Sequencing of genomic DNA by combined amplification and cycle sequencing reaction.
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 Dye 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 microM 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.
Murphy
Kathleen M
KM
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA. kmurphy@jhmi.edu
Berg
Karin D
KD
Eshleman
James R
JR
D024682:BRCA2 Protein / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005165:Factor V / Q000235:genetics; D016680:Genome, Bacterial; D015894:Genome, Human; D006801:Humans; D007711:Klebsiella pneumoniae / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D051901:Smad4 Protein; D013211:Staphylococcus aureus / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3
D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D005165:Factor V; C497970:FLT3 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3