Rapid DNA Sequencing of More Than 1000 Bases per Run by Capillary Electrophoresis Using Replaceable Linear Polyacrylamide Solutions

Abstract

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

“…There are many factors that govern the electrophoretic separation quality. Because such a topic has been detailed by others, [14][15][16][17][18][19][20][21][22]25,26 only a brief description is given here.…”

“…Recently, linear polymers have been popularly used in CE as the sieving matrix instead of the crosslinked gel, in terms of non-gel sieving or an entangled polymersolution CE, 13,14 due to its facilitation 15 of high separation speed, long read length and replaceable operation compared to CGE. Many polymers have proved to be useful, including linear polyacrylamide and its derivatives, 14,16,17 cellulose and its derivatives, 18,19 polyethene oxide 20,21 and agarose 22 etc. Currently, non-gel sieving CE is being extensively applied in DNA sequencing, characterizing the polymorphism of DNA, analyzing PCR products, gene diagnosis and therapy etc.…”

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

“…Zhang et al 1995Zhang et al , 1999Bashkin et al 1996a,b;Carrilho et al 1996;Kheterpal et al 1996;Dovichi 1997Dovichi , 1999Kheterpal and Mathies 1999;Pang et al 1999;Crabtree et al 2000;Gao and Yeung 2000;McWhorter and Soper 2000) to provide high throughput analysis of DNA fragments.…”

Effects of Polyols, pH and Electrolyte Concentrations in TBE Buffer o Separation of Double Strand DNA Fragments by Capillary Electrophoresis

“…1 This technique has been successfully applied to mutation detection, [2][3][4] genotyping, 5 DNA sequencing, 6 and geneexpression analysis. 7 Some investigators have examined what the polyols in the TBE buffer markedly improve the separation efficiency of DNA fragments using cellulose derivatives as sieving media.…”