Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.

Abstract

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with <1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method. The need for rapid, reliable automated sequencing with low cost per sequenced base has been addressed (3-7). Several automated approaches to DNA sequencing have been reported using both radioactive detection (8, 9) and fluorescent detection (10-17).The purpose of this report is to show an improved method for automated, continuous, on-line, real-time DNA sequencing (10-12). The method uses standard dideoxy reactions with a fluorescently tagged primer carrying two fluoresceins. A fluorescence detection system located at a fixed distance from the loading wells records the bands as a twodimensional image as they move past the detector. MATERIALS AND METHODS Synthesis of Fluorescently Labeled Primers. A deoxyuridineanalog with a primary amine "linker arm" of 12 atoms attached at C-5 was synthesized as published (18,19). Synthesis of the analog consists of derivatizing 2'-deoxyuridine through organometallic intermediates to give 5-(methyl propenoyl)-2'-deoxyuridine. Reaction with dimethoxytritylchloride produces the corresponding 5'-dimethoxytrityl adduct. The methyl ester is hydrolyzed, activated, and reacted with an appropriately monoacylated alkyl diamine. After purification, the resultant linker arm nucleosides are converted to nucleoside analogs suitable for chemical oligonucleotide synthesis. The structure of the linker arm analog is shown in Fig. 1 5610The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Fluorescent Oligonucleotides - Versatile Tools as Probes and Primers for DNA and RNA Analysis

“…16 The sensitivity of a fluorescent probe used for in situ hybridization experiments is determined not only by the spectroscopic properties of the dye itself but also by the target specificity of the probe and the stability of the probe-target duplex. 17 Incorporation of multiple fluorescent building blocks at specific sites of an oligonucleotide may increase its sensitivity, [18][19][20] but in some cases it is also known to quench the fluorescence and decrease the extinction coefficient of the dye. 21,22 Furthermore, since even one dye molecule can perturb the structure of the probe-target duplex, several bulky fluorescent residues often considerably reduce the melting point of a double stranded oligomer and thus decrease duplex stability.…”

“…The base, commonly a thymine, is functionalized with a C-6 or C-2 linker that ends with a primary amino group (Figure 9). [17][18][19][42][43][44] Chemical structure of an internal modifier according to Randolph et al 17 R= amino protecting group; n = 3, 6…”

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

“…This realisation underlies efforts at extending the sensitivity with the aim of extracting more data per sequencing run while reducing the amount of sample required. One can, on the one hand, simply incorporate multiple labels into a primer by using C-5 aminomodified uridines (Haralambidis et al, 1987;Brumbaugh et al, 1988) or an aminobutyl-1,3-propanediol modified backbone (Nelson et al, 1992) to achieve multiple dye substitutions. The addition of an internal fluorescent label, in addition to one at the 5' terminus was found to enhance priming efficiency, although the hybridisation stability was reduced (Mineno et al, 1993).…”

Sequencing and Analysis of a Calmodulin cDNA from Pea (Pisum sativum L. var Alaska)

“…The PCR products were then directly subcloned into pCR-Script SK(+). Plasmid dsDNA was then sequenced by the University of Nebraska-Lincoln Center for Biotechnology DNA Sequencing Facility using a modified dideoxy method (Brumbaugh et al, 1988).…”