Sequencing off Bacterial Genomic DNA with ABI's Big Dye Terminators

The ABI image file below is the data obtained using a synthetic 18mer under the above sequencing conditions and the 1.92Mbp Strep. pyogenes genomic DNA as the template. The 40 ul reaction was divided in half and purified by passage through G-50, but clearly we needed to do a better job of cleaning it up as too much terminator leaked through. However, the reaction really does work directly with genomic DNA as a template!!!!

Recent experiments with Genomic DNA Templates and Big Dyes

Reaction "size" Big Dye Mix (ul) 20-30mer
Primer (pmoles)
Purifed Bacterial Genomic DNA Template(ngrams) ThermoFidelase (ul) H20 to final volume of Number of Cycles
C. Heiner's 2x reaction 16 ul 15-30  3000-6000 1 ul 40 ul 45
Roe-Lab 1x reaction 8 ul 15-30  3000-6000 1 ul 20 ul 99

Modified Bacterial Thermo-Cycle Conditions :

Note #1: Increasing the number of cycles from 45 to 60 to 80 and eventually to 100, almost linearly improved both the signal and read length. Increasing the annealing temperature from 50 to 55degC and the extension temperature from 60 to 65degC also improved the signal and read length but to a lesser extent than increasing the number of cycles. Because of the increased number of cycles, these incubations only should be done overnight.

Note #2: ThermoFidelase improves the signal and read length with bacterial genomic templates for all primers tested.

Note #3: Although some primers work better than others and we have no clue why, it is clear that any proposed primer should be screened against the already known sequence data for the entire know portions of the bacterial genome, as well as sequencing vector sequences. Thus, to prevent the primer from binding to multiple places on the target clone, the primer sequence should be compared to all the contigs in the target clone database. Only those primers with a homology of less than 12/20 Smith Waterman Identity elsewhere on the target clone should be used for custom primer directed sequencing. The PrimOU computer program is available from our informatics group that has been modified from the SW Medical Center's Primo program, to allow screening against multiple contigs in a phred/phrap database at various stringencies. In addition, typically we choose primers with either a G or C on their 3'end and a roughly even distribution of each of the 4 bases or a slightly higher G/C content (i.e. 1-2 more G's or C's than A's or T's to shift the Tm slightly higher)

Note #4: As one increases the primer length, the annealing temp. can be increased so too can the extension temperature. We have tested 20,25,30,3 5 mers on bacterial genomic templates as well as increasing the extn temp from 60 to 65 to 72degC and the annealing temperature from 50 to 55, 60, 65, 70degC.

Our conclusion is that:

Cheryl Heiner and collegues at ABI have published a paper describing their work on sequencing directly off bacterial genomes. See:

Heiner CR, Hunkapiller KL, Chen SM, Glass JI, Chen EY
Sequencing multimegabase-template DNA with BigDye terminator chemistry
Genome Res 1998 May;8(5):557-61


Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of >700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial genome sequencing projects without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.

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