Search results for "Primer extension"
showing 4 items of 14 documents
DNA Polymerase Action on Oligonucleotide Templates from Human Ha-rasProtooncogene Containing N6-Deoxyadenosine Adducts Derived from Trans Addition of…
1996
Abstract In the present work we have used a DNA polymerase assay to investigate the primer extension with T7 DNA polymerase (Sequenase 2.0) and the Klenow fragment of Escherichia coli DNA polymerase I (exo − KF) on chemically synthesized 21mer templates representing partial sequences of the human Ha-ras protooncogene with site-specifically positioned trans-N 6-dA adducts of (-)- (adduct 1) and (+)-anti-benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (adduct 2) at codon 61 (CA∗G; A∗ indicates the adducted position). With Sequenase 2.0 a complete block of primer extension opposite both adduct 1 and 2 was noted using a 10mer primer reaching the (n-1)-position of the adduct. A detailed analys…
Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hor…
1992
0962-1075 (Print) Journal Article; The complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH2-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corre…
Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA
2011
International audience; Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups fre…
Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA.
2010
Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid–nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification.