Vol. 9, Issue 6, 588-595, June 1999

METHODS
Improved DNA Sequencing Accuracy and Detection of Heterozygous Alleles Using Manganese Citrate and Different Fluorescent Dye Terminators

¹ University of Colorado Cancer Center (UCCC) DNA Sequencing and Analysis Core and ² Division of Medical Oncology, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262 USA

The use of dideoxynucleotide triphosphates labeled with different fluorescent dyes (dye terminators) is the most versatile method for automated DNA sequencing. However, variation in peak heights reduces base-calling accuracy and limits heterozygous allele detection, favoring use of dye-labeled primers for this purpose. We have discovered that the addition of a manganese salt to the PE Applied Biosystems dye-terminator sequencing kits overcomes these limitations for the older rhodamine dyes as well as the more recent dichloro-rhodamine dyes (dRhodamine and BigDyes). Addition of manganese to reactions containing dRhodamine-based dye terminators produced the highest base-calling accuracy. This combination resulted in the most uniform electropherogram profiles, superior to those produced by BigDye terminators and published for dye primers, and facilitated detection of heterozygous alleles.