Methods for DNA sequencing

Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions (26) are modified from those presented earlier (25) by altering the absolute amounts and the relative deoxy/dideoxynucleotide ratios in the termination mixes. Two separate termination mixes provided optimal overlap for sequence data starting in the polylinker and extending to approximately 600 bases from the priming site. This two-step format eliminated the need for the chase required in the Bst one-step reaction (25).

Each extension reaction contained 500-750 ng of Biomek isolated single-stranded DNA, reaction buffer, nucleotide extension mix, oligonucleotide primer (typically M13 (-40) universal sequencing primer, see Appendix D), either [a-32-P]dATP or [a-35-S]dATP and Bst polymerase. After the reactions are extended for 2 minutes at 67℃ and briefly centrifuged, four aliquots are removed and added to the appropriate base-specific termination mix. All nucleotide mixes contained the guanosine nucleotide analog, 7-deaza-dGTP, but differed in their deoxy/dideoxynucleotide ratios to yield fragments ranging in size from the beginning of the polylinker to greater than 300 bases from the primer, or fragments from about 150 to greater than 600 bases from the primer for "short" or "long" mixes, respectively. Following an incubation at 67℃ for 10 min and a brief centrifugation, the reactions are stopped by the addition of dye/formamide/EDTA, and incubated at 100℃. When desired, sequencing reactions are stored at -70℃ prior to the addition of loading dye.

When double-stranded pUC-based subclones are used as templates, the amount of primer is doubled and a denaturing/annealing step is added. Here, 3 μg of plasmid DNA, isolated by either the mini- or midi-prep diatomaceous earth method, is mixed with primer, placed in a boiling-water bath, and rapidly cooled by plunging into an ethanol/dry-ice bath (28). Following an incubation on ice, the remaining sequencing extension reagents (reaction buffer, nucleotide extension mix, either [a-32-P]dATP or [a-35-S]dATP, and Bst polymerase) are added. Reactions are performed as described above for single-stranded sequencing.

Protocol

For single-stranded DNA sequencing:

1. Prepare the following extension reaction in a microcentrifuge tube:

750 ng  M13 template DNA

 2 μl  Bst reaction buffer

 2 μl  Bst nucleotide extension mix

 1 μl  oligonucleotide primer (2.5 ng/μl)

 0.5-1 μl [alpha]-32-P-dATP or [alpha]-35-S-dATP

 1 μl  diluted Bst polymerase (0.1 U/μl)

 q.s.  sterile ddH2O

 12 μl

[alpha]-32-P-dATP (PB 10384) and [alpha]-35-S-dATP (SJ 1304) from Amersham.

Dilute the Bst polymerase (BioRad 170-3406) in Bst dilution buffer.

2. Incubate the reactions for 2 minutes at 67℃, and briefly centrifuge to reclaim condensation.

3. Remove 2.5 μl aliquots for each reaction into the four base-specific termination mixes (either short or long), already pipetted into a V-bottomed microtiter plate (Dynatech).

4. Incubate the reactions for 10 minutes at 67℃, and briefly centrifuge to reclaim condensation. It is possible to store the reactions at -70℃ at this stage.

5. Stop the reactions by the addition of 4 μl of agarose gel loading dye and incubate for 5-7 minutes at 100℃.