Two-Step Cycle Sequencing Improves Base Ambiguities and Signal Dropouts in DNA Sequencing Reactions Using Energy-Transfer-Based Fluorescent Dye Terminators

2001 ◽  
Vol 17 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Long Wen
Keyword(s):  
Energy Transfer ◽  
Dna Sequencing ◽  
Fluorescent Dye ◽  
Step Cycle ◽  
Cycle Sequencing ◽  
Base Ambiguities ◽  
Dye Terminators

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

1999 ◽  
Vol 9 (6) ◽  
pp. 588-595
Author(s):  
Christopher Korch ◽  
Harry Drabkin
Keyword(s):  
Dna Sequencing ◽  
Fluorescent Dyes ◽  
Fluorescent Dye ◽  
Allele Detection ◽  
Base Calling ◽  
Automated Dna Sequencing ◽  
Dye Terminators ◽  
Rhodamine Dyes

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.

Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures

1998 ◽  
Vol 10 (3) ◽  
pp. 231-236
Author(s):  
Thomas Michael Prychitko ◽  
Elizabeth Ann Ries ◽  
William Samuel Moore
Keyword(s):  
Step Cycle ◽  
Cycle Sequencing ◽  
Annealing Temperatures ◽  
High Annealing

scholarly journals A Method For Parallel, Automated, Thermal Cycling of Submicroliter Samples

2001 ◽  
Vol 11 (3) ◽  
pp. 441-447
Author(s):  
Jonathan Nakane ◽  
David Broemeling ◽  
Roger Donaldson ◽  
Andre Marziali ◽  
Thomas D. Willis ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Thermal Cycling ◽  
High Throughput ◽  
Dna Analysis ◽  
Detection Efficiency ◽  
Large Fraction ◽  
Cycle Sequencing ◽  
High Throughput Dna Sequencing ◽  
Reaction Volumes ◽  
The Cost

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 μL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

DNA sequencing using four-color capillary array electrophoresis and energy-transfer primers

1996 ◽  
Author(s):  
Indu Kheterpal ◽  
Jingyue Ju ◽  
Arun Radhakrishnan ◽  
Gabriel S. Brandt ◽  
Charles L. Ginther ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Dna Sequencing ◽  
Capillary Array Electrophoresis ◽  
Capillary Array
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