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Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures
T M Prychitko1, E A Ries, W S Moore
1Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA. tprych@biology.biosci.wayne.edu
Molecular Biotechnology
|February 10, 1999
Summary
To improve DNA sequencing accuracy, researchers found that increasing annealing temperature prevents premature DNA polymerase termination. This method enhances reading length for double-stranded polymerase chain reaction (PCR) products.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products can yield shorter fragments than expected due to premature polymerase termination.
- Secondary structures in DNA templates can form during primer annealing, contributing to these premature terminations and reducing DNA sequence reading length.
Purpose of the Study:
- To develop a method for reducing premature terminations in DNA sequencing.
- To improve the reading length and accuracy of DNA sequence analysis.
Main Methods:
- Investigated the effect of annealing temperature on premature termination during cycle sequencing.
- Implemented a method where primer annealing and DNA extension occur at a single, elevated temperature (72°C).
- Utilized long primers (at least 27 mer) with high optimal annealing temperatures.
Main Results:
- Maintaining the annealing temperature above a critical threshold significantly reduces premature terminations.
- The described method effectively minimizes premature terminations in DNA sequences that previously exhibited them at 60°C.
- Achieved longer and more reliable DNA sequence readings.
Conclusions:
- Elevating annealing temperature is a simple and effective strategy to overcome secondary structure-induced premature terminations in cycle sequencing.
- This optimized cycle sequencing protocol enhances DNA sequence reading length and reliability.
- The method is particularly applicable for sequencing templates prone to secondary structure formation using long primers.