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RISS 인기검색어
- 검색키워드
- 저자 : Klimkait, (검색결과 5 건)
- 무료
- 기관 내 무료
- 유료
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'Restriction-PCR' - a Superior Replacement for Restriction Endonucleases in DNA Cloning Applications
Klimkait, Thomas 생화학분자생물학회 2001 BMB Reports Vol.33 No.2
Polymerise 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 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, $quot;Restriction-PCR$quot; 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, $quot;Restriction-PCR$quot; allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for those endonucleases involved in the cloning. Small site-specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties $quot;Restriction-PCR$quot; has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.
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'Restriction-PCR' - a Superior Replacement for Restriction Endonucleases in DNA Cloning Applications
Klimkait, Thomas 생화학분자생물학회 2000 Journal of biochemistry and molecular biology Vol.33 No.2
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.
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'Restriction-PCR' - a Superior Replacement for Restriction Endonucleases in DNA Cloning Applications
Klimkait, Thomas The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.2
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 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 those endonucleases involved in the cloning. Small site-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.
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Brennan, Lyndall E.,Sune, Carlos,Klimkait, Thomas 생화학분자생물학회 2002 Journal of biochemistry and molecular biology Vol.35 No.3
A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.
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(Lyndall E. Brennan),(Carlos Sune),(Thomas Klimkait) 생화학분자생물학회 2002 BMB Reports Vol.35 No.3
A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIVp24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.
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