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DNA sequencing by capillary electrophoresis using short oligonucleotide primer libraries
M C Ruiz-Martinez1, E Carrilho, J Berka
1Northeastern University, Boston, MA, USA.
Biotechniques
|June 1, 1996
Summary
This study demonstrates successful DNA sequencing using primer walking and capillary electrophoresis, achieving high accuracy for M13mp18 sequencing. The method yielded approximately 450-base reads with 97.8% accuracy.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Primer walking is a key technique for DNA sequencing.
- Capillary electrophoresis offers high-resolution separation for DNA fragments.
Purpose of the Study:
- To evaluate DNA sequencing efficiency using primer walking and capillary electrophoresis.
- To assess the accuracy and read length achievable with specific sequencing chemistry and analysis software.
Main Methods:
- DNA sequencing was performed using primer walking with short oligonucleotide primer libraries.
- Capillary electrophoresis with linear polyacrylamide polymer solutions and fluorescence detection was employed.
- T7 PRISM dye-terminator/Sequenase chemistry was used to sequence a 3.5-kb M13mp18 template.
- An in-house base-calling program was utilized for data analysis.
Main Results:
- Successful sequencing of a 3.5-kilobase (kb) M13mp18 DNA template was achieved.
- The method provided read lengths of approximately 450 bases.
- An average accuracy of 97.8% was obtained for the DNA sequence reads.
Conclusions:
- Primer walking combined with capillary electrophoresis is an effective strategy for DNA sequencing.
- The developed in-house base-calling program contributes to achieving high accuracy in DNA sequence analysis.