Nucleic Acids Research Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 x 10"8 mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples. INTRODUCTION Electrophoretic separations of macromolecules in gel sieving media have become a versatile technique in all laboratories. In a standard procedure, the sample is applied to the gel and macromolecules are separated due to their difference in molecular weight. Modern analytical micro-methods use very small amounts of materials and their fast and reliable detection becomes a limiting factor. Until recently, radioactive labelling has been the most sensitive technique used in DNA sequencing [1,2]. After the separation run, the gel mold is dismantled, gel dried, film is applied and exposed (for about a day or more) to obtain sufficient film darkening allowing detection of bands on the autoradiogram and determination of DNA sequence. Silver staining has been used extensively for detection of DNA and protein bands in gels [3] down to 1CT 1 0 1 4 mol per band, but this method fails at still lower concentrations in the range of 1 0 1 6 10^ 1 7 mol per band, which are required in the DNA sequencing methods. Time for detection of the separated macromolecules can be significantly shortened when they are fluorescently labelled and detected in the gel already during the electrophoresis, transferring the information directly into a computer, without gel drying, 11RL Press Limited, Oxford, England. 4593 Downloaded from https://academic.oup.com/nar/article-abstract/15/11/4593/2358859/Automated-DNA-sequencing-ultrasensitive-detection by guest on 15 September 2017 Nucleic Acids Research film exposure, handling and analysis. In one method [4], four fluorescent dyes are used, each to mark one of the bases which are then co-electrophoresed and detected in one gel tube. In spite of the obvious attraction of this method, there seem to be drawbacks associated with it, namely variations in the electrophoretic mobility due to different dyes, spectral overlap of the dyes, additional software needed to correct these problems and a need for moving parts in filtering or scanning. In our first automated device [5-7], tetramethylrhodamine has been chosen as a single fluorophore for the four bases. DNA sequences are determined in the gel during electrophoresis and read directly into a computer. The chemistry for preparation of the fluorescent primer is reliable and straightforward, and moreover the preparation of the sequencing samples is less expensive compared to the method using radioactive labels. The developed instrument includes several novel features such as detection of laser-induced fluorescence in conjunction with slab gel electrophoresis, excitation laser beam passing inside the gel throughout its entire width, efficient and simple light coupling in and out of the gel, continuous monitoring of the four tracks, no moving parts, and background reduction by adjustment of the plane of polarisation. Another important background reduction is achieved by eliminating the fluorescence of glass, inherent to all techniques where the excitation laser beam traverses glass within the light collecting field. In this paper we describe improvements in sensitivity, resolution and design of the instrument for the automated DNA sequencing method [5-7], with fluorescein as the fluorescent dye for the four bases. DISCUSSION AND RESULTS The structure of the fluorescent primer Is illustrated in Figure 1. Continuous monitoring of the four gel tracks makes the software required for sequence determination less complicated. In addition, besides the positive peak in one track output, the absence of peaks in the other three track outputs gives further information for the confirmation of a 0—R—0*—oligonucleotide Figure 1 Structure of the fluorescein labelled M13 sequencing primer. Oligonucleotide d[GTAAAACGACGGCCAGT].