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Clinical Chemistry 51: 35-39, 2005. First published October 28, 2004; 10.1373/clinchem.2004.039164
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(Clinical Chemistry. 2005;51:35-39.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

Kathleen M. Murphy1,a, Karin D. Berg1,2 and James R. Eshleman1,2

Departments of1 Pathology and 2 Oncology, Johns Hopkins Medical Institutions, Baltimore, MD.

aAddress correspondence to this author at: Johns Hopkins School of Medicine, Carnegie Bldg., Room 358, 600 North Wolfe St., Baltimore, MD 21287. Fax 410-614-7440; e-mail kmurphy4{at}jhmi.edu.

Background: Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one single reaction, enabling direct sequencing of genomic DNA.

Methods: Combined amplification and sequencing reactions were set up with Big DyeTM sequencing reagents (Applied Biosystems) supplemented with variable amounts of forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and input DNA. Reactions were thermal-cycled for 35 or 45 cycles. Products were analyzed by capillary electrophoresis to detect sequencing products.

Results: Reactions using two oligonucleotide primers at a ratio of 5:1 (500 nM primer 1 and 100 nM primer 2), 125 µM supplemental dNTPs, and 35–45 thermal cycles optimally supported combined amplification and cycle sequencing reactions. Our results suggest that these reactions are dominated by PCR during early cycles and convert to cycle sequencing in later cycles. We used this technique for a variety of sequencing applications, including the identification of germline mutations/polymorphisms in the Factor V and BRCA2 genes, sequencing of tumor DNA to identify somatic mutations in the DPC4/SMADH4 and FLT3 genes, and sequencing of 16S ribosomal DNA for bacterial speciation.

Conclusions: PCR amplification and cycle sequencing can be combined into a single reaction using the conditions described. This technique allows direct sequencing of genomic DNA, decreasing the cost and labor involved in gene sequencing.






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Copyright © 2005 by the American Association for Clinical Chemistry.