Method: Purification and Sequencing of PCR Product

Terry C. Lairmore

Principle:

This method is used to sequence directly a specific PCR product after large scale amplification. The reaction should be carried out in the usual fashion (see "PCR Method"), except that after optimization of annealing temperature and other conditions in the 5 µl volume, a large scale 50 µl (10X) total reaction volume should be used to generate sufficient PCR product for sequencing. The large scale reactions are carried out exactly as detailed in the polymerase chain reaction method, except that the reaction is scaled up 10 fold. One of the PCR primers or a new internal primer can serve as the sequencing primer. However, it is necessary to purify the PCR product from excess unreacted primers left over from the PCR reaction before sequencing. This is accomplished by spinning the PCR product on Sepharose exclusion columns. If the PCR product to be sequenced is very specific (a single strong band), it is possible to add the product directly to the columns for purification. However, if the PCR reaction generates a number of nonspecific light bands, the band of interest should be cut out of the gel and electroeluted or otherwise recovered before loading on the columns.

Time required:

3 hours

Special reagents:

• Linkers 6 quick-spin Sepharose columns CL-6B (Boehringer Mannheim, #100639)

Special equipment:

• Vacuum dessicator

Procedure:

1. Reduce total volume of the PCR reaction product (50 µl) to approximately 25 µl by placing in a vacuum dessicator for 30-60 minutes.
2. Pre-spin the Linkers 6 quick-spin Sepharose columns CL-6B (place columns within a 15 ml snap cap to spin), for 5-10 minutes at 1,100 x g in a swinging bucket rotor to get rid of excess buffer and discard the flow-through. Change the collection tube and pre-spin a second time for 5-10 minutes at 1,100 x g and again discard the buffer.
3. Load 25 µl (maximum volume for the columns) directly into the center of each column and spin at 1,100 x g for 10 minutes in Beckman TJ-6 (approximately 2500 rpm), collecting the product in a clean labeled tube. Yield is approximately 35-50 µl.
4. Remove 1/5 of purified product to a clean tube. To denature the DNA in the aliquot add 0.2 N NaOH (to total volume of 40 µl), mix and hold at room temperature for 5 minutes.
5. Neutralize the solution with 1/3 volume (13.3 µl) of 3 M KOAc and 2.5 volumes (100 µl) of 100% ethanol.
6. Chill to -20 degrees C for 20 minutes, then pellet the precipitate with a 15 minute, 12,000 rpm spin in a 4 degrees C microcentrifuge.
7. Wash the pellet with 70% ethanol and air dry or dry in a vacuum dessicator.

Sequencing may be carried out using the SequenaseR kit and a slight modification of the double stranded sequencing protocol:

1. Resuspend pellet (not always visible) in:

   6 µl ddH20
   2 µl 5X Sequencing buffer
   2 µl Sequencing primer (0.5 pmol/µl)
   -----
   10 µl

2. Place tube at 37 degrees C for 15 minutes for annealing of primer and template.
3. Add 2 µl diluted labeling mix (1:5 to 1:15)

   1 µl 100 mM DTT
   1 µl 32P dATP
   2 µl diluted Sequenase (diluted 1:8 in ice-cold TE immediately 
   before use).  Allow the reaction to extend at room temperature 
   (or at 4 degrees C) for 5 minutes.

4. Dispense 3.5 µl into each of 4 prelabeled tubes (G, A, T, C) containing 2.5 µl of appropriate termination mix (ddGTP, ddATP, ddTTP, ddCTP) and incubate tubes at 37 degrees C for 5 minutes or longer (volume of each tube is 6 µl).
5. Add 4 µl of Sequenase stop solution to each tube. Samples may be stored at -20 degrees C until they are run on a gel.
6. Heat the samples to 75-80 degrees C for 2 minutes and load 1-3 µl per lane immediately onto a sequencing gel.

method after B. Dubose (pers. comm., Hartl lab)