Studies on polynucleotides

Kleppe, Kjell; Ohtsuka, Eiko; Kleppe, Ruth K.; Molineux, Ian J.; Khorana, H. G.

doi:10.1016/0022-2836(71)90469-4

Cited by 300 publications

(50 citation statements)

References 11 publications

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“…Furthermore, the results illustrated that the last nucleotide was added in ovek 90% of the template strands. The procedure used here was similar to that of Kleppe et al (9) and confirmed their results.…”

Section: -V(b)supporting

confidence: 89%

Enzymatic multiplication of a chemically synthesized DNA fragment

Olson¹,

Gabriel²,

Michalewsky³

et al. 1975

Nucl Acids Res

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A synthetic DNA fragment of 19 residues was enlarged by the enzymatic addition of deoxyadenylate residues to its 3'-end with calf thymus terminal deoxynucleotidyl transferase. The 3'-terminus of this elongated DNA strand was blocked with 2',3'-dideoxyadenylate to prevent hydrolysis by the 3'-exonuclease function of E. coli DNA polymerase I. This elongated and 3'-blocked fragment was annealed to an oligomeric primer and used as a template for the synthesis of a complementary copy of the synthetic 19-mer. The product of such a repair synthesis was separated by gel filtration and analyzed by nearest neighbor techniques.All template strands were copied with complgte repair in over 90% of the chains. Facile recovery of the elongated template by virtue of its size permitted repetition of the copy process, thus allowing accumulation of the desired strand.

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Section: -V(b)supporting

confidence: 89%

Enzymatic multiplication of a chemically synthesized DNA fragment

Olson¹,

Gabriel²,

Michalewsky³

et al. 1975

Nucl Acids Res

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“…It is clear that there is very little change in the respective transcription patterns, although the longest transcripts contain 1270 nucleotides. 5'-end analysis of the two longer transcripts showed that they were capped for more than 90% designed to obtain transcripts starting with a cap structure and are similar in design to the procedure used for priming transcription by ribo-oligonucleotides (18)(19)(20)(21)(22). The structure of the cap primers, however, is more similar to a nucleoside triphosphate analogue.…”

Section: Resultsmentioning

confidence: 99%

Simple, efficientin vitrosynthesis of capped RNA useful for direct expression of cloned eukaryoti genes

Contreras¹,

Cheroutre²,

Degrave³

et al. 1982

Nucl Acids Res

151

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A simple and efficient method for direct in vitro synthesis of capped transcripts of cloned eukaryotic genes is described. As an example capped transcripts were made from a plasmid containing the human fibroblast interferon gene cloned under the control of a prokaryotic promoter. These transcripts were translated in vivo in XenoPus laevis oocytes and in vitro in reticulocyte and in wheat germ cellfree protein synthesizing systems.

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“…Total RNA isolated by using TRIzol reagent according to the manufacturer's protocol (Invitrogen) from either WT1 or ⌬508 cells treated with or without CF6-CF4 annealed duplex oligonucleotides (final concentration of duplex in reaction mixture 10 M) was used to perform the RT-PCR assay (37) in two steps by using a ThermoScript RT-PCR system (GIBCO͞BRL). Several sets of primers were used (see Table 1).…”

Section: Methodsmentioning

confidence: 99%

Reversal of cystic fibrosis phenotype in a cultured Δ508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion

Zamecnik

Raychowdhury

Tabatadze

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5 to the deletion), accounts for Ϸ75% of cases of the disease. In the present study, an oligonucleotide complex (CF4 -CF6 , 2 -0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in ⌬508 CFTR mRNA from a cultured (⌬508) cell line. cAMP-activated wholecell currents and Cl ؊ transport were detected in CF4 -CF6-treated, but not control ⌬508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of ⌬508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5 to the deletion site in ⌬508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.tissue culture ͉ functional restoration ͉ mRNA repair

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Studies on polynucleotides

Cited by 300 publications

References 11 publications

Enzymatic multiplication of a chemically synthesized DNA fragment

Enzymatic multiplication of a chemically synthesized DNA fragment

Simple, efficientin vitrosynthesis of capped RNA useful for direct expression of cloned eukaryoti genes

Reversal of cystic fibrosis phenotype in a cultured Δ508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion

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