• KSBB Journal
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• v.9 no.1
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• pp.63-71
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• 1994

During the last decade enzyme reaction in organic solvent has been studied to show that specificity in buffer is different from that in organic solvent. The specificity of restriction enzyme was effected by various factors such as ionic strength, salt organic solvent and temperature. In this study, restriction enzyme PvuII which is used most frequently in genetic engineering and the substrate was vector pGEM3 whose sequence was already known were used. As a result the recognition sequence site was changed in the presence of organic solvents whose Log P are -1.5∼0. Their specificities were contrast with activities were contrasted. Specificities were not changed in organic solvent easily in inactivating enzyme. We think that the enzyme recognition site was not changed randomly but by preferential order. A recombinant vector which does not contain typical cleavage site CAG↓CTG was cleaved in 20% ethanol solution. This result might show that restriction enzyme could be used to cleave at unusual sites by changing the reaction conditions.

• Journal of fish pathology
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• v.21 no.3
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• pp.181-188
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• 2008

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect and identify four fish viruses, fish iridovirus, viral hemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV). Four viruses were detected by PCR with each specific primers. Identification of iridovirus was achieved by digesting the PCR amplified fragment with a restriction enzyme ApaⅠ. It was possible to distinguish positive from false positive PCR amplicons of VHSV by RFLP of PstⅠ or HindⅢ restriction enzymes. VNNV was identified using RFLP of BamHⅠrestriction enzyme and HRV was identified by XbaⅠ restriction enzyme. This approach can be used for more rapid, simple and specific diagnosis of fish viral diseases.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.30 no.1 s.244
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• pp.26-31
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• 2006

This paper reports low-cost PDMS/glass based DNA microbiochip for the restriction enzyme reaction and its products detection using the capillary electrophoresis. The microbiochip ($25mm{\times}75mm$) has the heater integrated reactor ($5{\mu}{\ell}$) for DNA restriction enzyme reaction at $37^{\circ}C$ and the microchannel ($80\;{\mu}m{\times}100\;{\mu}m{\times}58mm$) for the capillary electrophoresis detection. It is experimentally confirmed that the digestion of the plasmid ($pGEM^{(R)}-4Z$) by the enzyme (Hind III and Sca I) is performed for less than 10 min and its electrophoresis detection is able to sequentially on the fabricated microbiochip.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.28 no.10
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• pp.1598-1602
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• 2004

In this paper, we report a microthermostat using PDMS (poly-dimethylsiloxane) and glass. This PDMS/glass chip is able to maintain constant temperature that is necessary for restriction enzyme reaction. Since PDMS is the low-cost and the mass-producible material and has very good biochemical compatibility, PDMS chip has more benefit than general Si chip. Heater was made of Au wiring patterned on Pyrex glass. A reaction chamber has a capacity of about 3 ${mu}ell$. We performed a restriction enzyme reaction by using the fabricated microthermostat and conventional method. Then, with the electrophoresis, we made a comparison between the result from the micro reactor and the one from conventional method.

• BMB Reports
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• v.33 no.2
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• pp.162-165
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• 2000

Polymerase chain reaction (PCR) is well established as an indispensable tool of molecular biology; and yet a limitation for cloning applications continues to be that products often require subsequent restriction to be that products often require subsequent restriction digests, blunt-end ligation, or the use of special linear vectors. Here a rapid, PCR-based system is described for the simple, restriction enzyme-free generation of synthetic, 'restriction-like' DNA fragments with staggered ends. Any 3'- or 5'-protruding terminus, but also non-palindromic overhangs with an unrestricted single strand length are specifically created. With longer overhangs, "Restriction-PCR" does not even require a ligation step prior to transformation. Thereby the technique presents a powerful tool e.g. for a successive, authentic reconstitution of sub-fragments of long genes with no need to manipulate the sequence or to introduce restriction sites. Since restriction enzyme-free and thereby devoid the limitations of partial DNA digests, "Restriction-PCR" allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties "Restriction-PCR" has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.40-45
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• 1987

The gene for the Bdi I modification enzyme, which is one of Bdi I restriction-modification system, fromBrevibacterium divaricatum FERM 5948 was cloned and expressed in E. coli. For cloning of the Bdi I methylase gene, we have initially used three cloning site(EcoRI, BamHI and Sal I) of plasmid vector pBR 322 and adopted the retransformation method after Bdi I restriction endonuclease cleavage. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by Bdi I restriction enzyme, and the recombinant plasmid pBDIM 116 containing 5.6kb EcoRI insery was proved to carry the gene. Crude cell extracts prepared from strains carrying the plasmid pBDIM 116 contained an S-adenosylmethionine-dependent methyltransferase activity specific for the Bdi I recognition site, ATCGAT. The restriction map was constructed with 11 restriction enzyme, and the Bdi I restriction-modification system was also discussed.

• BMB Reports
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• v.29 no.1
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• pp.17-21
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• 1996

Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers $Mn^{2+}$ to $Mn^{2+}$ as the cofactor. In $Mn^{2+}$ buffer the mutant is as active as the wild type enzyme in $Mn^{2+}$ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.73-79
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• 1987

A restriction endonuclease, Kpn I has been isolated from Klebsiella pneumonia. Cells were broken by sonication. After ultracentrifugation the supernatant containing Kpn I activity was further purified by Sepharose-6B gel filtration, DEAD-Cellulose, Heparin-Agarose, and Aminohexyl-Agarose column chromatography. Final enzyme preparation was essentially free of contamination exonuclease and phosphatase, as judged by ligation-recut test. Total activity of the enzyme recovered from 10 grams of cells was $4.7\times 10^5$ units.

• Journal of Plant Biology
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• v.33 no.4
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• pp.217-223
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• 1990

Restriction enzyme inhibiting substance (REIS) extracted from spinach chloroplast and Chlamydomonas seems not to be proteinaceous, because its inhibiting activity was not lost by heat or trypsin treatment. And it seems not to be lipid or polysacchride, because its inhibiting activity was not lost by lipase or $\alpha$-amylase treatment, respectively. In Chlamydomonas, putrescine, spermidine and spermine were present. The amount of putrescine was the smallest and that of spermie was the greatest. But only spermine was contained in REIS and the activity of REIS. It was proportional to the amount of spermine in REIS and it was hindered by Na+ ion. So, the inhibiting activity of REIS seems to be deeply related to spermine contained in REIS. But restriction enzyme inhibiting activity remained to the some extent although salts and spermine were eliminated by dialysis.

• Journal of Microbiology and Biotechnology
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• v.5 no.5
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• pp.269-273
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• 1995

A thermotolerable restriction endonuclease. Svil, found in Streptomyces violochromogenes D2-5 was purified. For the purification, streptomycin sulfate and ammonium sulfate precipitation was used. Ph osphocellulose P-ll, DEAE-Cellulose and Sephacryl-S200 HR colum chromatography were also performed. The purified enzyme was found to be homogeneous and the molecular weight of the enzyme estimated by polyacrylamide gel electrophoresis containing 0.1$%$ SDS was about 32, 000 daltons. The recognition sequence and cleavage site of the enzyme were determined to be $5^1$-$TT\downarrow CGAA$-$3^1$ which is the same sequence as that of Asull. Unlike Asull, however, the Svil shows high thermal stability.