Received on June 24, 2004
Accepted on September 27, 2004

Molecular Diagnostics and Genetics

Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

¹ Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD
² Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, and Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD

^* To whom correspondence should be addressed. 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, amplify the target sequence, purify the amplified product, and finally sequence the product. 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 Dye^TM 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.