Automated cycle sequencing of PCR templates: Relationships between fragment size, concentration and Strand renaturation rates on sequencing efficiency

Taylor G.O. and Dunn I.S. (1994) Automated cycle sequencing of PCR templates: Relationships between fragment size, concentration and Strand renaturation rates on sequencing efficiency. Mitochondrial DNA, 5 1: 9-15. doi:10.3109/10425179409039699


Author Taylor G.O.
Dunn I.S.
Title Automated cycle sequencing of PCR templates: Relationships between fragment size, concentration and Strand renaturation rates on sequencing efficiency
Journal name Mitochondrial DNA   Check publisher's open access policy
ISSN 1940-1744
Publication date 1994-01-01
Sub-type Article (original research)
DOI 10.3109/10425179409039699
Volume 5
Issue 1
Start page 9
End page 15
Total pages 7
Publisher Informa Healthcare
Subject 1311 Genetics
1312 Molecular Biology
1303 Specialist Studies in Education
1310 Endocrinology
Abstract With the Applied Biosystems 373A automated DNA sequencer, we have systematically investigated the amounts of double-stranded PCR fragments of varying size (200, 564, and 1126 bp) required to give sequence of defined lengths, up to the maximum possible. Sequencing was performed on purified double-stranded PCR products using the dye-terminator chemistry and a thermal cycling procedure. The minimal template concentrations allowing determination of short sequences (160 bases) were essentially identical for the fragments studied. Maximal possible sequence determination from the 200 bp fragment was achieved over a wide concentration range, despite the fact that within this range a significant fraction of the template renatured by the mid-point of the sequencing reaction time-course. We conclude that the cyclic sequencing process overcomes competitive strand reannealing of double-stranded PCR products. The sequencing concentration-response curves for the 564 bp and 1126 bp fragments were similar to each other, although the minimal template concentrations required to read >300 bases were slightly increased for the 564 bp fragment. Excess template is undesirable for optimal sequence length determination, but this is unlikely to be solely due to strand reannealing as single-stranded M13 templates in super-optimal concentrations also showed marked reduction in sequencing efficiency.
Keyword Automated DNA sequencing
Dye-terminator
PCR products
Sequencing efficiency
Strand renaturation
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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