계대의 상아질모세포 유전자의 특성을 알아보는 것이다. 전신 의과 병력이 없는 6 - 9세 사이의 남녀아이 12명에게서 서면동의를 얻고 모두 상악에 위치한 과잉치를 발거하여 당일 발거된 과잉치의 치수세포를 채취하였다. 12개의 세포를 각각 3계대와 10계대에서 골형성 유도 분화제를 처리한 군과 처리하지 않은 군을 나누어 실시간 중합효소 연쇄반응을 시행하여 상아질모세포의 특성을 알아보았다. 사용된 유전자는 osteonectin (ONT), alkaline phosphatase (ALP), osteocalcin (OCN), dentin matrix protein 1 (DMP-1), 그리고 dentin sialophosphoprotein (DSPP)였다. 유전자 발현양은, 분화제를 처리하지 않은 군 3계대에서는 ONT, ALP, OCN, DMP-1, DSPP순서로 많이 발현하였다. 분화제를 처리하지 않은 군 10계대에서는 ONT, DMP-1, OCN, ALP, DSPP순으로 ONT, OCN, DSPP의 순서에는 변화가 없지만 ALP, DMP-1의 순서는 서로 바뀌었다. 이상의 결과를 종합해 볼 때, ALP와 DMP-1은 3계대와 10계대 세포의 분화를 위한 중요한 표지자로 사용될 수 있다. 과잉치 치수유래 줄기세포는 상아질모세포의 특성을 가지며, 또한 과잉치가 어린 나이에 발거되고 10계대까지 소요되는 시간이 적게 걸린다는 것을 고려하면, 과잉치는 치아 유래 줄기세포의 공여부로서 훌륭한 활용가능성이 있음을 확인하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.2807-2811
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• 2015

Scorpion venom peptides recently have attracted attention as alternative chemotherapeutic agents that may overcome the limitations of current drugs, providing specific cytotoxicity for cancer cells with an ability to bypass multidrug-resistance mechanisms, additive effects in combination therapy and safety. In the present study, BmKn-2 scorpion venom peptide and its derivatives were chosen for assessment of anticancer activities. BmKn-2 was identified as the most effective against human oral squamous cells carcinoma cell line (HSC-4) by screening assays with an $IC_{50}$ value of $29{\mu}g/ml$. The BmKn-2 peptide killed HSC-4 cells through induction of apoptosis, as confirmed by phase contrast microscopy and RT-PCR techniques. Typical morphological features of apoptosis including cell shrinkage and rounding characteristics were observed in treated HSC-4 cells. The results were further confirmed by increased expression of pro-apoptotic genes such as caspase-3, -7, and -9 but decrease mRNA level of anti-apoptotic BCL-2 in BmKn-2 treated cells, as determined by RT-PCR assay. In summary, the BmKn-2 scorpion venom peptide demonstrates specific membrane binding, growth inhibition and apoptogenic activity against human oral cancer cells.

• Journal of Plant Biotechnology
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• 제45권1호
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• pp.63-70
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• 2018

Brazzein은 열대식물인 P. brazzeana Baillon의 과실에서 분리된 가장 작은 감미단백질로 토착민들의 단맛원료로 사용되어 왔다. Brazzein은 sucrose보다 분자량 기준으로 500 ~ 2000배, 몰 기준으로 9500배 당도가 높아 감미료로써 매우 높은 평가를 받고 있다. 그러나 이 감미단백질은 재배가 어렵고 생산 비용이 높아서 brazzein 단백질의 이용 가능성을 높이기 위한 대체 생산 시스템으로 형질전환 식물체 육성 하고자 하였다. 본 연구에서는 brazzein 관련 유전자를 벼에 도입하기 위하여 식물형질전환용 Ti-plasmid에 $2{\times}CaMV\;35S$ 프로모터에 의해 지배되어 발현하도록 하고, 선발 마커로 bar 유전자가 삽입된 식물발현 벡터를 구축하여 A. tumefaciens EHA105에 형질전환시켜 17개의 재분화 식물체를 육성하였다. 17개 재분화 식물체는 PCR 및 RT-PCR 분석을 통하여 유전자 도입 및 발현을 확인하였으며, TaqMan PCR을 통해 single copy로 도입된 T0 세대 9개체를 선발하였다. 또한 FST 분석을 통하여 도입 유전자가 intergenic으로 삽입된 개체 5개를 선발하였다. 이들 5개체를 이용하여 western blot 분석에 의해 단백질 발현량을 분석한 결과 선발된 모든 개체에서 발현 밴드를 확인하였다. 그 중 brazzein 단백질의 발현량이 높은 개체를 TG11으로 계통화하여 후대 종자를 육성하였다. TG11 계통은 천연 감미료 brazzein을 생산하는 새로운 벼 품종을 개발하기 위한 육종 소재로 활용 가능하다고 시사된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권9호
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• pp.1330-1338
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• 2008

This study was conducted to investigate the effects of Acanthopanax senticosus extract (ASE) as a dietary additive on gut microflora in weaned piglets. A total of sixty pigs were weaned at 21 d of age (BW = $5.64{\pm}0.23kg$) and allocated on the basis of BW and litter to three dietary treatments in a randomized complete block design. The dietary treatments were: control group (basal diet), antibiotics group (basal diet+0.02% colistin), and ASE group (basal diet+0.1% ASE). On d 7, 14 and 28 after consuming the experimental diets, five piglets per group were sacrificed and then the contents from the jejunum, ileum and cecum were collected to determine changes in the microbial community by using a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique and estimating the contents of Lactobacillus and E. coli by in vitro culturing methods. The results showed that the ASE promoted the microflora diversity in the cecum. Enumeration of bacteria in the gut contents showed that the number of Lactobacillus increased (p<0.05), while that of E. coli decreased (p<0.05) when compared with the other 2 groups as the days of age progressed post-weaning. These findings suggested that the ASE, as a substitute for dietary antimicrobial products, could improve the development of the normal gut microflora and suppress bacterial pathogens, and effectively promote a healthy intestinal environment.