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

The following protocol and cycle conditions courtesy of Cheryl Heiner-ABI

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Protocol:

	Big Dye Terminator Mix*		16ul
	ThermoFidelase** 		1ul
	Primer (18-24mer)		15-30pmoles
	Genomic DNA Template		3-6ug
	Final Reaction Volume		40ul

Cycle Conditions:

	95 degC - 5 min. initial hold
	45 cycles of 
		95 degC - 30 sec.
		55 degC - 20 sec.
		60 degC - 4 min.
		4 degC hold

Purify through G-50 spin columns and load under standard gel conditions

				 *PE-ABI #4303150 for the 1000 reaction kit
					 Description:  TF,KIT BTD RR-1000
				**Fidelty Systems Inc.  (301) 527-8250
                                  http://www.fidelitysystems.com

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

Modified Bacterial Thermo-Cycle Conditions :

• 95 degC for 5 min. initial hold required for ThermoFidelase
• Followed by 100 cycles of:
• Rapid thermal ramp to 95degC
• 95 degC for 30 sec.
• Rapid thermal ramp to 50degC
• 55 degC for 20 sec.
• Rapid thermal ramp to 65degC
• 65 degC for 4 min.
• or 100 cycles of:
• Rapid thermal ramp to 95degC
• 95 degC for 30 sec.
• Rapid thermal ramp to 65degC
• 65 degC for 5 min.
• Followed by rapid thermal ramp to 4degC and hold until ready to purify through G-50 microtiter plate spin columns.

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:

• the amount of product increases as the extension temperature is increased to 65 degrees but decreaces dramatically after that, i.e. at 70 or 72degC.
• as the signal strength also increases approximately proportional to the increased primer length, but peaks at a 30 mer and then decreases with a 35 mer.

Abstract:
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.