Analysis of oligonucleotides by capillary gel electrophoresis

Paulus, Aran; Ohms, Jonas

doi:10.1016/s0021-9673(01)84187-x

Cited by 106 publications

(31 citation statements)

References 29 publications

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“…Perhaps one of the earliest reports was that of Paulus and Ohms [4], who showed rapid and reliable analysis of oligonucleotides, 20-50 nucleotides in length, in gel-filled capillaries. Analysis was typically performed in 7.s0/oT, 3.3%c columns, in presence of 50 mM Tris-borate buffer, pH 8.3, 7 M urea, 3% polyethylene glycol (PEG) with voltage gradients from 250 to 450 V/cm.…”

Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers

Gelfi¹,

Perego

Righetti

1996

Electrophoresis

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Analysis of oligonucleotides (especially in regard to assessing the purity of antisense compounds) is typically performed in 18% T sieving liquid polyacrylamide, in 30% formamide and 7 M urea. Up to 600 V/cm have been reported, with transit times, for 20 to 25 oligomers, of 15-20 min. We show that the same analysis can be performed in isoelectric buffers, typically histidine (His), and in more dilute linear polyacrylamides, e.g. 10% T (at 0% C), with much reduced analysis times. A series of His concentrations has been explored, ranging from 25 to 150 mM. Best performance is obtained in 100 mM His (at pH = pI, i.e., 7.47 at 25 degrees C), dissolved in 7 M urea, in presence of 10% sieving liquid linear polyacrylamide. Such a buffer allows delivering 800 V/cm without any loss of resolution due to Joule heating, with retention of very high resolving power down to fragments as short as tetranucleotides. Under these conditions, the analysis time for an antisense oligonucleotide containing fragments from a 10-mer to 18-mer is in a time window of 4-5 min. It is shown that the smallest fragment (10-mer) migrates in the capillary at the remarkable speed of 5 cm/min.

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Section: Introductionmentioning

confidence: 99%

Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers

Gelfi¹,

Perego

Righetti

1996

Electrophoresis

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“…The solutions were prepared by dissolving F127 in the 16TBE buffer containing different urea concentrations. In order to make a comparison, peak resolution and efficiencies were calculated by the equations of R = 2(t 2 -t 1 )/ (1.699(w 2 1 w 1 ))/Db and N = 5.54(t/w) 2 , where R, N, t, w, and Db are resolution, efficiency, migration time, peak width at half height, and base difference, respectively. The calculations, using data from Fig.…”

Section: Effect Of Ureamentioning

confidence: 99%

“…CE with LIF is one of the better alternatives for the fast separation of oligos [2][3][4][5][6][7]. DNA separations can be easily achieved by microchip electrophoresis combined with hydrophilic polymer media.…”

Section: Introductionmentioning

confidence: 99%

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen® as fluorescence inducing agent

Zhang

Liang

et al. 2005

Electrophoresis

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The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.

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“…Coating of capillary inner walls was accomplished according to the methods of Paulus and Ohms (1990).…”

Section: Capillary Coating Proceduresmentioning

confidence: 99%

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

Arakawa

Watanabe

Kashiwazaki

et al. 2001

Biomedical Chromatography

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Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.

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Analysis of oligonucleotides by capillary gel electrophoresis

Cited by 106 publications

References 29 publications

Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers

Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen® as fluorescence inducing agent

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

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