Automated DNA sequencing

We will be using a sequencing procedure called PCR sequencing to sequence DNA. In PCR sequencing, we have a single primer that is complementary to the beginning of the sequence that we wish to determine. To sequence the DNA, we mix together one primer, plasmid DNA, and a mix that contains Taq DNA polymerase, dNTP, and ddNTP covalently linked to fluorescent dyes. We then put the samples into a PCR machine and run it just like a standard PCR procedure. This means that we cycle between three temperatures: (1) a high temperature to denature the DNA, (2) a low temperature to anneal the primers onto the plasmid DNA, and (3) a medium temperature that allows the Taq DNA polymerase to make DNA. When the DNA is made, if a ddNTP is added the chain ends, the chain is terminated, and ends with a colored base (hence they are called dye terminators). Since each of the four ddNTP has a different color, each DNA molecule that is made ends in a different color. We then separate the pieces via gel electrophoresis. As the DNA pieces pass near the bottom of the gel, the sequence is read using a laser. A computer then analyzes the colored DNA to determine the sequence. This handout details the sequencing.

Materials
DNA: from previous experiment(s). It can be plasmid or PCR generated PCR mix (Taq polymerase, ddNTP, dNTP, salts) [Note that this is very expensive] SeqSaver Primers (normally Forward and Reverse) Loading dye: Prepared by adding 40 l of blue dextran (prepared in EDTA) and 160 l of formamide.

Methods
ExoSAP procedure (needed only if you are sequencing PCR products) If you prepare DNA by PCR, the primers are still in your sample. To remove them, we use ExoSAP. ExoSAP is an exonuclease that removes ssDNA. If you have more than one band in your PCR reaction, you have to gel purify the band instead of using this procedure. Note that you need a nice band on your agarose gel in order to obtain a good sequence. a) In a PCR tube, mix together 5 l of your PCR reaction and 2 l of ExoSAP. b) Incubate for 15 minutes at 37C, and then 15 minutes at 80C. This is the ExoSAP program on the iCycler. If they are being used, program 25 on the Robocycler PCR machine is set up to do this (it uses 0.5 ml PCR tubes). Otherwise, set up two water baths with the correct temperatures. After the procedure, the sample is ready for sequencing. Use 1 or 2 l in your sequencing reactions (1 l for short PCR pieces and 2 l for longer ones).

Last printed on: September 30, 2004

PCR sequencing 1) For each of your samples, mix together the following (in order): Chemical Primer (e.g., forward or reverse) [1/30 dilution of stock] DNA Seq saver PCR mix H2O Volume (plasmid DNA) 1.0 l Up to 10.0 l [Normally 8 for minpreps] 3.0 l 1.0 l Add to 15 l total

Notes: (a) Keep the PCR mix and your samples on ice until they are put into the PCR machine! (b) If you are sequencing PCR products, be sure to first use the ExoSAP procedure to remove the primers from the PCR reaction, then use 2 l of the ExoSAPed DNA instead of 8 l of the DNA. The ExoSAP procedure is above. 2) Put the samples into the PCR machine and set it as follows: Set it at 95C for 5 minutes for one cycle. Set it to do 30 cycles of: 95C for 15 seconds 45C for 15 seconds 60C for 240 seconds Set it at 60C for an additional cycle of 240 seconds: Note: This is the iCycler Sequence program 3) Later that day or the next day: place your samples in the freezer. Removing primers We will now remove any unused primers from our samples. This is done by centrifugation through a matrix. Large DNA elutes while short DNA, including the primers, stays in the column. 4) Get one CentriSep column per sample. 5) Tap the column on the counter to get all the powder on the bottom of the column. 6) Remove the top and add 0.8 ml dH2O. Put the cap back on and invert the column or vortex it to hydrate all the gel. 7) Let the columns sit for 30 minutes. Obtain wash tubes while you are waiting. 8) Remove bubbles from the column by inverting the column or sharpely tapping it on the counter. Then
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take off the top cap. Next remove the bottom cap and place the column in a wash tube. 9) Discard any fluid that enters the tube. Then centrifuge at 1800 rpm for 2 minutes. 10) Discard the fluid in the wash tube. 11) Add 15 l of dH2O to each sample. 12) Place a sequence sample, all of it, in the center of the column (not on the edge of it), and place the column in a centrifuge tube. 13) Centrifuge again at 1800 rpm for 2 minutes. 14) Discard the column and place the sample on ice. 15) Put the sample in the concentrator (or Speed Vac) and let it dry. It takes 60 minutes or more. Adding dye 16) Place 1.25 l of the loading dye into each of the dry sample tubes. These were prepared by drying down the Centrisep purified samples. 17) Heat the samples at 85-90C for 2 minutes. Putting the sample onto the gel 18) Next, place the samples onto the comb for the sequencing gel. We use a 96-well comb, but put our samples into every other lane (tooth) in order to prevent contamination by the adjacent lane. Start loading samples at the 3rd tooth. Carefully pipet 0.5 l of each sample onto a tooth of the comb. Be sure to put each sample at the end (bottom) of the tooth. 19) Fill out the sample sheet on the computer by entering the sample name in all capital letters, with the name or plate (e.g., R1 or R11), then, if this is a sample from a plate, the row and column, then F (forward) or R (reverse). For example R2D2F (plate R2, row D, column 2, forward primer). Do not use spaces or dashes. This format allows us to better use the sequence analysis program. 20) Run the gel. It takes 3.5 hours. After the gel is run, the samples are tracked and analyzed by the computer to find the sequence of bases.

Last printed on: September 30, 2004