Worm Breeder's Gazette 12(3): 18 (June 15, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have begun DNA sequence analysis of 50 recessive mec-4 mutations. By combining aspects of a few different protocols we have come up with a method for sequencing of PCR products that is reliable and gives high quality data. We hope that this protocol may be of use to others involved in sequencing projects!
A. Generation of the template DNA for sequence analysis
1. Amplify genomic DNA in standard PCR reaction. We use 2 µg genomic DNA and approximately .5 µg each primer (our primers average 21 bp in length) in 100 µl reaction volume.
2 Run an agarose gel to assay the concentration of amplified DNA. Ideally, the concentration of DNA should be at least 1 µg in 10 µl. If the DNA concentration is very low, either ethanol precipitate to concentrate it for the next step or reamplify. Also if large visible amounts of primers remain, it is good to remove them as outlined in Step 4 below.
3 Amplify one strand of DNA. In separate experiments we have used either a new primer internal to the original boundaries of the PCR product or we reused on of the original primers for the single stranded amplification. Both approaches work quite well. Usually we use 10 µl of the DNA generated in Step 1 above (About 1 µg if possible) and about 200 ng of primer in 100 µl volume.
4. Purify the ss amplified DNA away from unincorporated primers by passing over Miniprep Spun Columns form Pharmacia. Alternatively, make homeade columns with Sephacryl S-400.
5. Check DNA concentration on an agarose gel. For certain success, about 2 µg of single-stranded DNA is used in each sequencing reaction. If the DNA concentration is too low, DNA can be ethanol precipitated to concentrate the sample or a second single-stranded amplification step is suggested. The amount of DNA template is very important.
B. Denaturation of the template and annealing of sequencing primer
1. Add 8 µl of template DNA and 1 µl of 1 M NaOH to a 1.5 ml microcentrifuge tube; incubate at room temperature for 10 minutes
2. Add 1 µl of 1 M HCl to neutralize the solution and then immediately add 2 µl (about 200 ng) of sequencing primer (from the complementary strand, of course) and 2 µl of sequencing buffer (as in Sequenase kit from US Biochemical). Note: perform this step on each tube individually, ie., if you are doing 10 reactions do not add HCl to each tube and then go back and add primers. Neutralize the first tube and add the appropriate primer and buffer and then start the next tube. This is important.
3. Incubate at 37° C for 15 minutes to allow the primer to anneal to template DNA
C. Sequencing reactions
Reactions are according to instructions in the USB Sequenase kit with a few changes:
Labelling reaction:
-14 µl annealed DNA (the entire reaction prepared above)
-1 µl DMSO (added fresh)
-1 µl DTT. 0.1 M
-2 µl diluted dITP mix (1:5 dilution)
-0.5 µl 35S
-2 µl diluted sequenase*
*When diluting Sequenase for reactions the dilution mix is made as follows:
enzyme dilution buffer, 6.5 µl pyrophosphatase, 0.5 µl Sequenase, 1 µl
as usual, do this just before starting reactions
Termination reaction:
Although the starting volume is slightly larger than in the USB protocol, still add 4 µl of labelled DNA into 2.5 µl of each dITP for termination reactions, add 4 µl stop solution.
Notes:
1. We can read at least 400 bp for each reaction.
2. This procedure works very well for sequencing double stranded plasmid DNA.