Direct PCR amplification of various modified DNAs having amino acids: Convenient preparation of DNA libraries with high-potential activities for in vitro selection

Kuwahara, Masayasu; Hanawa, Kazuo; Ohsawa, Kazuomi; Kitagata, Rina; Ozaki, Hiroaki; Sawai, Hiroaki

doi:10.1016/j.bmc.2005.11.030

Cited by 50 publications

(30 citation statements)

References 33 publications

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“…The results are similar to previous reports that showed that the family B-type DNA polymerases display superior incorporation efficiencies of a wide variety of dNTP analogues. [24][25][26][27][28][29] An incorporation ratio of Z-QG-deoxyuridine (Z-QGdU) in each Z-QG-DNA was quantified by using reversedphase HPLC after fragmentation of the Z-QG-DNA prepared with KOD FX to nucleosides ( Figure S3 in the Supporting Information). The incorporation ratio increased as the percentage of Z-QG-dUTP increased in a PCR mixture (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection

Kitaoka

Tsuruda

Tanaka

et al. 2011

Chemistry A European J

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A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

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Section: Resultsmentioning

confidence: 99%

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection

Kitaoka

Tsuruda

Tanaka

et al. 2011

Chemistry A European J

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“…Recently, several types of modified DNA bearing diverse groups, for example, aminoalkynyl or aminoalkyl [4 6] and several types of attached functional molecules, for example, biotin, [6] acridones, [7] fer rocene, [8] amino acids, [9] carbohydrates, [10] or fluorescein labels, [11] have been prepared from the corresponding modi fied dNTPs by this methodology. The dNTP building blocks are usually prepared [4 9] by troublesome and laborious tri phosphorylation of the corresponding modified nucleosides, in which the functional groups usually have to be protected and deprotected.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross‐Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

Čapek¹,

Cahová²,

Pohl³

et al. 2007

Chemistry A European J

134

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Single‐step aqueous cross‐coupling reactions of nucleobase‐halogenated 2′‐deoxynucleosides (8‐bromo‐2′‐deoxyadenosine, 7‐iodo‐7‐deaza‐2′‐deoxyadenosine, or 5‐iodo‐2′‐deoxy‐uridine) or their 5′‐triphosphates with 4‐boronophenylalanine or 4‐ethynylphenylalanine have been developed and used for efficient synthesis of modified 2′‐deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8‐substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7‐substituted 7‐deazaadenine and 5‐substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two‐step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.

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“…33 The biotinylated amino acids BG, BE, BL and BD were synthesised by coupling 6-[6-(biotinylamino)hexanoylamino]-hexanoic acid-N-hydroxysuccinimide (Biotin-(AC5)2-OSu) with glycine t-butyl ester, DL-glutamic acid, L-glutamic acid-α,γ-di-tbutyl ester and D-glutamic acid-α,γ-di-t-butyl ester, respectively. This was followed by treatment with 50% trifluoroacetic acid in dichloromethane for BG, BL and BD but not for BE (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Arginine-modified DNA Aptamers That Show Enantioselective Recognition of the Dicarboxylic Acid Moiety of Glutamic Acid

et al. 2008

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Direct PCR amplification of various modified DNAs having amino acids: Convenient preparation of DNA libraries with high-potential activities for in vitro selection

Cited by 50 publications

References 33 publications

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross‐Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

Arginine-modified DNA Aptamers That Show Enantioselective Recognition of the Dicarboxylic Acid Moiety of Glutamic Acid

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Direct PCR amplification of various modified DNAs having amino acids: Convenient preparation of DNA libraries with high-potential activities for in vitro selection

Cited by 50 publications

References 33 publications

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross‐Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

Arginine-modified DNA Aptamers That Show Enantioselective Recognition of the Dicarboxylic Acid Moiety of Glutamic Acid

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Product

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Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)_n Probe for Highly Sensitive DNA Detection