Biotechnology
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Simultaneous Bi-Directional Cycle Sequencing.
Stephen C. Roemer, Kurt A. Brumbaugh, Vince Boveia,
Mary Jensen, and John Gardner1
LI-COR, inc., Biotechnology Division, 4647 Superior St., Lincoln, NE 68504
and 1CEPRAP, University of California, Davis, CA
SBS Overview
Simultaneous bi-directional sequencing (SBS) is a method for sequencing both strands of double-stranded DNA by including two strand-specific primers (each carrying a unique label) in the same Sanger chain termination reaction (1,2).
The principle of SBS is now applied to a standard cycle sequencing reaction using a thermostable polymerase and two opposite primers tagged with different NIR fluorescent dyes. The method can be used with four commercial sequencing kits:
- SequiTherm Long-Read Cycle Sequencing Kit-LC (Cat # S36100LC), Epicentre Technologies, Madison, WI.
- SequiTherm Excel Long-Read DNA Sequencing Kit-LC (Cat # SE6100LC), Epicentre Technologies, Madison, WI.
- Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Cat # RPN 2438), Amersham Life Science, Cleveland, OH.
- CircumVent Thermal Cycle Sequencing Kit (Cat # 430-100), New England Biolabs, Beverly, MA.
*The amount of template (plasmid DNA) used in a bi-directional cycle sequencing reaction must be adjusted for different insert sizes (i.e. the distance in nucleotides between the forward and reverse primer annealing sites).
| Insert Size | Template Amount |
| 300-500 bp | 50-100 fmol |
| 600-1200 bp | 125-200 fmol |
| 1300-1800 bp | 225-300 fmol |
| > 1800 bp | 300-500 fmol |
Four µl of the premix is added to an appropriate volume of the termination mix (2 µl in the case of SequiTherm Long Read Cycle Sequencing Kit). Thermal cycling conditions for the elongation/termination reactions are 2 min at 92 °C for initial denaturation and (92 °C 30 sec, 50 °C 30 sec, 70 °C 30 sec) x 30 cycles. Four µl of stop solution is added to the completed sequencing reaction. Samples are then denatured at 92 °C for 3 min and 1 - 2 µl loaded on a sequencing gel. LI-COR's new dual-dye automated DNA sequencer (Model 4200 IR2 System) is used to electrophorese and analyze two sequence ladders from a single bi-directional sequencing reaction.
