• Journal of the Korean Society of Tobacco Science
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• v.37 no.1
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• pp.25-33
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• 2015

Genome walking is a par ticular application for identifying sequences of unknown genomic regions adjacent to a known region. Many genome walking methods based on polymerase chain reaction (PCR) are available. Even if earlier techniques suffer from low reproducibility, inefficiency, and non-specificity, improved strategies have been developed. In this study, we present an alternative strategy: the genomic DNA is digested with restriction enzymes. After cytosine overhangs at 5' ends, the fragments are ligated to linker adaptor s had guanine overhang at 3' ends. Then nested PCR is performed. The improvements in this strategy focus on two points. The first is the C tailing method using Pfu polymerase instead of the A tailing method based on nontemplate-dependent terminal transferase activity of Taq polymerase. Therefore unintended modification of target DNA can be prevented without A tailing error. The second point is the use of C/G-specific ligation had advantage in the ligation efficiency compared with A/T-specific ligation. Therefore, the C-G linker PCR method increases ligation efficiency between digested genomic DNA and adaptor DNA. As a result, the quantity of target DNA to amplify by PCR is enriched. We successfully used G-C linker PCR to retrieve flanking regions bordering the phophinothricin resistance gene in genetically modified tobacco (GMO).

• Korean Journal of Food Science and Technology
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• v.28 no.6
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• pp.1001-1008
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• 1996

Staphylococcal food poisoning is the major cause of bacterial food poisoning occurring in this country. Therefore government regulates commercial foods through Official Dictionary of Food that there should be free of enterotoxigenic Staphylococcus aureus in Korean rice cakes, bread, and a box lunch. Since at least 5 days are required to identify the S. aureus by the official method in the Dictionary it is difficult to prevent the food poisoning and the investigation of the outbreaks. In this report an improved determination method of the S. aureus has been developed using polymerase chain reaction (PCR) technique. Sense and antisense primers for specific amplification of genes encoding staphylococcal enterotoxins were designed and synthesized for the PCR. Rapid chromosomal DNA isolation method was also developed from S. aureus using lysostaphin. The PCR condition was developed as follows. Reaction solution $(50\;{\mu}l)$ consisted of target DNA $2\;{\mu}l$ (about 20ng), 10X buffer $5\;{\mu}l$, primer 100pmole, dNTP (10 mM) $4\;{\mu}l$ and Taq DNA polymerase 2.5 unit in a thin-wall tube. Operation condition of the PCR was 5 min pre-denaturation at $94^{\circ}C$, 15 sec denaturation at $94^{\circ}C$, 15 sec annealing at $50^{\circ}C$, 20 sec extension at $72^{\circ}C$, and 5 min post-extension at $72^{\circ}C$, and 30 cycles of denaturation-annealing- extension. Using the PCR with Perkin Elmer GeneAmp PCR system 2400, types of enterotoxigenic S. aureus could be identified from Ddok or bread in a day.

• Analytical Science and Technology
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• v.12 no.3
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• pp.260-264
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• 1999

A simple and rapid procedure, called FoLT-PCR(Formamide Low Temperature-Polymerase Chain Reaction) was applied to amplifying DNA directly from various forensic biological evidences including human blood, saliva, hair root, or semen without any DNA preparative steps. We added washing step with non-ionic detergent, 1% Triton X-100, and used Taq DNA polymerase instead of Tth DNA polymerase to amplify 3 STR loci and gender allele simultaneouly. Optimal concentration of formamide and annealing temperature were determined empirically to 8%(v/v), and $48^{\circ}C$ respectively. We also compared this method with standard PCR.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.233-234
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• 1994

A general improved procedure for preparation of a cosmid library based on the use of a pBLcosT vectorwas described. The vector was modified to contain 2 tandem Xcml sites and was digested with Xcml to yield 2 terminal 3 T overhangs, capable of ligation with the insert that contains 2 terminal complementary 3 A overhangs. The resultant ligation mixture was packaged and a cosmid library in Escherichia coli was established.

• Parasites, Hosts and Diseases
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• v.34 no.1
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• pp.15-20
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• 1996

Cysts of Entamoebn histoIMtica are still found from humans in Korea, but notall of the cysts are known as pathogenic. The non-pathogenic strain is regarded as a differenL species, E. nispnr. In this study, Korean isolates of conventional E. histolvticn were subjected for the differentiation by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Human stools were screened by routine microscopic examination, and cyst or trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoites were prepared for DNA extraction, and the DNAs were used for PCR with common primers of Pl gene. The PCR products were divested with 3 restriction enzymes and RFLP was observed. Also anti-sense primers containing the cleavage site of each restriction eWe were designed for differentiation only by PCR. The PCR products of Korean isolates 59,512, YS-6, and YS-27 were spliced by Taq I and Xmnl but not byAccl, and the isolates S1, S3, S11, S15, S16, S17, S20, YS- l7, and YS-44 were spliced by Acc I but not by Taq I and Xmn I. These RFLP pattern correlated well with PCR products by the species specific primers. The findings confirm that the Korean isolates 59,512, YS-6, and YS-27 are E. histolwtico and others are E. dispar. In Korea, most of the asymptomatic cyst carriers are infected by E. dispar, not by E. histolytica. Key words: Entcmoebc histolytica, Entcmoebn dispar Korean isolates, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.35-44
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• 1990

