DNA Sequencing by Capillary Electrophoresis Using Short Oligonucleotide Primer Libraries

Ruiz‐Martinez, Marie C.; Carrilho, Emanuel; Berka, Jan; Kieleczawa, Jan; Miller, Arthur W.; Foret, František; Carson, Spencer; Karger, Barry L.

doi:10.2144/96206rr03

Cited by 15 publications

(14 citation statements)

References 27 publications

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“…The design of a single-capillary instrument with detection based on laser-induced fluorescence (LIF) has been described previously. 30 Briefly, the fluorescence emission was collected with a microscope objective (model 13600, Oriel, Stamford, CT), and the spectra of the labeled sequencing fragments were acquired continuously in the range from 500 to 660 nm by a photodiode array (model 1461, EG&G Princeton Applied Research, Princeton, NJ). The CE columns were fused-silica capillaries of 50-, 75-, and 100-µm i.d, 365-µm o.d.…”

Section: Methodsmentioning

confidence: 99%

Routine DNA Sequencing of 1000 Bases in Less Than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions

et al. 1998

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Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

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Section: Methodsmentioning

confidence: 99%

Routine DNA Sequencing of 1000 Bases in Less Than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions

et al. 1998

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“…As an example of the high-throughput possible with these gels, one DNA sequencing run demonstrated separation of 350 oligonucleotides in about 30 min, which corresponds to about 11 compounds per minute . Several groups have also demonstrated similar fast sequencing separations. − …”

Section: Rapid Ce Of Dnamentioning

confidence: 98%

“…181 Several groups have also demonstrated similar fast sequencing separations. [182][183][184] Carrilho et al have designed a system capable of sequencing more than 1000 bases in 80 min. 185 The effect of size and concentration of polyacrylamide gel, temperature, and electric field on separation was studied in this work.…”

Section: Rapid Ce Of Dnamentioning

confidence: 99%

Fast Analytical-Scale Separations by Capillary Electrophoresis and Liquid Chromatography

et al. 1999

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“…The widely used technique of PCR always requires two primers at a time and multiplexed PCRs (3–5) use numerous primer pairs in a single tube. It is also possible to use either pairs of forward and reverse primers (6,7), duplex primers (8,9) or strings of more than one oligonucleotide (10–12) in DNA sequencing. Sets of cooperative or helper oligonucleotides may also be used as probes (13–19) or antisense oligonucleotides (20).…”

Section: Introductionmentioning

confidence: 99%

Tandem oligonucleotide synthesis using linker phosphoramidites

Pon

2005

Nucleic Acids Research

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Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

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DNA Sequencing by Capillary Electrophoresis Using Short Oligonucleotide Primer Libraries

Abstract: ABSTRACT

Cited by 15 publications

References 27 publications

Routine DNA Sequencing of 1000 Bases in Less Than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions

Routine DNA Sequencing of 1000 Bases in Less Than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions

Fast Analytical-Scale Separations by Capillary Electrophoresis and Liquid Chromatography

Tandem oligonucleotide synthesis using linker phosphoramidites

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