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Fluorescence-based automated DNA sequencing by limited primer labeling
G De Bellis1, R Pergolizzi, S Debernardi
1Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche, Avanzate, LITA, Segrate, Italy.
Biotechniques
|July 1, 1995
Summary
This study introduces an economical method for fluorescence-based DNA sequencing using unlabeled primers. The novel technique simplifies DNA sequencing, offering a cost-effective alternative for large-scale primer applications.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Context:
- Automated DNA sequencing relies on expensive, fluorescence-labeled oligonucleotides, limiting its application in strategies requiring numerous primers.
- Current fluorescence labeling protocols can be costly and complex.
Purpose:
- To develop a cost-effective and simplified method for fluorescence-based DNA sequencing.
- To enable the use of unlabeled oligonucleotides in automated DNA sequencing.
Summary:
- A novel procedure for fluorescence-based DNA sequencing using unlabeled oligonucleotides is presented.
- The method involves limited primer extension incorporating a single fluorescently modified deoxynucleotide.
- The elongated, fluorescently labeled primer is then utilized in standard T7 sequencing reactions.
Impact:
- This approach offers an economical and straightforward alternative to existing fluorescence-labeling protocols.
- The protocol demonstrates reliability across various DNA templates, enhancing the accessibility of automated DNA sequencing.
- Reduces the cost associated with large-scale DNA sequencing projects.