Poly (A) + RNA was isolated from mouse submaxillary gland and renin mRNA was isolated by poly (U)-sepharose chromatography and sucrose linear densiW gradient centifugation. And renin mRNA was identified by in vitro translation and immunoprecipitation. In order to construct recombinant plasmid, renin cDNA was synthesized by using reverse transcriptase and inserted into EcoRi site of PUC19. In addition, the cDNA was also synthesized using polymerase chain reaction and inserted into HindlIl site of PUC19. The recombinant plasmid was transformed into JMlO3 and the expression of the inserted renin cDNA was examined. The transformant produced renin protein having a molecular weight of 45, 000 dolton, which showed hypertensive effect upon injecting it into rabbit ear vein. A renin genomic library was prepared by inserting rabbit kidney DNA into EMBL3 phage, and was screeined for the isolation of renin gemomic DNA using renin cDNA probe.

• Journal of the Korean Society of Environmental Restoration Technology
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• v.7 no.5
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• pp.94-99
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• 2004

This study was carried out to find out what is the optimum conditions for RAPD of Zelkova serata. We changes the factors what affect to PCR band patterns, as a result, we established the optimum conditions as follows; template DNA 100mg, Primer 0.25uM, dNTP 100mM, Taq polymerase 1.0u, and total reaction volume was filled up to 10uL with distilled water. As the amount of primers went higher, PCR reaction rates were lowered. This reason was cause by exhaustion of primers during initial reaction. The amount of dNTP didn't showed noticable differtations between the range, but the optimum amount was 100mM for efficiency. Taq polymerase 1.0 unit was the best in the range. As the concentration of polymerase were increased, many non-specific bands were appeared, In primer selection, most Openron Random Primers are amplified in this experiment. The primers GC contents were 60, and set A, B, C, D, E, X were tested. Thermal cycler(ASTEC PC808, Japan) condition was, $95^{\circ}C$, 5min, initial denaturation, $94^{\circ}C$, 20sec, denaturation, $37^{\circ}C$, 40sec, annealing, $72^{\circ}C$, 1min, extention, 45cycle repeated and final extention $72^{\circ}C$10min.

• Korean Journal of Medicinal Crop Science
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• v.8 no.3
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• pp.266-273
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• 2000

The optimal conditions of PCR components for the random amplification of genomic DNA were $20\;ng/20{\mu}l$ in template DNAs, 250 mM in dNTP, 10 pM in primer $1.0unit/20{\mu}l$ in Taq DNA polymerase respectively with the annealing temperature at $36^{\circ}C$, respectively. Twelve local lines were divided into 3 groups by the coefficients of 107 polymophic bands by Jaccard and Nei. The coefficients value of group I including Chongup # 1, Seochon # 1, Andong # 1, Chinan # 1, and Danyang # 1 ranged from 0.27 to 0.05 and those of group II including Suwon # 2, Chunchon # 1, Japan # 3, Danyang#2 and $F_1$ (Variety Jihwang $1{\times}$ Seohchon) ranged from 0.29 to 0.11. While, Jihwang 1 originated from China and Japan # 1 in group III showed a distant genetic relationship to Korean local lines.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.91-97
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• 1990

Complementary DNA (cDNA) coding for human cytoplasmic superoxide dismutase (SOD1) (superoxide: superoxide oxidoreductase E.C.1.15.1.1) was isolated from human liver cDNA library of $\lambda$gt11 by in situ plaque hybridization. The insery cDNA gas the 5' untranslational region (UTR) and 3'UTR of SOD1 gene. Polymerase Chain Reaction (PCR) method was used fro subcloning of SOD1 structural gene. Using synthetic sense strand primer (24mer) containing a start codon and antisense strand primer (24mer), SOD1 structural gene was selectively amplified. Amplified DNA was directly cloned into the HincII site of pUC19 plasmid. Insery cDNA was subcloned into M13 mp19 and sequenced by dideowy chain termination method with Sequenase. The nucleotide sequence of insert cDNA had an open reading frame (ORF) coding for 153 amino acid residues. The structural gene of cytoplasmic SOD was placed under the control of bacteriophage $\lambda P_{L}$ regulatory sequences, generating a highly efficient expression plasmid. The production of human SOD1 in E. coli cells was about 7% of total cellular proteins and recombinant human SOD1 possessed its own enzymatic acitivity.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.248-255
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• 2012

In order to develop a protocol to quantify cyanobacteria and Microcystis simultaneously, the primers and probe were designed from the conserved regions of 16S rRNA gene sequences of cyanobacteria and Microcystis, respectively. Probe match analysis of the Ribosomal Database Project showed that the primers matched with over 97% of cyanobacterial 16S rRNA genes, indicating these can be used to amplify cyanobacteria specifically. The TaqMan probe, which is located between two primers, matched with 98.2% of sequences in genus GpXI, in which most Microcystis strains are included. The numbers of cyanobacterial genes were estimated with the emission of SYBR Green from the amplicons with two primers, whereas those of Microcystis spp. were measured from the fluorescence of CAL Fluor Gold 540 emitted by exonuclease activity of Taq DNA polymerase in amplification. It is expected that this method enhances the accuracy and reduces the time to count cyanobacteria and potential toxigenic Microcystis spp. in aquatic environmental samples.