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New dye-labeled terminators for improved DNA sequencing patterns
B B Rosenblum1, L G Lee, S L Spurgeon
1PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. rosenbbb@perkin-elmer.com
Nucleic Acids Research
|February 12, 1998
Summary
New dye sets improve DNA sequencing accuracy by providing more even peak heights and reducing weak G peaks. This advancement enhances automated base-calling for complex DNA templates.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Automated DNA sequencing relies on dye-labeled terminators.
- Existing rhodamine dyes can produce uneven peak heights, particularly weak G peaks.
- This unevenness can reduce the accuracy of automated base-calling software.
Purpose of the Study:
- To develop and evaluate new dye sets for automated dye-labeled terminator DNA sequencing.
- To improve peak evenness and signal-to-noise ratio compared to existing dyes.
- To enhance the accuracy of DNA sequencing, especially for challenging templates.
Main Methods:
- Utilized two novel dye sets: 4,7-dichlororhodamine (d-rhodamine) dyes and energy-transfer dyes.
- Employed a new linker chemistry for dye-nucleotide conjugation.
- Compared performance against unsubstituted rhodamine dyes using automated sequencing platforms.
Main Results:
- Both new dye sets demonstrated more even peak heights than unsubstituted rhodamine dyes.
- The incidence of weak G peaks was significantly reduced or eliminated.
- Energy-transfer dyes showed an improved signal-to-noise ratio.
- Successful sequencing of high molecular weight DNA, including bacterial artificial chromosomes, was achieved.
Conclusions:
- The new d-rhodamine and energy-transfer dye sets offer superior performance for terminator DNA sequencing.
- Improved peak evenness and signal quality enhance automated base-calling accuracy.
- These advancements enable reliable sequencing of complex and high molecular weight DNA templates.